Interleukin-2 variants and uses thereof in treatment of cancer

Abstract

The present application generally relates to an isolated interleukin-2 (IL-2) variant that exhibits a reduced affinity for the alpha-subunit of the IL-2 receptor (IL-2R), which can be used as an immunotherapeutic agent. In addition, the present application also encompasses a pharmaceutical composition comprising the isolated IL-2 variant, and the use of the pharmaceutical composition to treat cancer in an individual.

Description

[0001] Related Applications

[0002] This application claims the filing date of U.S. Provisional Application No. 63 / 398,290, filed on August 16, 2022, the entire contents of which are incorporated into this specification by reference.

[0003] Sequence Listing

[0004] This application also proposes an electronic file of the sequence listing XML, which was created on August 4, 2023, with the file name "P4261-PCT_SeqList_20230804_filed.xml" and a size of 36kb. The information in the electronic file of the sequence listing XML is incorporated into this specification by reference. Technical Field

[0006] The present application generally relates to the technical field of cancer treatment. More specifically, the present application relates to interleukin-2 (IL-2) variants and their use in treating cancer. Background Art

[0008] Cancer is a disease in which abnormal cells in a certain part of the body divide uncontrollably and may invade or spread to other parts of the body. Cancer has long been a serious threat to the health of all mankind and has become a heavy burden on the social medical and health system. According to statistics provided by the World Health Organization, in 2020 alone, there were 19,292,789 new cases of all cancers in men and women of all ages, and 9,958,133 deaths from cancer, causing significant human life and economic losses.

[0009] Therefore, scientists around the world are committed to developing effective cancer treatment methods to improve the efficacy of cancer treatment. So far, current treatment methods include surgery, chemotherapy, radiotherapy, hormone therapy, thermotherapy, palliative care, etc. for treating cancer. However, such traditional methods are often accompanied by serious side effects, for example, damage to normal tissues or cytotoxicity. In view of the above problems, treatment methods with fewer side effects have been developed in recent years, some of which are in the stage of research or preclinical trials. Among these treatment methods, immunotherapy using biomacromolecules (e.g., peptides, nucleic acids, carbohydrates, etc.) is considered to be the most promising treatment method because it eliminates tumors by regulating the function of the host immune system, thereby minimizing the side effects that may be caused to patients.

[0010] IL-2 is a potential immunotherapeutic agent for the treatment of cancer. Wild-type IL-2 (also known as proleukin, a T cell growth factor (TCGF)) is a 15.5 kilodalton (kDa) globular glycoprotein that is involved in the generation, survival, and homeostasis of lymphocytes. IL-2 is primarily produced by activated T cells (especially CD4 + It is synthesized by T helper cells. It stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTLs), induces the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes the expression of cytokines and cytolytic molecules by T cells, promotes the proliferation and differentiation of B cells and the synthesis of immunoglobulins by B cells, and stimulates the generation, proliferation, and activation of natural killer (NK) cells. IL-2 is considered an attractive therapeutic option for the treatment of cancer because it can expand the lymphocyte population in the body and increase the effector function of these cells, thereby producing anti-tumor effects.

[0011] However, IL-2 has a dual function in immune responses. It not only mediates the expansion and activity of effector cells, but also plays a key role in maintaining peripheral immune tolerance. IL-2 mediates its effects by binding to the IL-2 receptor (IL-2R), which consists of an α-subunit (CD25), a β-subunit (CD122), and a γ-subunit (CD34). c -subunit (γ c , CD132) and other subunits to form a dimer (containing β / γ c -subunits) or trimer (containing α / β / γ c Of the above subunits, the α-subunit is the one that confers high affinity for binding to its receptor, while the β-subunit and γ-subunit c -subunit is crucial for signal transduction. In the immune tolerance part, IL-2 binds to regulatory T (T reg ) cells express IL-2R trimers (i.e., α / β / γ c -subunit) to participate, and after binding, activate T regThe activity of cells, which in turn triggers the immunosuppression of the surrounding environment, thereby impairing the host's anti-tumor immunity. Based on this, wild-type IL-2 is not the best choice for inhibiting tumor growth, and optimized IL-2, by screening a series of IL-2 variants, to find a variant with reduced affinity for the α-subunit of IL-2R and with the β-subunit and γ-subunit of IL-2R. c -subunit affinity is not affected, which may help alleviate T reg The immunosuppression caused by IL-2 cells can enhance the overall anti-tumor activity of IL-2.

[0012] In view of this, the relevant field urgently needs to find the best IL-2 variant to overcome the problems associated with IL-2 immunotherapy, by cutting out the unwanted immune tolerance activity in the IL-2 protein, thereby enhancing its therapeutic applicability. Summary of the invention

[0013] The following is a brief summary of the present application to help readers have a basic understanding of the present application. This summary is not a comprehensive overview of the present application, nor is it used to identify the key / determining components of the present application, or to outline the scope of the present application. Its only purpose is to present certain concepts of the present application in a simplified form as a prelude to a more detailed description that will be presented later.

[0014] As embodied and broadly described herein, one aspect of the present application is directed to an isolated IL-2 variant that exhibits reduced affinity for the α-subunit of IL-2R and increased affinity for the β-subunit and γ-subunit of IL-2R. c -subunit affinity is not affected, the isolated IL-2 variant has an amino acid sequence with a sequence number of at least 85% similarity to SEQ ID NO: 1, wherein the isolated IL-2 variant comprises an amino acid substitution at the 42nd, 45th, 50th, 72nd or 125th position corresponding to SEQ ID NO: 1, wherein:

[0015] The amino acid substitution at position 42 in SEQ ID NO: 1 is substitution of phenylalanine (F) with alanine (A), cysteine ​​(C), glutamic acid (E), histidine (H), valine (V), or tryptophan (W);

[0016] The amino acid substitution at position 45 corresponding to SEQ ID NO: 1 is substitution of tyrosine (Y) with alanine (A), aspartic acid (D), glycine (G), methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S), or threonine (T);

[0017] The amino acid substitution at position 50 corresponding to SEQ ID NO: 1 is substitution of alanine (A) with isoleucine (I);

[0018] The amino acid substitution at position 72 corresponding to SEQ ID NO: 1 is substitution of leucine (L) with aspartic acid (D), isoleucine (I), lysine (K), asparagine (N), or valine (V); and

[0019] The amino acid substitution at the position corresponding to the 125th position in SEQ ID NO: 1 is substitution of cysteine ​​(C) with glycine (G).

[0020] According to certain embodiments of the present application, the isolated IL-2 variant comprises an amino acid sequence having a sequence similarity of at least 85% to sequence number: 1 and the following amino acid substitutions: the phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is replaced by tryptophan (W), and the leucine (L) at the 72nd position corresponding to sequence number: 1 is replaced by lysine (K).

[0021] According to other embodiments of the present application, the isolated IL-2 variant comprises an amino acid sequence having a sequence similarity of at least 85% to sequence number: 1 and the following amino acid substitutions: the phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with valine (V), the tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with alanine (A), the leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N), and the cysteine ​​(C) at the 125th position corresponding to sequence number: 1 is substituted with glycine (G).

[0022] According to another embodiment of the present application, the isolated IL-2 variant comprises an amino acid sequence having a sequence similarity of at least 85% with sequence number: 1 and the following amino acid substitutions: the phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is replaced by glutamic acid (E), and the tyrosine (Y) at the 45th position corresponding to sequence number: 1 is replaced by glycine (G).

[0023] Optionally, the isolated IL-2 variant comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 1 and the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with valine (V), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with serine (S), leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N), and cysteine ​​(C) at the 125th position corresponding to sequence number: 1 is substituted with glycine (G).

[0024] Alternatively or additionally, the isolated IL-2 variant comprises an amino acid sequence having a sequence similarity of at least 85% to sequence number: 1 and the following amino acid substitutions: the phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with histidine (H), the tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with aspartic acid (D), the leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with isoleucine (I), and the cysteine ​​(C) at the 125th position corresponding to sequence number: 1 is substituted with glycine (G).

[0025] In certain further embodiments of the present application, the isolated IL-2 variant comprises an amino acid sequence having a sequence similarity of at least 85% to sequence number: 1 and the following amino acid substitutions: the phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with cysteine ​​(C), the tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with asparagine (N), and the leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with valine (V).

[0026] Alternatively, in a specific embodiment of the present application, the isolated IL-2 variant comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 1 and the following amino acid substitution: alanine (A) at the 50th position corresponding to sequence number: 1 is replaced by isoleucine (I).

[0027] According to certain specific embodiments of the present application, the isolated IL-2 variant comprises an amino acid sequence having a sequence similarity of at least 85% to sequence number: 1 and the following amino acid substitutions: the phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with alanine (A), the tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with aspartic acid (D), and the leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with aspartic acid (D).

[0028] In certain further embodiments of the present application, the isolated IL-2 variant comprises an amino acid sequence having a sequence similarity of at least 85% to sequence number: 1 and the following amino acid substitutions: the phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with alanine (A), the tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with methionine (M), and the leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N).

[0029] Still further in certain embodiments of the present application, the isolated IL-2 variant comprises an amino acid sequence having a sequence similarity of at least 85% to sequence number: 1 and the following amino acid substitutions: the phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with alanine (A), the tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with arginine (R), and the leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N).

[0030] According to certain preferred embodiments of the present application, the isolated IL-2 variant comprises an amino acid sequence having at least 90% sequence similarity to sequence number: 1; more preferably, the isolated IL-2 variant comprises an amino acid sequence having at least 95% sequence similarity to sequence number: 1; even more preferably, the isolated IL-2 variant comprises the amino acid sequence of sequence number: 1.

[0031] Accordingly, the present application also encompasses a pharmaceutical composition comprising the above-mentioned isolated IL-2 variant and a pharmaceutically acceptable carrier.

[0032] Another aspect of the present application is a method for treating cancer in an individual by using the IL-2 variant isolated by the present application or the pharmaceutical composition of the present application containing the variant, the method comprising the step of administering to the individual an effective amount of the IL-2 variant isolated by the present application or the pharmaceutical composition of the present application.

[0033] According to certain preferred embodiments of the present application, the isolated IL-2 variant of the present application or the pharmaceutical composition of the present application is administered to the individual in an amount of 1-500 μg / kg body weight.

[0034] The cancer suitable for treatment by the IL-2 variant isolated by the present application, the pharmaceutical composition by the present application, or the method by the present application can be bladder cancer, bile duct cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, epidermal cancer, gastric cancer, gastrointestinal stromal tumor (GIST), glioma, lymphoid hematopoietic tissue tumor, liver cancer, Kaposi's sarcoma, leukemia, lung cancer, lymphoma, intestinal cancer, melanoma, myeloid leukemia, pancreatic cancer, prostate cancer, retinoblastoma, ovarian cancer, renal cell carcinoma, spleen cancer, squamous cell carcinoma, thyroid cancer, or thyroid follicular carcinoma.

[0035] According to certain advanced embodiments of the present application, the method of the present application further comprises the step of subjecting the individual in need of treatment to surgery, radiotherapy, chemotherapy, immunotherapy, hormonal therapy, or a combination thereof before, simultaneously with, or after administering the isolated IL-2 variant of the present application or the pharmaceutical composition of the present application.

[0036] In certain preferred embodiments of the present application, the IL-2 variant isolated by the present application or the pharmaceutical composition of the present application is used in combination with immunotherapy, wherein the immunotherapy comprises administering to the individual an effective amount of CC motif chemokine ligand 3 (CCL3), CC motif chemokine ligand 26 (CCL26), CXC motif chemokine ligand 7 (CX-C motif chemokine ligand 7). 7, CXCL7); granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α); interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15); apremilast, imiquimod (imiquimod), lenalidomide, pomalidomide, sipuleucel-T, thalidomide; anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody; anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD11a antibody, anti-CD20 antibody, anti-CD25 antibody, anti-CD52 antibody, anti-EGFR antibody, anti-HER2 antibody, anti-PCDP1 antibody, anti-SLAMF7 antibody, or anti-Trop-2 antibody. According to a better embodiment of the present application, the immunotherapy used in combination with the IL-2 variant separated by the present application or the pharmaceutical composition of the present application in the method of the present application comprises administering an effective amount of IL-15 to the individual.

[0037] The individual that can be treated by the IL-2 variant isolated by the present application, the pharmaceutical composition by the present application, or the method by the present application is a mammal, for example, a human, a mouse, a rat, a guinea pig, a hamster, a monkey, a pig, a dog, a cat, a horse, a sheep, a goat, a cow, and a rabbit. Preferably, the individual is a human.

[0038] After referring to the following detailed description and drawings, many of the accompanying features and advantages of the present application will be easily understood. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The present application and other features, aspects and advantages will become more apparent after reviewing the following detailed description, claims and drawings, in which:

[0040] Figures 1A-1E The results of characteristic analysis of the IL-2 variants of the present application prepared according to the embodiments of the present application are described. Figure 1A Results from Western blots illustrate the expression of specific IL-2 variants. Figure 1B-1E is the result of flow cytometry, illustrating the expression of specific IL-2 variants (in Figure 1B and 1D -1E, 30 nanomolar concentration (nM) was used; Figure 1C After treatment with 4 nM of IL-2R, CTLL-2 cells (expressing α / β / γ c -subunit; Figure 1B ) or HH cells (expressing IL-2R β / γ c -subunit; Figure 1C-1E ) induced by the phosphorylated STAT-5 (pSTAT-5) expression. MFI, mean fluorescence intensity; pSTAT-5 + The percentage of cells (%) shows the percentage of pSTAT-5-positive cells.

[0041] Figures 2A-2F are flow cytometry results illustrating the effects of specific IL-2 variants on CTLL-2 cells ( Figure 2A , 2C and 2E) or HH cells ( Figure 2B , 2D IL-2v(AAG), a mutant IL-2 (AAG: IL-2 with F42A, Y45A, and L72G substitutions).

[0042] Figures 3A-3D is the relative organ weight of mice after a specific treatment ( Figure 3A ,lung; Figure 3B ,liver; Figure 3C , spleen; relative to body weight), and its body weight ( Figure 3D ). **, p<0.01; ***, p<0.005; ****, p<0.0001.

[0043] Figures 4A-4C Flow cytometry results showing active CD8 in the spleen of mice after specific treatment + T cells ( Figure 4A), active NK cells ( Figure 4B ), and T reg cell( Figure 4C ) percentage. **, p<0.01; ***, p<0.005; ****, p<0.0001. DETAILED DESCRIPTION

[0044] In order to make the description of this application more detailed and complete, the following is an illustrative description of the implementation aspects and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation covers the features of multiple specific embodiments and the method steps and sequences used to construct and operate these specific embodiments. However, other specific embodiments can also be used to achieve the same or equivalent functions and step sequences.

[0045] I. Definitions

[0046] For convenience, the specific proper nouns used in this specification, embodiments and claims are collected here. Unless otherwise defined in this specification, the meaning of scientific and technical terms used herein is the same as that understood and used by those skilled in the art. In addition, in the case of not conflicting with the context, the singular noun used in this specification covers the plural form of the noun, and the singular form of the noun is also covered when the plural noun is used. Specifically, in this specification and the scope of the patent application, the singular form "one" (a, an and the) includes plural reference values, except where otherwise indicated by the context. In addition, in this specification and the scope of the patent application, the expressions "at least one" and "one or more" have the same meaning, both of which represent the inclusion of one, two, three or more. Unless otherwise specified, the practice of this application will adopt conventional techniques in the fields of molecular biology, tumor biology, microbiology, and immunology, which are all within the technical scope of this field. Such techniques have been explained in detail in the public literature.

[0047] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate values, the relevant numerical values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value will inevitably contain standard deviations due to individual test methods. Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range. Alternatively, the word "about" means that the actual value falls within the acceptable standard error of the mean value, depending on the consideration of those skilled in the art. Except for the examples, or unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used herein (for example, to describe the amount of material, the length of time, temperature, operating conditions, quantitative ratios, etc.) are modified by "about". Accordingly, unless otherwise indicated to the contrary, the numerical parameters disclosed in this specification and the attached patent scope are all approximate values ​​and can be changed as needed. At least, these numerical parameters should be understood as the indicated significant digits and the values ​​obtained by applying the general rounding method.

[0048] Unless otherwise indicated, the term "interleukin-2" or "IL-2" as used herein refers to any native IL-2 polypeptide from any vertebrate source, including IL-2 polypeptides from mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed IL-2 as well as any form of IL-2 produced by intracellular processing. The term also encompasses naturally occurring IL-2 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is shown in SEQ ID NO: 1. Unprocessed human IL-2 also contains a 20 amino acid signal peptide at the N-terminus, which is not present in the mature IL-2 molecule.

[0049] As used herein, the term "IL-2 variant" is intended to encompass any mutant form of various forms of IL-2 polypeptides, including full-length IL-2, truncated forms of IL-2, and forms in which IL-2 is linked to another molecule by fusion or chemical conjugation. When referring to IL-2, "full-length" means a mature, native-length IL-2 molecule. For example, full-length human IL-2 refers to a molecule having 133 amino acids (see, e.g., SEQ ID NO: 1). Various forms of IL-2 variants are characterized by having at least one amino acid mutation that affects the interaction of IL-2 with the α-subunit (CD25) of IL-2R. The mutation may involve substitution, deletion, truncation, or modification of a wild-type amino acid residue normally located at that position. Preferably, the mutant is obtained by amino acid substitution. Unless otherwise indicated, IL-2 variants may be referred to herein as IL-2 mutants, IL-2 mutant peptide sequences, IL-2 mutant polypeptides, IL-2 mutant proteins, or IL-2 mutant analogs. The naming of various forms of IL-2 herein is relative to the sequence shown in SEQ ID NO: 1. Various nomenclatures may be used herein to refer to the same mutation. For example, a mutation in which phenylalanine at position 42 is replaced with alanine may be represented as 42A, A42, F42A, or Phe42Ala.

[0050] As used herein, a "wild-type" form of IL-2 refers to a form of IL-2 that is otherwise identical to an IL-2 variant, but has a wild-type amino acid at each mutated amino acid position of the IL-2 variant. For example, if the IL-2 variant is full-length IL-2 (i.e., IL-2 that is not fused or conjugated to any other molecule), the wild-type form of the mutant is full-length native IL-2. If the IL-2 variant is a fusion between a mutant IL-2 and another encoded polypeptide (e.g., an antibody chain) located downstream of IL-2, the wild-type form of the IL-2 variant is a fusion of the wild-type IL-2 amino acid sequence to the same downstream polypeptide. In addition, if the IL-2 variant is a truncated form of IL-2 (having a mutation or modified sequence in the non-truncated portion), the wild-type form of the IL-2 variant is a form of IL-2 that is also truncated, but has a wild-type sequence. For the purpose of comparing the binding affinity or biological activity of various forms of IL-2 variants compared to their corresponding wild-type IL-2 forms to the IL-2 receptor, the term "wild-type" also encompasses a form of IL-2 that contains one or more amino acid mutations that do not affect its binding to the IL-2 receptor compared to naturally occurring native IL-2, such as replacement of the cysteine ​​residue at position 125 of human IL-2 with alanine.

[0051] As used herein, the term "high-affinity IL-2R" refers to the heterotrimeric form of IL-2R, which consists of the receptor α-subunit (also known as CD25 or P55), the receptor β-subunit (also known as CD122 or P70), and the receptor γ-subunit (also known as CD122 or P70). c -subunit (also known as common cytokine receptor gamma c -subunit, γ c , or CD132). In contrast, the term "intermediate-affinity IL-2R" refers to a protein that consists of only the β-subunit and the γ-subunit. c -subunit, but not the α-subunit of IL-2 receptor.

[0052] As used herein, the term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise specified, the term "binding affinity" as used herein refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). The affinity of a molecule X for its partner Y can usually be expressed in terms of the dissociation constant (K). D ), which is the dissociation rate constant (K off ) and the binding rate constant (K on ). Thus, "equivalent affinity" may include different rate constants as long as the ratio of the rate constants remains constant. Affinity can be measured by any established method known in the art, such as surface plasmon resonance (SPR) analysis using standard instrumentation such as a BIAcore instrument, where each subunit of IL-2R can be obtained by expressing recombinant protein. Alternatively, cell lines known to express certain IL-2R forms can be used to assess the binding affinity of IL-2 variants to different IL-2R forms.

[0053] As used herein, the term "effector cell" refers to a population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells, such as CD8 + Cytotoxic T cells, NK cells, LAK cells, and macrophages / monocytes.

[0054] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly connected by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain structure composed of two or more amino acids, and does not specify a specific product length. Therefore, "peptide", "dipeptide", "tripeptide", "oligopeptide", "protein", "amino acid chain", or other words used to refer to any two or more amino acid chains are included in the definition of "polypeptide", and the term "polypeptide" can replace or be used interchangeably with these words. "Polypeptide" also means a post-expression modification product of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be from a natural biological source or produced by recombinant technology, but it is not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including by chemical synthesis. The size of the polypeptides of the invention may be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are referred to as folded, while those that do not have a defined three-dimensional structure, but can adopt a large number of different configurations, are referred to as unfolded.

[0055] An "isolated" polypeptide or variant or derivative thereof refers to a polypeptide that is not present in its native source. No particular degree of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. For purposes of the present invention, polypeptides and proteins produced by recombinant means expressed in host cells are considered isolated, as are native polypeptides or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0056] As discussed herein, slight changes in the amino acid sequence in the IL-2 variants of the present application are still considered to be included in the inventive concepts disclosed and claimed in the present application, as long as these changes occur at positions other than the 42nd, 45th, 50th, 72nd or 125th positions in SEQ ID NO: 1 (i.e., changes occur at positions 1-41st, 43-44th, 46-49th, 51-71st, 73-124th, or 126-133th positions in SEQ ID NO: 1), and maintain at least 85% sequence similarity with SEQ ID NO: 1 (e.g., having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence similarity with SEQ ID NO: 1), and do not affect the binding affinity of the IL-2 variants of the present application to the α-subunit of IL-2R.

[0057] "Percentage (%) of sequence similarity" is defined as the percentage of identity of the amino acid residues in the candidate sequence with the amino acid residues in the specific polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage of sequence similarity, and without considering any conservative substitutions as part of the sequence similarity. The sequence similarity percentage can be compared and confirmed using various methods well known to those skilled in the art, for example, using public computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate measurement alignment parameters, including any desired algorithm to achieve the maximum alignment value for the full-length sequence being compared. To achieve this purpose, the sequence alignment between the two amino acid sequences is analyzed using the computer software Blastp (protein-protein BLAST) provided by the National Center for Biotechnology Information (NCBI) on the Internet. The percentage of amino acid sequence similarity between a given amino acid sequence A and a given amino acid sequence B (or, a given amino acid sequence A has a certain % sequence similarity with a given amino acid sequence B) is calculated using the following formula:

[0058] Where X is the number of amino acid residues, which is the number of identical matches obtained by the sequence alignment program BLAST after aligning the A and B sequences, and Y is the total number of amino acid residues in the A or B sequence, whichever is shorter.

[0059] The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredients contained therein to be effective, and which contains no additional ingredients which would be unacceptably toxic to a subject to which the composition would be administered.

[0060] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, carrier, solvent, or encapsulating material, which is involved in carrying or transporting a subject drug from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients in the formulation. The pharmaceutical formulation contains the IL-2 variant of the present invention and one or more pharmaceutically acceptable ingredients. The carrier can be in the form of a solid (e.g., a capsule), a semisolid (e.g., a cream), or a liquid (e.g., a diluent). These pharmaceutical formulations are also the subject of the present invention. Generally speaking, the amount of active ingredient is about 0.1-95% by weight of the formulation, preferably about 0.2-20% by weight of the formulation in a formulation for parenteral use, and preferably about 1-50% by weight of the formulation in a formulation for oral administration. For clinical use of the method of the present application, the pharmaceutical composition of the present invention can be formulated into a formulation suitable for a predetermined route of administration.

[0061] The term "treatment" as used herein may refer to a curative or palliative measure. Specifically, the term "treatment" as used herein refers to the application or administration of the IL-2 variant of the present application, or a pharmaceutical composition comprising them, to an individual suffering from cancer, or suffering from a symptom or disease associated with cancer, or suffering from a secondary disease or condition secondary to cancer, in order to partially or completely alleviate, ameliorate, relieve, delay the onset, inhibit the progression, reduce the severity, and / or reduce the incidence of one or more symptoms or conditions associated with cancer.

[0062] The terms "administered, administering or administration" are used interchangeably herein to refer to the direct administration of the IL-2 variant of the present application, the pharmaceutical composition comprising the IL-2 variant of the present application, and / or the method of the present invention.

[0063] As used herein, the term "an effective amount" refers to an amount effective, at the dosage and for the period of time necessary, to achieve the desired therapeutic outcome for the treatment of cancer. For therapeutic purposes, an effective amount may also refer to an amount in which any toxic or deleterious effects of an ingredient are outweighed by the therapeutically beneficial effects. The specific effective amount or sufficient amount will vary depending on a variety of factors, such as the specific condition being treated, the physical condition of the patient (e.g., the patient's weight, age, or sex), the species of mammal or animal being treated, the duration of treatment, the nature of concurrent therapy (if any), and the specific formulation and structure of the compound or its derivatives employed. An effective amount can be expressed, for example, as the total mass of active ingredient (e.g., in grams, milligrams, or micrograms), or as the ratio of the mass of active ingredient to body weight (e.g., in milligrams per kilogram (mg / kg)). Alternatively, the effective amount can be expressed as the concentration of the active ingredient (e.g., the IL-2 variant of the present application), for example, molar concentration, mass concentration, volume concentration, molality, mole fraction, mass fraction, and mixing ratio. Those skilled in the art can calculate the human equivalent dose (HED) of the drug (e.g., the IL-2 variant of the present application) based on the dose determined by the animal model. For example, the HED can be estimated according to the industry guide "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" issued by the U.S. Food and Drug Administration (FDA) to estimate the maximum safe dose for human individuals.

[0064] The term "subject" or "patient" refers to animals, including human species, that can be treated with the IL-2 variants, pharmaceutical compositions, and / or methods of the present application. Unless a gender is explicitly indicated, the term "subject" or "patient" means both male and female. Accordingly, the term "subject" or "patient" includes any mammal that may benefit from cancer treatment. In an exemplary embodiment, the subject is a human.

[0065] II. Detailed Description of the Invention

[0066] The present application is based at least in part on the discovery that the IL-2 variants of the present application have reduced or ablated affinity for the α-subunit of IL-2R, resulting in improved immunotherapy properties for the IL-2 variants of the present application. As described above, different forms of IL-2R are composed of different subunits and exhibit different affinities for IL-2. The intermediate affinity IL-2R has a receptor β / γ c -subunit and is expressed in resting effector cells (e.g., natural killer cells and T cells) and is sufficient for IL-2 signaling upon binding to IL-2, thereby inducing proliferation and activation of these effector cells. In contrast, the high-affinity IL-2R additionally contains the receptor α-subunit and is primarily expressed in T cells. reg When combined with IL-2, it can promote T cells and activated effector cells. reg Therefore, reducing the affinity of IL-2 for the α-subunit of IL-2R may reduce the T cell-mediated immunosuppression induced by IL-2. reg The present invention provides a novel IL-2 variant with a lower affinity for the α-subunit of IL-2R to confer the desired immunotherapy properties to IL-2.

[0067] 1. IL-2 variants

[0068] Accordingly, the first aspect of the present application relates to an isolated IL-2 variant, which comprises at least one amino acid mutation, which causes the IL-2 variant to have a reduced or eliminated affinity for the α-subunit of IL-2R compared to wild-type IL-2, while retaining affinity for IL-2R with moderate affinity. The isolated IL-2 variant has a reduced affinity for the α-subunit of IL-2R, and comprises an amino acid sequence having a sequence similarity of at least 85% to SEQ ID NO: 1, wherein at least one amino acid substitution in the isolated IL-2 variant occurs at a position corresponding to the 42nd, 45th, 50th, 72nd or 125th position in SEQ ID NO: 1, wherein:

[0069] The amino acid substitution at position 42 in SEQ ID NO: 1 is substitution of phenylalanine (F) with alanine (A), cysteine ​​(C), glutamic acid (E), histidine (H), valine (V), or tryptophan (W);

[0070] The amino acid substitution at position 45 corresponding to SEQ ID NO: 1 is substitution of tyrosine (Y) with alanine (A), aspartic acid (D), glycine (G), methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S), or threonine (T);

[0071] The amino acid substitution at position 50 corresponding to SEQ ID NO: 1 is substitution of alanine (A) with isoleucine (I);

[0072] The amino acid substitution at position 72 corresponding to SEQ ID NO: 1 is substitution of leucine (L) with aspartic acid (D), isoleucine (I), lysine (K), asparagine (N), or valine (V); and

[0073] The amino acid substitution at position 125 corresponding to SEQ ID NO: 1 is substitution of cysteine ​​(C) with glycine (G);

[0074] These variants exhibit substantially similar binding affinity to the intermediate affinity IL-2R as compared to wild-type IL-2, and have substantially reduced affinity to the α-subunit of IL-2R or the high affinity IL-2R.

[0075] According to an embodiment of the present application, the isolated IL-2 variant may have: an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 1, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence similarity to SEQ ID NO: 1; preferably, the isolated IL-2 variant comprises an amino acid sequence having at least 90% sequence similarity to SEQ ID NO: 1; more preferably, the isolated IL-2 variant comprises an amino acid sequence having at least 95% sequence similarity to SEQ ID NO: 1. preferably, the isolated IL-2 variant comprises the amino acid sequence of SEQ ID NO: 1; wherein the at least one amino acid substitution occurs at position corresponding to the 42nd, 45th, 50th, 72nd or 125th position in SEQ ID NO: 1, and the amino acid substitution is selected from the group consisting of F42A, F42C, F42E, F42H, F42V and F42W; Y45A, Y45D, Y45G, Y45M, Y45N, Y45Q, Y45R, Y45S and Y45T; A50I; L72D, L72I, L72K, L72N and L72V; and C125G.

[0076] According to certain embodiments of the present application, the IL-2 variant of the present application named IL-2_#2 may be: having an amino acid sequence with at least 85% sequence similarity to sequence number: 1; preferably, having an amino acid sequence with at least 90% sequence similarity to sequence number: 1; more preferably, having an amino acid sequence with at least 95% sequence similarity to sequence number: 1; more preferably, having the amino acid sequence of sequence number: 1; wherein there are the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with tryptophan (W), and leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with lysine (K). In an operating embodiment, the IL-2 variant IL-2_#2 of the present application has the amino acid sequence of sequence number: 3.

[0077] According to other embodiments of the present application, the IL-2 variant of the present application named IL-2_#3 may be: having an amino acid sequence with a sequence similarity of at least 85% to sequence number: 1; preferably, having an amino acid sequence with a sequence similarity of at least 90% to sequence number: 1; more preferably, having an amino acid sequence with a sequence similarity of at least 95% to sequence number: 1; even more preferably, having an amino acid sequence of sequence number: 1; wherein the following amino acid substitutions are present: the phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with valine (V), the tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with alanine (A), the leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N), and the cysteine ​​(C) at the 125th position corresponding to sequence number: 1 is substituted with glycine (G). In one working example, the IL-2 variant IL-2_#3 of the present application has the amino acid sequence of sequence number: 4.

[0078] According to still other embodiments of the present application, the IL-2 variant of the present application named IL-2_#7 may be: having an amino acid sequence with at least 85% sequence similarity to sequence number: 1; preferably, having an amino acid sequence with at least 90% sequence similarity to sequence number: 1; more preferably, having an amino acid sequence with at least 95% sequence similarity to sequence number: 1; more preferably, having the amino acid sequence of sequence number: 1; wherein there are the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with glutamic acid (E), and tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with glycine (G). In an operating embodiment, the IL-2 variant IL-2_#7 of the present application has the amino acid sequence of sequence number: 8.

[0079] Alternatively or in addition, the IL-2 variant of the present application named IL-2_#8 may be: having an amino acid sequence with at least 85% sequence similarity to sequence number: 1; preferably, having an amino acid sequence with at least 90% sequence similarity to sequence number: 1; more preferably, having an amino acid sequence with at least 95% sequence similarity to sequence number: 1; even more preferably, having the amino acid sequence of sequence number: 1; wherein there are the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with valine (V), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with serine (S), leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N), and cysteine ​​(C) at the 125th position corresponding to sequence number: 1 is substituted with glycine (G). In one working example, the IL-2 variant IL-2_#8 of the present application has the amino acid sequence of sequence number: 9.

[0080] Optionally, the IL-2 variant of the present application named IL-2_#9 can be: having an amino acid sequence with at least 85% sequence similarity to sequence number: 1; preferably, having an amino acid sequence with at least 90% sequence similarity to sequence number: 1; more preferably, having an amino acid sequence with at least 95% sequence similarity to sequence number: 1; and even more preferably, having the amino acid sequence of sequence number: 1; wherein there are the following amino acid substitutions: the phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with histidine (H), the tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with aspartic acid (D), the leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with isoleucine (I), and the cysteine ​​(C) at the 125th position corresponding to sequence number: 1 is substituted with glycine (G). In one working example, the IL-2 variant IL-2_#9 of the present application has the amino acid sequence of sequence number: 10.

[0081] In a further embodiment of the present application, the IL-2 variant of the present application, named IL-2_#12, may be: having an amino acid sequence with at least 85% sequence similarity to sequence number: 1; preferably, having an amino acid sequence with at least 90% sequence similarity to sequence number: 1; more preferably, having an amino acid sequence with at least 95% sequence similarity to sequence number: 1; more preferably, having the amino acid sequence of sequence number: 1; wherein there are the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with cysteine ​​(C), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with asparagine (N), and leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with valine (V). In an operating embodiment, the IL-2 variant IL-2_#12 of the present application has the amino acid sequence of sequence number: 13.

[0082] In certain embodiments of the present application, the IL-2 variant of the present application, named IL-2_#25, may have an amino acid sequence with at least 85% sequence similarity to SEQ ID NO: 1; preferably, an amino acid sequence with at least 90% sequence similarity to SEQ ID NO: 1; more preferably, an amino acid sequence with at least 95% sequence similarity to SEQ ID NO: 1; more preferably, an amino acid sequence with SEQ ID NO: 1; wherein there is the following amino acid substitution: alanine (A) at the 50th position corresponding to SEQ ID NO: 1 is substituted with isoleucine (I). In an operating embodiment, the IL-2 variant IL-2_#25 of the present application has an amino acid sequence with SEQ ID NO: 21.

[0083] In other embodiments of the present application, the IL-2 variant of the present application, named IL-2_#28, may be: having an amino acid sequence with at least 85% sequence similarity to sequence number: 1; preferably, having an amino acid sequence with at least 90% sequence similarity to sequence number: 1; more preferably, having an amino acid sequence with at least 95% sequence similarity to sequence number: 1; more preferably, having the amino acid sequence of sequence number: 1; wherein there are the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with alanine (A), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with aspartic acid (D), and leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with aspartic acid (D). In an operating embodiment, the IL-2 variant IL-2_#28 of the present application has the amino acid sequence of sequence number: 24.

[0084] Sometimes, the IL-2 variant of the present application named IL-2_#31 may be: having an amino acid sequence with at least 85% sequence similarity to sequence number: 1; preferably, having an amino acid sequence with at least 90% sequence similarity to sequence number: 1; more preferably, having an amino acid sequence with at least 95% sequence similarity to sequence number: 1; more preferably, having the amino acid sequence of sequence number: 1; wherein there are the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with alanine (A), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with methionine (M), and leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N). In an operating embodiment, the IL-2 variant IL-2_#31 of the present application has the amino acid sequence of sequence number: 27.

[0085] In still other embodiments of the present application, the IL-2 variant of the present application, named IL-2_#31_Y45R, may be: having an amino acid sequence with at least 85% sequence similarity to sequence number: 1; preferably, having an amino acid sequence with at least 90% sequence similarity to sequence number: 1; more preferably, having an amino acid sequence with at least 95% sequence similarity to sequence number: 1; more preferably, having the amino acid sequence of sequence number: 1; wherein there are the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with alanine (A), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with arginine (R), and leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N). In an operating embodiment, the IL-2 variant IL-2_#31_Y45R of the present application has the amino acid sequence of sequence number: 34.

[0086] The IL-2 variants of the present application can be prepared by deletion, substitution, insertion, or modification using genetic engineering or chemical synthesis methods well known in the art. Genetic engineering methods may include site-specific mutagenesis, polymerase chain reaction (PCR), gene synthesis, CRISPR / cas9 gene editing and other methods of coding DNA sequences. Correct nucleotide changes can be verified by methods such as sequencing. In this regard, the nucleotide sequence of human native IL-2 can be obtained from public databases such as GenBank (GenBank Accession Number: S77834.1). The amino acid sequence of human native IL-2 is shown in sequence number: 1. Substitution or insertion may involve natural and non-natural amino acid residues. Amino acid modifications include chemical modification methods known in the art, such as adding glycosylation sites or carbohydrate appendages.

[0087] The IL-2 variants of the present application can be obtained by, for example, solid phase peptide synthesis or recombinant production. In the recombinant production part, one or more polynucleotides encoding the IL-2 variants will be isolated and then inserted into one or more vectors for further replication and / or expression in host cells. Such polynucleotides can be easily isolated and sequenced using conventional procedures. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the IL-2 variants of the present application and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo recombination / genetic recombination technology. The expression vector can be a part of a plasmid, a virus, or a nucleic acid fragment. The expression vector includes an expression cassette, into which the polynucleotides encoding the IL-2 variants of the present application are cloned and operably connected to a promoter and / or other transcription or translation control components. If the promoter can affect the transcription of the nucleic acid, the promoter can be operably connected to the nucleic acid encoding the polypeptide. The promoter can be a cell-specific promoter that only guides a large amount of DNA transcription in a predetermined cell. In addition to the promoter, other transcription control elements (eg, enhancers, operators, repressors, and transcription termination signals, etc.) may be operably linked to the polynucleotide to direct cell-specific transcription.

[0088] In certain embodiments, the amino acid mutations present in the IL-2 variant may reduce the affinity of the IL-2 variant to the α-subunit of the IL-2 receptor by at least 5 times, for example, by at least 10 times, by at least 25 times, by at least 30 times, by at least 50 times, or even by at least 100 times. In one embodiment, the amino acid mutations present in the IL-2 variant may eliminate the affinity of the IL-2 variant to the α-subunit of the IL-2 receptor. At the same time, when the IL-2 variant exhibits an affinity of more than about 70% compared to the wild-type IL-2 for the IL-2R with moderate affinity, for example, an affinity of more than about 80%, or an affinity of more than about 90%, the IL-2 variant achieves a substantially similar degree of binding to the receptor with moderate affinity, that is, the IL-2 variant of the present application retains affinity for the receptor.

[0089] In a specific embodiment, the IL-2 variant of the present application may cause one or more cellular responses, for example, promoting the proliferation of activated T lymphocytes, promoting the differentiation of activated T lymphocytes, promoting the activity of cytotoxic T cells (CTL), promoting the proliferation of activated B cells, promoting the differentiation of activated B cells, promoting the proliferation of NK cells, promoting the differentiation of NK cells, promoting the secretion of cytokines by activated T cells or NK cells, or promoting the anti-tumor cytotoxicity of NK / LAK. In one embodiment, the IL-2 variant of the present application may reduce T cell proliferation compared to wild-type IL-2. reg IL-2 signaling in cells, reduces activation-induced cell death (AICD) of T cells, has a reduced in vivo toxicity profile, or possesses a prolonged serum half-life.

[0090] The IL-2 variants of the present application can be identified, screened, or characterized by their physical / chemical properties and / or biological activities using various detection methods known in the art.

[0091] For example, physical / chemical properties can be measured, such as binding affinity tests. Specifically, the binding affinity of the IL-2 variants of the present application to the IL-2 receptor can be easily confirmed by the following detection methods: enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), using a BIAcore instrument (GE Healthcare), or using a cell line known to express a certain IL-2 receptor form and measuring according to procedures known and established in the art.

[0092] In the biological activity test, the activity of the IL-2 variant of the present application can be indirectly measured by detecting the immune activation effects that occur downstream after receptor binding, which may include, for example, the ability to induce proliferation of T cells and / or NK cells with IL-2 receptors, the ability to induce IL-2 signaling in T cells and / or NK cells with IL-2 receptors, the ability to induce NK cells to produce IFN-γ, the ability to reduce the ability to induce peripheral blood mononuclear cells (PBMC) to express IL-10 or TNF-α, the ability to reduce the ability to induce T cell apoptosis, the ability to induce tumor regression and / or improve survival rate, and the ability to reduce toxicity distribution (for example, reducing vascular permeability in vivo). These detection methods are well known in the art.

[0093] Alternatively, activity testing can be performed by measuring downstream signaling triggered by IL-2, including the detection of Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling molecules. c After the subunit interaction, the phosphorylation of JAK1 and JAK3, as well as the phosphorylation of STAT-5, can be evaluated as an indicator of activated IL-2 signaling. The details of this method are disclosed in the Examples. T cells are treated with the IL-2 variants of the present application, and the amount of phosphorylated STAT-5 is detected by flow cytometry.

[0094] 2. Pharmaceutical compositions

[0095] The present application also encompasses a pharmaceutical composition comprising the above-mentioned IL-2 variant for use in treating cancer and / or alleviating or improving symptoms associated with or caused by cancer. Specifically, the pharmaceutical composition comprises an effective amount of the IL-2 variant as described in any aspect or embodiment of the present application; and optionally, a pharmaceutically acceptable carrier.

[0096] The amount of the IL-2 variant of the present invention is about 0.1-99% (weight %) of the total weight of the pharmaceutical composition. In certain embodiments, the amount of the IL-2 variant of the present invention is about at least 1% (weight %) of the total weight of the pharmaceutical composition. In a specific embodiment, the amount of the IL-2 variant of the present invention is about at least 5% (weight %) of the total weight of the pharmaceutical composition. In other embodiments, the amount of the IL-2 variant of the present invention is about at least 10% (weight %) of the total weight of the pharmaceutical composition. In yet other embodiments, the amount of the IL-2 variant of the present invention is about at least 25% (weight %) of the total weight of the pharmaceutical composition.

[0097] The pharmaceutical composition of the present application is prepared according to pharmaceutical procedures well known in the art, and can be formulated into solid, semisolid, or liquid forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, and injections, etc., to adapt to a predetermined route of administration. These pharmaceutical preparations are also further objects of the present invention. Those skilled in the art are familiar with various dosage forms suitable for each route of administration. It should be noted that, in any given case, the most appropriate route of administration will depend on the nature or severity of the disease or condition to be treated.

[0098] A pharmaceutically acceptable carrier is any carrier that is relatively nontoxic and harmless to the patient at a concentration consistent with the effective activity of the active ingredient, so that any side effects caused by the carrier do not impair the beneficial effects of the active ingredient. Pharmaceutically acceptable carriers according to the present invention are, for example, disintegrants, binders, lubricants, fillers, plasticizers, surfactants and wetting agents, film formers and coating materials, and colorants (e.g., pigments).

[0099] Disintegrants include, but are not limited to, crosslinked sodium carboxymethylcellulose, crosslinked polyvinylpyrrolidone, alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium, microcrystalline cellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, polacrilin potassium, crosslinked polyvinyl pyrrolidone, sodium alginate, sodium starch glycolate, partially hydrolyzed starch, sodium carboxymethyl starch, and starch.

[0100] Binders include, but are not limited to, hydroxypropyl cellulose, hypromellose, hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose, acacia gum, alginic acid, carboxymethyl cellulose, ethyl cellulose, methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, polyvinyl alcohol, polyacrylates, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium, compressed sugar, ethyl cellulose, gelatin, liquid glucose, methyl cellulose, polyvinyl pyrrolidone, and pregelatinized starch.

[0101] Lubricants include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, stearic acid, fumaric acid, sodium stearylfumarate, zinc stearate, and polyethylene glycol.

[0102] Fillers include, but are not limited to, dibasic calcium phosphate, kaolin, lactose, mannitol, microcrystalline cellulose, silicate microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, maltitol, sorbitol, xylitol, lactose (e.g., in anhydrous form or in hydrated form, such as monohydrate form), dextrose, maltose, sucrose, glucose, fructose or maltodextrin, powdered cellulose, precipitated calcium carbonate, sodium carbonate, sodium phosphate, and starch.

[0103] Surfactants and wetting agents include, but are not limited to, heptadecaethyleneoxycetanol, lecithin, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polyoxyethylene stearate, polyoxyethylene sorbitan monolaurate, benzalkonium chloride, nonoxynol-10, octoxynol-9, polysorbates (e.g., polysorbate 20, 40, 60, or 80), sorbitan monopalmitate, sodium lauryl sulfate, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, glyceryl monostearate, sorbitan monolaurate, polyethylene glycol sorbitan monolaurate, polyethylene glycol sorbitan monostearate, polyethylene glycol sorbitan monooleate, copolymers of ethylene oxide and propylene oxide. and ethoxylated triglycerides.

[0104] Film formers and coating materials include, but are not limited to, liquid glucose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose (HPMC), methylcellulose, ethylcellulose, celluloseacetatephthalate, shellac, polyvinylpyrrolidone, copolymers of vinylpyrrolidone and vinyl acetate (e.g., ), copolymers of dimethylaminomethacrylic acid and neutral methacrylate, polymers of methacrylic acid or methacrylate, copolymers of ethyl acrylate and methyl methacrylate, and copolymers of acrylic acid and methyl acrylate.

[0105] Plasticizers include, but are not limited to, polyethylene glycol, diethyl phthalate, and glycerin.

[0106] Colorants include, but are not limited to, pigments, inorganic pigments, FD&C Red No. 3, FD&C Red No. 20, FD&C Yellow No. 6, FD&C Blue No. 2, D&C Green No. 5, D&C Orange No. 5, D&C Red No. 8, caramel, red iron oxide, yellow iron oxide, and titanium dioxide.

[0107] Other commonly used pharmaceutical carriers that may be used as appropriate to formulate the composition for the intended route of administration include, but are not limited to, acidifiers (e.g., acetic acid, citric acid, fumaric acid, hydrochloric acid, and nitric acid); alkalizers (e.g., aqueous ammonia, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, and triethanolamine); adsorbents (e.g., powdered cellulose and activated carbon); stabilizers and antioxidants (e.g., ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, phosphinothricin, acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, and sodium metabisulfite); other binding materials (e.g., block polymers, natural and synthetic rubbers, polyacrylates, polyurethanes, silica gels, polysiloxanes, and styrene-butadiene copolymers); buffers (e.g., potassium metaphosphate, dipotassium phosphate, sodium acetate, anhydrous sodium citrate, and sodium citrate hydrate); encapsulating agents (e.g., gelatin, starch, and cellulose derivatives); flavoring agents, masking agents (e.g., agents), and flavoring agents (e.g., anise oil, cinnamon oil, cocoa, menthol, citrus oils, peppermint oil, and vanillin); humectants (e.g., glycerol, propylene glycol, and sorbitol); sweeteners (e.g., aspartame, dextrose, glycerol, mannitol, propylene glycol, saccharin sodium, sorbitol, and sucrose); anti-adherents (e.g., magnesium stearate and talc); direct compression excipients (e.g., calcium hydrogen phosphate, lactose, and microcrystalline cellulose); tablet polishing agents (e.g., palm wax and white wax).

[0108] The pharmaceutical composition of the present application may further comprise other known pharmaceutically active agents (eg, chemotherapeutic agents, immunotherapeutic agents, or hormonal therapeutic agents) to treat diseases and disorders caused by or associated with cancer.

[0109] The chemotherapeutic agent suitable for the pharmaceutical composition of the present application can be one or more of the following: actinomycin D, altretamine, aminoglutethimide, amsacrin, anastrozole, anthracycline, asparaginase, bendamustine, bexarotene, bleomycin, bortezomib, buserelin, busulfan, camptothecin, cin), capecitabine, carboplatin, carmustine, chlorambucil, chlormethine, cisplatin, cladribine, clofarabine, clomifene, curcumin, cyclophosphamide, cytarabine or cytosinarabinoside, dacarbazine dacarbazine, dactinomycin, daunorubicin, dexamethasone, docetaxel, doxorubicin, epirubicin, estramustine, estrone, estradiol, estriol, etoposide, exemestane, fludarabine, fluorouracil fluorouracil, formestane, foxuridine, gemcitabine, glucocorticoid, goserelin, hycamtin, hydroxyurea, idarubicin, ifosfamid, imatinib, indirubin, irinotecan, ixabepilone,letrozole, leuprorelin, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, miltefosin, mitomycine, mitoxantrone, nintedanib, nimustine, oxaliplatin, paclitaxel, pentostatin, plicamycin, prednisone, procarbazine, progesterone progesterone, raloxifene, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, testosterone, thiotepa, thioguanine, topotecan, treosulfan, tretinoin, triptorelin, trofosfamide, vinblastine, vincristine, vindesine, or vinorelbine.

[0110] The immunotherapeutic agent is selected from the group consisting of CCL3, CCL26, CXCL7; G-CSF, GM-CSF, IFN-α, IFN-β, IFN-γ, TNF-α; IL-4, IL-5, IL-7, IL-10, IL-12, IL-13, IL-15; Apremilast, Imiquimod, Lenalidomide, Pamalidomide, Siprussot-T, Thalidomide; Anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody; Anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD11a antibody, anti-CD20 antibody, anti-CD25 antibody, anti-CD52 antibody, anti-EGFR antibody, anti-HER2 antibody, anti-PCDP1 antibody, anti-SLAMF7 antibody, and anti-Trop-2 antibody. According to a preferred embodiment of the present application, the additional immunotherapeutic agent used in the pharmaceutical composition of the present application is IL-15.

[0111] Examples of hormonal therapeutic agents include, but are not limited to, androgen receptor agonists (e.g., androstenediol dipropionate, boldenone undecylenate, clostebol, or cloxotestosterone acetate); gonadotropins (e.g., bicalutamide, tamoxifen, clomifene, gonadorelin, or leuprorelin); androgen receptor antagonists (e.g., abiraterone acetate, canrenone, chlormadinone acetate, cyproterone acetate, or delmadinone acetate); acetate); steroidogenesis inhibitors (e.g., alfatradiol, dutasteride, epristeride, finasteride, flutamide, ketoconazole, or nilutamide); antigonadotropins (e.g., domperidone, metoclopramide, risperidone, haloperidol, chlorpromazine, sulpiride, or testosterone); estrogen receptor agonists (e.g., alpha-estradiol, testosterone, methyltestosterone, metandienone, or estradiol); estrogen receptor antagonists (e.g., acolbifene, anordrin, bazedoxifene, broparestrol, clomiphene, or cyclofenil); aromatase inhibitors (e.g., fentanyl, testolactone, fadrozole, formestane, anapol, exemestane, or letrozole);Progesterone receptor agonists (e.g., allylestrenol, dienogest, norethisterone, progesterone, quingestrone, anagestone acetate, chlormadinone acetate, or chlormethenmadinone acetate); Progesterone receptor antagonists (e.g., aglepristone or mifepristone).

[0112] 3. Methods for treating cancer

[0113] Another aspect of the present application is a method for treating cancer in an individual, wherein the method comprises administering to the individual an effective amount of the IL-2 variant of the present application or a pharmaceutical composition comprising the IL-2 variant.

[0114] The effective dose of the IL-2 variant or pharmaceutical composition of the present invention administered to the subject can be in the range of about 0.01 μg / kg to 1,000 mg / kg of the subject's body weight, for example, about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3 0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62 , 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000 micrograms per kilogram of body weight (= 1 mg / kg body weight), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 7, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 mg / kg body weight of a subject; preferably, between about 0.1 to 1000 micrograms / kg of individual body weight; more preferably, about 1 to 500 micrograms / kg of individual body weight; even more preferably, about 10 to 200 micrograms / kg of individual body weight. In certain operating embodiments, the effective dose of the IL-2 variant or pharmaceutical composition of the present application administered to the individual is 15, 50, or 150 micrograms / kg of individual body weight. The dose can be administered in a single aliquot or in more than one aliquot. A person skilled in the art or a clinical practitioner can adjust the dose or treatment regime according to the patient's physical condition or the severity of the disease. .

[0115] It is understood that in the case where the individual suffers from cancer, the method of the present application can be administered to the individual alone, or in combination with other additional therapies that have certain benefits for cancer treatment (e.g., surgery, radiotherapy, chemotherapy, immunotherapy, hormonal therapy, or a combination thereof). Depending on the intended therapeutic purpose, the method of the present application can be administered to the individual before, at the same time, or after the additional therapy is administered to the individual. In the above-mentioned additional therapies, chemotherapy, immunotherapy, and hormonal therapy refer to administering an effective amount of a chemotherapeutic agent, an immunotherapeutic agent, or a hormonal therapeutic agent to the individual, as described above; for the sake of brevity, a detailed description of these agents is omitted here.

[0116] The cancer can be any of the following: bladder cancer, bile duct cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, epidermal cancer, gastric cancer, gastrointestinal stromal tumor (GIST), glioma, lymphoid hematopoietic tissue tumor, liver cancer, Kaposi's sarcoma, leukemia, lung cancer, lymphoma, intestinal cancer, melanoma, myeloid leukemia, pancreatic cancer, prostate cancer, retinoblastoma, ovarian cancer, renal cell carcinoma, spleen cancer, squamous cell carcinoma, thyroid cancer, or thyroid follicular carcinoma.

[0117] It should be noted that during the treatment period of the present method, different therapies or treatments may be administered to the individual via different routes, at different doses, time intervals, etc. The doses and time intervals may vary due to factors such as those described above and depend on the professional considerations of the practitioner; and the routes may be via oral, enteral, buccal, nasal, transdermal, transmucosal, intravenous, intraperitoneal, intraarterial, intradermal, subcutaneous, and intramuscular routes.

[0118] Basically, the subject suitable for treatment by the method of the present application is a mammal; preferably, the subject is a human.

[0119] A number of embodiments are provided below to illustrate certain aspects of the present invention, so that those skilled in the art can practice the present invention, and these embodiments should not be regarded as limiting the scope of the present invention. It is believed that those skilled in the art can fully utilize and practice the present invention without excessive interpretation after reading the description provided herein. All public documents cited herein are considered part of this specification in their entirety.

[0120] Example

[0121] Materials and Methods

[0122] 1. Design and cloning of IL-2 variants

[0123] To design the IL-2 variants used in this study, the structures of the active and inactive monomeric forms of IL-2 when bound to the receptor were first obtained from the online database Protein Data Bank (PDB), where the active form uses the PDB number 2B5I or 2ERJ, and the inactive form uses the PDB number 1M4C or 3INK. The structures of the active and inactive forms of IL-2 were aligned using the commercial software PyMOL, and the distances between the spatial positions of specific atoms of specific amino acids on helices A, C, and D in the active and inactive forms were calculated. Therefore, by comparing the structures of the active and inactive forms of IL-2, the atoms with the largest and smallest changes in spatial position were determined. Based on this, the distances between atoms with significant movement and atoms with relatively no movement were calculated to create a signature between the active and inactive forms of IL-2. In this study, the distances between the 50 atoms with the largest changes in spatial position and the 60 atoms with the smallest changes in spatial position were calculated, resulting in a total of 3000 (50×60) distances. Among these calculated distances, the largest distance was selected as the characteristic of IL-2 changing from an inactive form to an active form. On the other hand, there are 7,999 (20×20×20–1 (wild type)) variants of human IL-2 at positions F42, Y45, and L72. Among them, the structure of each IL-2 variant was predicted by online software (AlphaFold Colab). Based on the structures of the known IL-2 active form and IL-2 variant, the spatial distance between the two was calculated according to the calculation method previously performed between the active and inactive forms of IL-2. The IL-2 variant with the largest number of atoms with a distance of <10% from the active form of IL-2 was selected as the IL-2 variant of the present invention (sequence number: 1-29) for further study; then these IL-2 variant sequences were artificially synthesized and cloned.

[0124] 2. Expression and Validation of IL-2 Variants

[0125] The constructs for expressing each of the above IL-2 variants (with a hexahistidine (his6) tag) in prokaryotic cells (his6-IL-2, in pET) were transformed into E. coli strain BL21(DE3) and grown at OD 600 When the pH reached 0.5-0.9, 0.1 mM IPTG was added to the culture containing lysogeny broth (LB) / ampicillin (50 μg / ml) to induce the expression of IL-2 variants, and then cultured at room temperature (RT) for 16 hours. The bacteria were harvested and lysed with 0.1 ml of lysis buffer (containing 20 mM HEPES (pH 7.4), 250 mM KCl, 25 μg / ml DNase, 25 μg / ml lysozyme, 10 mM PMSF, and 10 mM β-mercaptoethanol), and the resulting mixture was centrifuged to collect the supernatant containing the expressed protein. The soluble his6-IL-2 in the supernatant was separated by SDS-PAGE (containing 10% polyacrylamide) gel and confirmed by Western blot using anti-his6 antibody. The supernatant was then filtered and purified using a nickel-resin column (Qiagen, Valencia, CA) according to the manufacturer's instructions. The amount of purified IL-2 variants was detected using an ELISA kit (Human IL-2 Quantikine, R&D Systems).

[0126] The above IL-2 variants were also expressed in the mammalian protein expression system (ExpiCHO TM The constructs containing the above IL-2 variants (in pcDNA3.4) were transfected into ExpiCHO-S cells, and on the first day after transfection, ExpiFectamine TM CHO booster was added to the culture. On day 7, ExpiCHO-S cells were removed by centrifugation and the supernatant containing IL-2 variant protein was collected and its concentration was measured by ELISA.

[0127] 3. Cell Culture

[0128] CTLL-2 cells (mouse cytotoxic T lymphocytes) and HH cells (human T lymphoblasts) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum and 2 mM glutamine, while KPC-luc cells (mouse pancreatic adenocarcinoma cells stably transfected with plasmids encoding blasticidin-S deaminase and firefly luciferase) were cultured in the above medium and further supplemented with 4 nM / ml blasticidin-S. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

[0129] 4. Measurement of pSTAT-5 produced by cells stimulated with IL-2 variants

[0130] In order to evaluate the activation degree of CTLL-2 cells or HH cells after stimulation with the IL-2 variants described herein, flow cytometry was used to detect the activation of stimulated CTLL-2 cells and HH cells (2×10 5 The amount of phosphorylated STAT-5 (pSTAT-5) in 10 cells / well) was measured. Depending on the purpose of the experiment, CTLL-2 cells and HH cells were cultured in a medium containing: wild-type IL-2 (proleukin) at a single concentration of 30 nanomolar concentration (nM) or a series of concentrations between 0-30 nM, a specific IL-2 variant, or mutant IL-2 (i.e., IL-2v(AAG), where AAG refers to IL-2 with F42A, Y45A, and L72G substitutions; F. Hoffmann-La Roche Ltd) for 4 hours. The treated CTLL-2 cells and HH cells were placed in a 96-well microtiter plate, in which each well had been pre-seeded with KPC-luc cells (8×10 3 Cells / well) were added and reacted for 30 minutes, followed by fixation with 2% formaldehyde (volume / volume) at room temperature for 15 minutes. The fixed cells were permeabilized, rehydrated, reacted with anti-phosphorylated STAT5 (pSTAT-5) antibody at 4°C for 16 hours, and then reacted with FITC-labeled anti-rabbit IgG antibody in the dark for 60 minutes. Fluorescence signals were detected using a flow cytometer (BD FACS Calibur flow cytometer), and data were analyzed using software (CellQuest Pro and Cytexpert software; Beckman Coulter).

[0131] 5. Animal Experiments

[0132] Female Balb / c mice (6 weeks old) were housed in an environment with a temperature of 23±1°C and a relative humidity of 45-70%, and provided with a 12-hour / 12-hour light / dark cycle. The mice were randomly divided into three groups (n=5 in each group): a PBS control group, a human recombinant IL-2 (IL-2) control group, and an IL-2_#7 experimental group. For the PBS group, mice were intraperitoneally administered according to the following schedule: once a day for five consecutive days, followed by two days of rest; this cycle was repeated three times in total. For IL-2 group and IL-2_#7 group, mice were intraperitoneally administered according to the following schedule: (week 1) once a day, each mouse received a dose of 3 micrograms (about 0.15 mg / kg) for five consecutive days, followed by a two-day rest; (week 2) once a day, each mouse received a dose of 10 micrograms (about 0.5 mg / kg) for five consecutive days, followed by a two-day rest; and (week 3) once a day, each mouse received a dose of 30 micrograms (about 1.5 mg / kg) for five consecutive days, followed by a two-day rest. After completing the above-mentioned dosing schedule, the animals were weighed and killed, and their lungs, livers, and spleens were harvested for further analysis.

[0133] 6. Flow Cytometry Analysis

[0134] The spleen of mice was lysed to extract the cells. The cells were collected and fixed with 2% formaldehyde (vol / vol) at room temperature for 15 minutes and then analyzed by flow cytometry. To identify specific cell populations in the spleen, the following antibody combination was used: (1) to identify active CD8 + T cells: FITC-labeled anti-CD45 antibody (anti-CD45-FITC), APC-labeled anti-CD3 antibody (anti-CD3-APC), PE-cy7-labeled anti-CD8 antibody (anti-CD8-PE-cy7), and PE-labeled anti-Tbet antibody (anti-Tbet-PE); (2) identification of active NK cells: PE-labeled anti-CD45 antibody (anti-CD45-PE), APC-labeled anti-CD3 antibody (anti-CD3-APC), FITC-labeled anti-KN1.1 antibody (anti-KN1.1-FITC), and AlexaFluor 700-labeled anti-granzyme B (GrB) antibody (anti-GrB-AlexaFluor700); and (3) identification of T regCells: APC-cy7 labeled anti-CD45 antibody (anti-CD45-APC-cy7), FITC labeled anti-CD4 antibody (anti-CD4-FITC), APC labeled anti-CD25 antibody (anti-CD25-APC), and PE labeled anti-Foxp3 antibody (anti-Foxp3-PE). Fluorescence signals were detected using a flow cytometer (BD FACS Calibur flow cytometer), and data were analyzed using software (CellQuest Pro and Cytexpert software; Beckman Coulter).

[0135] 7. Statistical Analysis

[0136] All experiments were performed in triplicate. Experimental data are presented as mean ± standard deviation (SD). Statistical significance was analyzed using Student's t test. For all statistical analyses, a two-tailed p value ≤ 0.05 was considered significant. Calculation results and graphs were generated using GraphPad Prims 6.01 software.

[0137] Example 1 Preparation and Characterization of IL-2 Variants

[0138] 1.1 Preparation of IL-2 variants

[0139] The purpose of this example is to identify novel IL-2 variants that have strong binding ability to IL-2R with moderate affinity (rather than IL-2R with high affinity), thereby reducing the unwanted immunosuppressive effect caused by binding to the α-subunit of IL-2R. To achieve the above purpose, several IL-2 variants were designed and prepared according to the procedures described in the "Materials and Methods" section. The nucleic acid sequence encoding each IL-2 variant has been confirmed by sequencing (data not shown), and the amino acid sequence of each IL-2 variant is provided in the sequence table. These IL-2 variants were further confirmed for their biological activity in the following manner.

[0140] 1.2 Biological activity of IL-2 variants

[0141] First, the expression of the IL-2 variants (named IL-2_#1 to IL-2_#14) in Example 1.1 was confirmed by Western blot analysis. The experimental results are shown in Figure 1A In the experiment, each IL-2 variant (approximately 15 kDa) was successfully expressed.

[0142] Next, the biological activity of each IL-2 variant was evaluated by evaluating its ability to activate CTLL-2 cells or HH cells in the presence of KPC-luc cells, and the amount of phosphorylated STAT-5 induced in the cells was used to indicate the degree of activation of CTLL-2 cells or HH cells. Figure 1B-1C As shown, IL-2 variants can activate CTLL-2 cells ( Figure 1B ) or HH cells ( Figure 1C ); Among them, IL-2_#2, IL-2_#3, IL-2_#7, IL-2_#8, IL-2_#9, and IL-2_#12 consistently showed higher activation ability in both CTLL-2 cells and HH cells (i.e., higher amounts of pSTAT-5), while IL-2_#1, IL-2_#4-6, IL-2_#10-11, and IL-2_#13-14 had lower activation abilities. Similarly, IL-2 variants (named IL-2_#19 to IL-2_#34) also had varying degrees of ability to activate HH cells. IL-2_#25, IL-2_#28, and IL-2_#31 showed higher activation ability in HH cells (i.e., a higher percentage of pSTAT-5-positive cells) compared to other IL-2 variants; Figure 1D ).

[0143] In addition, IL-2_#31 (IL-2 with F42A, Y45M, and L72N substitutions) was selected as an exemplary IL-2 variant for further studying the effects of amino acid substitutions at specific positions of IL-2 on the activation of HH cells. It should be noted that in the following variants related to IL-2_#31 (hereinafter referred to as "IL-2_#31 related variants"), the tyrosine (Y) at the 45th position of IL-2 is replaced with alanine (A) (Y45A), aspartic acid (D) (Y45D), glycine (G) (Y45G), glutamine (Q) (Y45Q), arginine (R) (Y45R), serine (S) (Y45S), or threonine (T) (Y45T), while F42A and L72N in IL-2_#31 are still retained in the above-mentioned IL-2_#31 related variants (sequence numbers: 30-36). The experimental results are as follows. Figure 1E As shown, compared with other variants, only IL-2_#31 related variant Y45R (i.e. IL-2_#31_Y45R) shows improved activity in HH cells. These experimental results show that in specific IL-2 variants, the amino acid changes on specific sites may drastically affect the activation of IL-2 for HH cells. These IL-2 variants (i.e. variants with the ability to activate cells) are subsequently further analyzed.

[0144] 1.3 Binding preference of IL-2 variants

[0145] The purpose of this example is to illustrate the binding preference of the IL-2 variants of Example 1.1 for high affinity IL-2R or medium affinity IL-2R. For this purpose, the amount of pSTAT-5 in stimulated CTLL-2 cells and HH cells (after treatment with a range of concentrations of specific IL-2 variants) was used as an indicator of cell activation, wherein CTLL-2 cells exhibit high affinity IL-2R (containing IL-2R α / β / γ c -subunit), while HH cells express intermediate affinity for IL-2R (containing IL-2R β / γ c -subunit). The results of IL-2_#2, IL-2_#3, IL-2_#7, IL-2_#8, IL-2_#9, and IL-2_#12 activation of CTLL-2 cells and HH cells are provided in Figure 2A-2B The experimental results are expressed as corrected MFI (%), which is corrected relative to IL-2 (designated as 100%); the EC of the above IL-2 variants for activated CTLL-2 cells and HH cells 50 Values ​​(EC 50 (CTLL-2) and EC 50 (HH)) were calculated accordingly and summarized in Tables 1 and 2. The binding preference of IL-2 variants for different forms of IL-2R is expressed as EC 50 (HH / CTLL-2) and are summarized in Table 3.

[0146] Table 1 EC of IL-2_#2 to IL-2_#12 on CTLL-2 cells 50 name IL-2 IL-2_#2 IL-2_#3 IL-2_#7 IL-2_#8 IL-2_#9 IL-2_#12 <![CDATA[EC 50 (nM)]]> 0.1700 0.3416 44.11 11.50 120.2 10.01 2.323

[0147] Table 2 EC values ​​of IL-2_#2 to IL-2_#12 for HH cells 50 name IL-2 IL-2_#2 IL-2_#3 IL-2_#7 IL-2_#8 IL-2_#9 IL-2_#12 <![CDATA[EC 50 (nM)]]> 14.24 0.6122 9.600 1.356 90.18 17.91 0.5563

[0148] Table 3 Relative EC values ​​of IL-2_#2 to IL-2_#12 for CTLL-2 cells and HH cells 50

[0149] According to Table 3, the EC of IL-2 50The (HH / CTLL-2) value was 83.76, indicating that IL-2 prefers to bind to the high affinity IL-2R rather than the medium affinity IL-2R. In contrast, the EC values ​​of IL-2_#2 to IL-2_#12 were 50 The (HH / CTLL-2) values ​​were smaller than those for IL-2, indicating that these IL-2 variants tend to bind to the intermediate affinity IL-2R rather than the high affinity IL-2R.

[0150] Figure 2C-2D The activation abilities of IL-2_#25, IL-2_#28, and IL-2_#31 for CTLL-2 cells and HH cells were shown; and the binding preference results are summarized in Tables 4 to 6.

[0151] Table 4 EC of IL-2_#25 to IL-2_#31 for CTLL-2 cells 50 name IL-2 IL-2_#25 IL-2_#28 IL-2_#31 <![CDATA[EC 50 (nM)]]> 0.2309 0.002908 9.562 2.018

[0152] Table 5 EC of IL-2_#25 to IL-2_#31 for HH cells 50 name IL-2 IL-2_#25 IL-2_#28 IL-2_#31 <![CDATA[EC 50 (nM)]]> 19.66 9.933 8.949 0.1099

[0153] Table 6 Relative EC of IL-2_#25 to IL-2_#31 for CTLL-2 cells and HH cells 50 name IL-2 IL-2_#25 IL-2_#28 IL-2_#31 <![CDATA[EC 50 (HH)]]> 19.66 9.933 8.949 0.1099 <![CDATA[EC 50 (CTLL-2)]]> 0.2309 0.002908 9.562 2.018 <![CDATA[EC 50 (HH / CTLL-2)]]> 85.48 3424.14 0.94 0.05

[0154] According to Table 6, IL-2 tends to bind to high affinity IL-2R rather than to medium affinity IL-2R. In contrast, both IL-2_#28 and IL-2_#31 tend to bind to medium affinity IL-2R rather than to high affinity IL-2R, and the EC values ​​of IL-2_#28 and IL-2_#31 are 50 The (HH / CTLL-2) values ​​were 0.94 and 0.05, respectively. 50 The (HH / CTLL-2) value was 3424.14, much higher than the corresponding value of IL-2, indicating that IL-2_#25 prefers to bind to the high-affinity IL-2R.

[0155] The binding preference of IL-2_#7 for CTLL-2 cells and HH cells was compared with that of a commercially available mutant IL-2 (IL-2v(AAG); F. Hoffmann-La Roche Ltd). Figure 2E-2FAs shown in Table 7. 50 (HH / CTLL-2) = 0.027) showed better binding preference than wild-type IL-2 (EC 50 (HH / CTLL-2)=10.817) or IL-2v(AAG)(EC 50 (HH / CTLL-2) = 0.037). This indicates that IL-2_#7 is superior to commercially available IL-2v(AAG) in binding to medium-affinity IL-2R (rather than high-affinity IL-2R).

[0156] Table 7 EC of IL-2_#7 and IL-2v(AAG) 50 and relative EC 50

[0157] Example 2 In vivo toxicity and immunomodulatory effects of IL-2 variants

[0158] 2.1 Toxicity of IL-2 variants

[0159] In this example, the in vivo toxicity and immunomodulatory effects of IL-2 variants were studied. Specific IL-2 variants were administered to mice according to the treatment schedule described in "Materials and Methods". After completing the treatment plan, the mice were killed. Considering that IL-2 has a deleterious effect on endothelial cells, it can cause organ edema, for example, lung, liver, and spleen edema, which in turn causes changes in body weight, so the weights of these organs were measured and corrected for individual body weight ( Figures 3A-3C ). In addition, the weight changes during the experiment were shown in Figure 3D In the study, the relative weights of the liver and spleen of mice treated with wild-type IL-2 increased significantly compared to those of mice treated with IL-2_#7, indicating that IL-2_#7 had a lower toxic effect on the treated mice. In this study, the relative weights of the lungs in the three treatment groups remained roughly the same. In addition, the weights of mice in these treatment groups remained consistent throughout the study. In summary, these experimental data indicate that IL-2_#7 exhibits better safety compared to wild-type IL-2.

[0160] 2.2 Immunomodulatory effects of IL-2 variants

[0161] Next, the present study evaluated the immunomodulatory capacity of IL-2 variants, which can be reflected by changes in the number of immune cells in peripheral lymphoid tissues (e.g., spleen). For this purpose, active CD8+ T cells, activated NK cells, and T reg The number of cells. Figures 4A-4B The experimental results showed that compared with the PBS treatment group and the IL-2 treatment group, the active CD8 + T cells (labeled with CD45 + 、CD3 + 、CD8 + , and T-bet + ), and active NK cells (labeled by CD45 + , GrB + 、NK1.1 + , and CD3 - ) were significantly increased. In contrast, the IL-2_#7 treatment group did not show T reg Cell population (labeled with CD45 + 、Foxp3 + 、CD4 + , and CD25 + In summary, these experimental data indicate that IL-2_#7 can stimulate active CD8 + The generation of T cells and NK cells promotes beneficial immune responses without activating T reg Therefore, IL-2_7 has the potential to enhance an individual's immune system and is therefore a potential candidate for development as an anti-cancer treatment.

[0162] In summary, the present invention provides a variety of IL-2 variants, many of which show a preference for binding to IL-2R with moderate affinity, thereby triggering biological activities associated therewith. Therefore, these IL-2 variants can not only eliminate unwanted immunosuppressive properties, but also retain the desired biological activity. Therefore, these IL-2 variants have great potential for the development of drugs for treating IL-2-related diseases.

[0163] It should be understood that the description of the above embodiments is only for illustrative purposes, and those skilled in the art may make various changes and modifications thereto. The above description, examples and experimental data provide a complete description of the structure and use of the present application as an illustrative embodiment. Although the various embodiments of the present application have been described above with a certain degree of characteristics, or with reference to one or more individual embodiments, those with ordinary knowledge in the art of the present invention may still make numerous modifications to the disclosed embodiments without departing from the spirit and scope of the present application.

Claims

1. An isolated interleukin-2 variant comprising an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 1, wherein the isolated IL-2 variant comprises an amino acid substitution at position corresponding to position 42, 45, 50, 72 or 125 in SEQ ID NO: 1, wherein: The amino acid substitution at position 42 in SEQ ID NO: 1 is substitution of phenylalanine (F) with alanine (A), cysteine ​​(C), glutamic acid (E), histidine (H), valine (V), or tryptophan (W); The amino acid substitution at position 45 corresponding to SEQ ID NO: 1 is substitution of tyrosine (Y) with alanine (A), aspartic acid (D), glycine (G), methionine (M), asparagine (N), glutamine (Q), arginine (R), serine (S), or threonine (T); The amino acid substitution at position 50 corresponding to SEQ ID NO: 1 is substitution of alanine (A) with isoleucine (I); The amino acid substitution at position 72 corresponding to SEQ ID NO: 1 is substitution of leucine (L) with aspartic acid (D), isoleucine (I), lysine (K), asparagine (N), or valine (V); and The amino acid substitution at the position corresponding to the 125th position in SEQ ID NO: 1 is substitution of cysteine ​​(C) with glycine (G).

2. The isolated IL-2 variant of claim 1, wherein the isolated IL-2 variant comprises the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with tryptophan (W), and leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with lysine (K).

3. The isolated IL-2 variant of claim 1, wherein the isolated IL-2 variant comprises the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with valine (V), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with alanine (A), leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N), and cysteine ​​(C) at the 125th position corresponding to sequence number: 1 is substituted with glycine (G).

4. The isolated IL-2 variant of claim 1, wherein the isolated IL-2 variant comprises the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with glutamic acid (E), and tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with glycine (G).

5. The isolated IL-2 variant of claim 1, wherein the isolated IL-2 variant comprises the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with valine (V), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with serine (S), leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N), and cysteine ​​(C) at the 125th position corresponding to sequence number: 1 is substituted with glycine (G).

6. An isolated IL-2 variant as described in claim 1, wherein the isolated IL-2 variant comprises the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with histidine (H), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with aspartic acid (D), leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with isoleucine (I), and cysteine ​​(C) at the 125th position corresponding to sequence number: 1 is substituted with glycine (G).

7. An isolated IL-2 variant as described in claim 1, wherein the isolated IL-2 variant comprises the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with cysteine ​​(C), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with asparagine (N), and leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with valine (V).

8. The isolated IL-2 variant of claim 1, wherein the isolated IL-2 variant comprises the following amino acid substitution: alanine (A) at position corresponding to position 50 in SEQ ID NO: 1 is substituted with isoleucine (I).

9. An isolated IL-2 variant as described in claim 1, wherein the isolated IL-2 variant comprises the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with alanine (A), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with aspartic acid (D), and leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with aspartic acid (D).

10. An isolated IL-2 variant as described in claim 1, wherein the isolated IL-2 variant comprises the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with alanine (A), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with methionine (M), and leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N).

11. An isolated IL-2 variant as described in claim 1, wherein the isolated IL-2 variant comprises the following amino acid substitutions: phenylalanine (F) at the 42nd position corresponding to sequence number: 1 is substituted with alanine (A), tyrosine (Y) at the 45th position corresponding to sequence number: 1 is substituted with arginine (R), and leucine (L) at the 72nd position corresponding to sequence number: 1 is substituted with asparagine (N).

12. The isolated IL-2 variant of claim 1, wherein the isolated IL-2 variant comprises the amino acid sequence of SEQ ID NO:

1.

13. A pharmaceutical composition comprising the isolated IL-2 variant according to claim 1, and a pharmaceutically acceptable carrier.

14. A method for treating cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of claim 13.

15. The method of claim 14, wherein the pharmaceutical composition comprising the isolated IL-2 variant is administered to the subject in an amount of 1-500 μg / kg body weight.

16. The method of claim 14, wherein the cancer is any one of the following: bladder cancer, bile duct cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, epidermal cancer, gastric cancer, gastrointestinal stromal tumor, glioma, lymphoid hematopoietic tumor, liver cancer, Kaposi's sarcoma, leukemia, lung cancer, lymphoma, intestinal cancer, melanoma, myeloid leukemia, pancreatic cancer, prostate cancer, retinoblastoma, ovarian cancer, renal cell carcinoma, spleen cancer, squamous cell carcinoma, thyroid cancer, or thyroid follicular carcinoma.

17. The method of claim 14, further comprising subjecting the individual to surgery, radiation therapy, chemotherapy, immunotherapy, hormonal therapy, or a combination thereof before, simultaneously with, or after administering the pharmaceutical composition of claim 13.

18. The method of claim 17, wherein the immunotherapy comprises administering to the subject an effective amount of CC motif chemokine ligand 3 (CCL3), CC motif chemokine ligand 26 (CCL26), CXC motif chemokine ligand 7 (CXCL7); granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α); interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), leukocyte interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15); apremilast, imiquimod, lenalidomide, pomalidomide, siprude-T, thalidomide; anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody; anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD11a antibody, anti-CD20 antibody, anti-CD25 antibody, anti-CD52 antibody, anti-EGFR antibody, anti-HER2 antibody, anti-PCDP1 antibody, anti-SLAMF7 antibody, or anti-Trop-2 antibody.

19. The method of claim 18, wherein the immunotherapy comprises administering to the subject an effective amount of IL-15.

20. The method of claim 14, wherein the subject is a human.

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