IL-2 Conjugate

Abstract

To provide conjugates of IL-2 that provide safer treatment.SOLUTION: An IL-2 conjugate or a pharmaceutically acceptable salt thereof of formula (Ia) or (Ib), where -D is a biased IL-2 moiety; -L1- is a linker moiety covalently and reversibly attached to -D; -L2- is a chemical bond or a spacer moiety; -Z is a polymeric moiety or a substituted fatty acid moiety; x is an integer selected from the group consisting of 1 to 16; and y is an integer selected from the group consisting of 2, 3, 4 and 5.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to IL-2 conjugates, pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising such IL-2 conjugates or pharmaceutically acceptable salts thereof, and the use thereof. [Background technology]

[0002] In healthy individuals, the immune system can often distinguish between healthy cells and cancer cells. When it identifies a given cell as cancerous, the immune system typically eliminates it. However, if the immune system is impaired or overwhelmed, for example due to acute or chronic defects, cancer can develop because the impaired immune system is unable to distinguish and eliminate cancer cells. In patients with cancer, administering immunomodulatory proteins can help activate the patient's immune system to enhance its ability to eliminate cancer cells. In this way, further cancer growth may be delayed, the potential spread of cancer may be suppressed, or the cancer may even be eliminated.

[0003] One such immunomodulatory protein used to treat patients with certain cancers is interleukin-2. Interleukin-2 (IL-2) is a naturally occurring cytokine that has activity as a stimulant for both natural killer (NK) cells and T cells, as well as functional activity. IL-2 plays a central role in the generation, differentiation, survival, and homeostasis of immune effector cells. IL-2 is synthesized by activated CD4+ helper T cells, and through different receptor interactions, IL-2 can modulate the immune response toward immunity or tolerance.

[0004] IL-2 acts by binding to the IL-2 receptor (IL-2R). The association of the α-(CD25), β-(CD122), and common γ-(γc,CD132) subunits results in the trimer-type high-affinity IL-2R. The dimer-type medium-affinity IL-2Rβγ consists of the β- and γ- subunits and binds to IL-2 with 1 / 50th the affinity. CD25 is not required for IL-2 signaling but confers high-affinity binding to the trimer receptor, while the β- and γ- subunits mediate signaling. IL-2Rβγ is expressed on NK cells, monocytes, macrophages, and resting CD4+ and CD8+ T cells, while IL-2Rαβγ is transiently induced on activated T and NK cells and constitutively expressed on regulatory T cells. IL-2's ability to proliferate and activate innate and adaptive effector cells is the basis of its antitumor activity.

[0005] In patients, IL-2 can stimulate antitumor efficacy, characterized by an increase in cytotoxic lymphocytes, including effector T and NK cells, when administered at high doses (i.e., 600,000–720,000 IU / kg body weight three times daily, up to 14 doses / cycle in humans). Presumably, during this treatment, all T cells are stimulated by IL-2 after high-dose administration, and once the treatment cycle ends and IL-2 levels decline at some point, IL-2 becomes limited, and regulatory T (Treg) cells expressing IL-2Rαβγ outperform effector T cells expressing IL-2Rβγ with respect to the remaining wild-type IL-2.

[0006] Aldesleukin, a recombinant human IL-2, was the first cancer immunotherapy approved by the FDA in 1992. With appropriate supportive care, aldesleukin resulted in complete cancer regression in approximately 10% of patients treated with it for metastatic melanoma and renal cancer. Approximately 70% of patients who achieved a complete response were cured and have maintained complete regression for more than 25 years after initial treatment.

[0007] Based on its antitumor efficacy, high-dose IL-2 (aldesleukin) is approved for patients with metastatic renal cell carcinoma and malignant melanoma. However, its antitumor immunity is limited by doses due to severe cardiovascular, pulmonary, hepatic, gastrointestinal, neurological, and hematological side effects, and therefore it is administered only to patients in specialized facilities. Furthermore, when administered IL-2 levels fall below the level required for IL-2Rβγ activity, it becomes favorable for the activation of Tregs expressing high-affinity IL-2Rαβγ, which can limit antitumor immunity.

[0008] Preclinical studies have shown that IL-2-induced pulmonary edema (as a model of vasoleap syndrome) can be caused by the interaction between IL-2 and CD25 on pulmonary endothelial cells, and that this can be suppressed by the use of CD25 blocking antibodies, gene disruption, or IL-2-antibody conjugates. Another proposed mechanism by which IL-2 induces vasoleap syndrome is related to eosinophil activation, because these cells can express IL-2Rαβγ, and IL-2 treatment in patients is accompanied by systemic increases in eosinophil and IL-5 levels.

[0009] CD4+ regulatory T cells (Treg cells), which are involved in suppressing the immune response and promoting immune tolerance, preferentially express the IL-2Rαβγ type of IL-2R. Therefore, it can be expected that the administration of compounds that bind to IL-2Rαβγ and are its agonists will suppress the immune response and thereby interfere with the antitumor response in cancer patients.

[0010] Effector CD4+ T cells, CD8+ T cells, and NK cells that significantly enhance the antitumor immune response preferentially express the IL-2Rβγ form of IL-2R. Therefore, administration of compounds that bind to IL-2Rβγ and are its agonists can be expected to enhance the immune response against tumors (for example, by increasing the proliferation and activity of CD4+ T cells, CD8+ T cells, and NK cells).

[0011] Therefore, the administration of IL-2Rβγ selective agonists (with reduced or no binding to IL-2Rα, or enhanced binding to IL-2Rβγ) is expected to be beneficial for patients with certain cancers, as it is anticipated to reduce systemic vascular leakage side effects such as pulmonary edema and provide an improved treatment window. In addition, IL-2Rβγ selective agonists have the advantage of avoiding the selective activation of immunosuppressive regulatory T cells at low doses, and are equally potent against Tregs, CD4+ effector T cells, cytotoxic CD8+ effector T cells, and NK cells, thereby enhancing the patient's immune system and providing more opportunities to eliminate cancer cells.

[0012] Optimally, such IL-2Rβγ selective agonists also exhibit relatively long exposure after administration, thereby further improving the patient's response to treatment. Enhancing the effector arm of the immune system in cancer patients via the administration of IL-2βγ selective agonists can be further enhanced through the administration of immunosuppressive pathway antagonists (e.g., CTLA-4 and PD-1 antagonists), or through the administration of immune agonists, such as TLR ligands, or agonists that stimulate immune-activating receptors, such as 41BB (CD137), OX40, ICOS, CD40, CD28, NKG2D, NKp30, NKp44, NKp46, LFA1, CD16, CD64, CD32A, and CD3-activated receptor agonists, or through the administration of antibody-targeted cytotoxicity (ADCC)-inducing antibodies.

[0013] Adoptive transfer of tumor-reactive T cells is evolving into a clinically useful therapy capable of inducing anti-tumor immunity in patients. However, widespread application of adoptive T cell transfer (ACT) therapy for cancer treatment faces several limitations, including the production of sufficient cell volume for injection and the inability of transferred T cells to remain viable and functional in vivo. In clinical settings, co-administration of the T cell growth factor interleukin-2 (IL-2) improves the survival, function, and antitumor activity of transplanted T cells. However, the use of IL-2 to enhance ACT is complicated by the multifaceted nature of IL-2, which induces both immunostimulatory and immunosuppressive T cell responses, as well as potentially severe toxicity.

[0014] Attempts have been made to address concerns about the toxicity of IL-2. One example is a formulation approach, see, for instance, U.S. Patent No. 6,706,289 and International Publications 02 / 00243 and 99 / 60128. Other approaches suggest specific conjugates of IL-2, see, for example, U.S. Patents No. 4,766,106, 5,206,344, 5,089,261 and 4,902,502. In addition, specific reversible conjugates of IL-2 have been proposed, see, for example, International Publication No. 12065086A1.

[0015] However, despite these approaches, there is still a need for IL-2 conjugates to provide safer treatments for cancer patients. [Overview of the project] [Problems that the invention aims to solve]

[0016] Therefore, an object of the present invention is to overcome the above-mentioned drawbacks at least partially. [Means for solving the problem]

[0017] The object of the present invention is formula (Ia) or (Ib) [ka] (In the formula, -D is the biased IL-2 portion, and the biased IL-2 portion includes the IL-2 portion, and with respect to the biased IL-2 portion, K of the biased IL-2 with respect to IL-2Rβ D K for the bias IL-2 with respect to IL-2Rαβ D The ratio of aldesleukin to IL-2Rβ is K D K for aldesleukin against IL-2Rαβ D Larger than the ratio, -L 1 - is a linker portion covalently and reversibly bonded to -D. -L 2 - represents a chemical bond or spacer portion. -Z is a polymer portion or a substituted fatty acid portion. x is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. (y is an integer selected from the group consisting of 2, 3, 4, and 5.) This is achieved by the IL-2 conjugate or its pharmaceutically acceptable salt. [Modes for carrying out the invention]

[0018] In this invention, the terms are used with the following meanings.

[0019] As used herein, the terms “interleukin-2” or “IL-2” refer to all IL-2 proteins, preferably derived from mammalian species, more preferably from primate species, most preferably from humans, characterized by their central role in lymphocyte generation, survival, and homeostasis, as well as their variants, analogs, orthologues, homologs, derivatives, and fragments thereof. The term “IL-2” also encompasses naturally occurring variants of IL-2, such as splice variants or allele variants.

[0020] Human IL-2 has the sequence of sequence number 1: JPEG0007857984000002.jpg19164

[0021] Unprocessed human IL-2 also contains a 20-amino acid signal peptide at its N-terminus, which is not present in mature IL-2 molecules.

[0022] Preferably, unless otherwise specified, the term "IL-2" refers to aldesleukin, i.e., a variant of human IL-2 in which the amino acid at position 1 (alanine) present in SEQ ID NO: 1 is deleted (desA1), and the cysteine ​​at position 125 of human IL-2 in SEQ ID NO: 1 is replaced with serine (C125S). The sequence of aldesleukin is shown in SEQ ID NO: 2: JPEG0007857984000003.jpg21164

[0023] As used herein, the terms “IL-2 protein variant” or “IL-2 variant” refer to a protein of the same species that differs from a reference IL-2 protein. Preferably, such a reference IL-2 protein sequence is the sequence of SEQ ID NO: 2. Generally, the differences are limited, and therefore the amino acid sequences of the reference and the variant are very similar as a whole and identical in many regions. Preferably, the IL-2 protein variant is at least 70%, 80%, 90%, or 95% identical to the reference IL-2 protein, preferably the IL-2 protein of SEQ ID NO: 2. A protein having an amino acid sequence that is at least, for example, 95% “identical” to the query amino acid sequence means that the amino acid sequence of the target protein is identical to the query sequence, except that it may contain no more than five amino acid changes per 100 amino acids of the query amino acid sequence. These changes in the reference sequence may occur at the amino-terminus (N-terminus) or carboxy-terminus (C-terminus) of the reference amino acid sequence, or at any location between such terminal positions, individually scattered among residues in the reference sequence, or scattered within one or more consecutive groups in the reference sequence. The query sequence may be the complete amino acid sequence of the reference sequence or any of the identified fragments described herein. Preferably, the query sequence is the sequence of SEQ ID NO: 2.

[0024] Such IL-2 protein variants may be isoforms encoded by native variants, such as native allele variants encoded by one of several alternative forms of IL-2 occupying a specific locus on a chromosome or organism, or by native splice variants derived from a single primary transcript. Alternatively, the IL-2 protein variant may be a variant not known to exist naturally and which can be produced by mutagenesis techniques known in the art.

[0025] It is known in the art that one or more amino acids can be deleted from the N-terminus or C-terminus of a bioactive peptide or protein, or from an internal position, i.e., between the N-terminal and C-terminal amino acids, without substantially losing biological function. Such N-terminal, C-terminal, and / or internal deletions are also included in the term IL-2 protein variant.

[0026] It is also known to those skilled in the art that the amino acid sequence of a portion of the IL-2 protein can be altered without significantly affecting the structure or function of the peptide. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to principles known in the art so as to have little effect on activity. For example, guidance on methods for making phenotypically silent amino acid substitutions is given in Bowie et al. (1990), Science 247:1306-1310 (which is incorporated herein by reference in its entirety), where the authors show that there are two main approaches to considering tolerance for changes in the amino acid sequence.

[0027] As used herein, the term "IL-2 analog" refers to IL-2 from different, unrelated organisms that perform the same function in their respective organisms but do not originate from a common ancestral structure shared by those organisms' ancestors. Instead, analog IL-2s arose independently and subsequently evolved to perform the same or similar functions. In other words, analog IL-2 proteins are proteins that have very different amino acid sequences but achieve the same biological activity.

[0028] As used herein, the term "IL-2 ortholog" refers to IL-2 present in two different species whose sequences are related to each other through a common homologous IL-2 in the ancestral species, but which have evolved to be distinct from each other.

[0029] As used herein, the term "IL-2 homolog" refers to IL-2 from different organisms that perform the same function in each organism and originate from an ancestral structure common to the ancestors of those organisms. In other words, homologous IL-2 proteins are proteins with very similar amino acid sequences that achieve the same biological activity. Preferably, an IL-2 protein homolog can be defined as a protein that shows at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% identity with a reference IL-2 protein, preferably the IL-2 protein of SEQ ID NO: 2.

[0030] As used herein, the term “IL-2 protein fragment” refers to any peptide comprising a contiguous range of a portion of the amino acid sequence of the IL-2 protein, preferably the protein of SEQ ID NO: 2. More specifically, an IL-2 protein fragment comprises at least 50, for example, at least 60, at least 70, or at least 80 consecutive amino acids of the IL-2 protein, preferably the protein of SEQ ID NO: 2.

[0031] The term "IL-2" also includes poly(amino acid) conjugates, such as depsipeptides, which have the above sequence but a backbone containing both amide and non-amide bonds, such as ester bonds. A depsipeptide is a chain of amino acid residues whose backbone contains both amide (peptide) and ester bonds. Therefore, as used herein, the term "side chain" refers to the portion attached to the α-carbon of the amino acid moiety when the amino acid moiety is linked through amine bonds, as in a peptide or protein, or to any carbon-carbon-containing portion attached to the backbone of a poly(amino acid) conjugate, as in the case of a depsipeptide. Preferably, the term "IL-2" refers to a protein having a backbone formed through amide (peptide) bonds.

[0032] The term "IL-2" includes the above-mentioned variants, analogs, orthologs, homologs, derivatives and fragments of IL-2. Therefore, all references to specific positions within the reference sequence include the equivalent positions in these variants, analogs, orthologs, homologs, derivatives and fragments of the IL-2 portion, even if not specifically mentioned.

[0033] As used herein, the term "biased IL-2" refers to a modified IL-2 in which the ratio of the K of the biased IL-2 for IL-2Rβ D to the K of the biased IL-2 for IL-2Rαβ D is greater than the ratio of the K of Aldesleukin of SEQ ID NO: 2 for IL-2Rβ D to the K of Aldesleukin for IL-2Rαβ D This is described by the following formula:

Number

Number

[0034] The K of the biased IL-2 for IL-2Rαβ D and the K of the biased IL-2 for IL-2Rβ D D ​, K for aldesleukin against IL-2Rαβ D , and K for aldesleukin against IL-2Rβ D The binding affinity / kinetics necessary to determine this can be evaluated using surface plasmon resonance (SPR) measured in a Biacore instrument (GE Healthcare) as follows: A human Fc capture surface on a CM5 (or alternatively C1 or CM4) chip is prepared by covalent coating with an anti-human Fc antibody, or alternatively, a Protein A chip is used. Next, IL-2Rβ-Fc, or a suitable mixture of IL2-Rα-Fc and IL2-Rβ-Fc, e.g., a 1:1 mixture, is immobilized on the chip. To measure the affinity / kinetic constant, serial dilutions of the sample are prepared with respect to the IL-2 compound, for example, between 1.5 nM and 2 μM, or starting from 100 nM and 1 μM (e.g., 0.03 nM to 100 nM, or 0.1 nM to 1 nM). Each sample is exposed to the receptor-modified chip for an appropriate time, e.g., 1 to 30 minutes (e.g., 2 minutes or 3 minutes), and then rinsed for an appropriate time, e.g., 2 to 60 minutes (e.g., 10 minutes). The binding curves obtained from the dilution series are fitted to a 1:1 kinetic model, and the observed response unit (R) is associated with the binding rate constant and dissociation rate constant k. a and k d To associate:

number

[0035] When determined via a kinetic 1:1 model, the ratio of dissociation rate to binding rate is given by the equilibrium dissociation constant K. D To provide.

[0036] Alternatively, the binding curve obtained from the dilution series can be used to determine the steady-state binding level (R) relative to the sample concentration (C). eq ) plot regarding K for 1:1 interactionD A 1:1 steady-state interaction model to calculate:

number

[0037] It should be understood that not every calculation method is necessarily possible for every biased IL-2 molecule. For example, if the reaction is too fast, it may not be possible to use a 1:1 kinetic model, and a 1:1 steady-state interaction model may be used instead. For example, if equilibrium is not obtained, it may not be possible to use a 1:1 interaction model, and a 1:1 kinetic model may be used instead.

[0038] K for IL2Rαβ and IL2Rβ of bias IL-2 D It is understood that this is measured for bias IL-2, but not for the IL-2 conjugate of the present invention. Thus, K D Measurement is partial-L 1 -, -L 2 - and -Z are absent, preferably before the synthesis of the IL-2 conjugate of the present invention, provided that the IL-2 conjugate is a translation fusion protein, K D This is measured after bias IL-2 is released, for example, after protease cleavage or chemical cleavage.

[0039] Such biased IL-2 is an IL-2 moiety, preferably one amino acid mutation, or at least one deletion, or at least one modified moiety M mod For example, modifying parts M 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mod The protein moiety is an IL-2 moiety having the amino acid sequence of SEQ ID NO: 2, or any combination thereof.mod This can be a protein portion or a non-protein portion. Bias IL-2 has one or more parts M mod If they include, these may be the same or different. In a particular embodiment, the IL-2 portion has the sequence of sequence number 1.

[0040] In the IL-2 conjugate of the present invention, bias IL-2 exists in the form of a corresponding bias IL-2 portion, and when all portion -Z is emitted, i.e., the bias IL-2 portion, i.e., -D and -L, the IL-2 conjugate of the present invention emits 1 When the reversible bond between - and - is broken, bias IL-2 is released.

[0041] As used herein, the term “affinity” refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated herein, “affinity” refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., between a receptor and a ligand). The affinity of a molecule X for its partner Y is generally expressed by the dissociation rate constant and binding rate constant (k, respectively) measured at equilibrium. d and k a The equilibrium dissociation constant (K) is the ratio of (K) to (K). D It can be expressed by ). Therefore, equivalent affinity may include different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by well-established methods known in the art, including the methods described herein.

[0042] As used herein, the terms “IL-2 receptor α subunit” and “IL-2Rα” refer to human CD25.

[0043] As used herein, the terms “beta subunit of the IL-2 receptor” and “IL-2Rβ” refer to human CD122.

[0044] As used herein, the terms “α and β subunits of the IL-2 receptor,” “α and β subunits of IL-2R,” and “IL-2Rαβ” refer to a mixture of IL-2Rα and IL-2Rβ, for example, a 1:1 mixture.

[0045] The IL-2 conjugate of the present invention releases biased IL-2, i.e., the IL-2 conjugate of the present invention is a prodrug of biased IL-2. As used herein, the term “prodrug” refers to a bioactive moiety, e.g., a biased IL-2 moiety, reversibly and covalently linked to a specialized protecting group through a linker moiety (which may also be called a “reversible prodrug linker”), the linker moiety comprising a reversible bond to the bioactive moiety, and the specialized protecting group altering or removing undesirable properties in the parent molecule of the bioactive moiety, i.e., the corresponding drug. This also includes enhancing desirable properties and suppressing undesirable properties in the drug. The specialized non-toxic protecting group is called a “carrier”. The prodrug releases a bioactive moiety, reversibly and covalently linked, in the form of its corresponding drug. In other words, a prodrug is a conjugate comprising a bioactive moiety covalently and reversibly conjugated to a carrier moiety via a reversible prodrug linker moiety, where the covalent and reversible conjugation between the carrier and the reversible prodrug linker moiety is either direct or through a spacer moiety. Another term for "bioactive moiety" is "drug moiety."

[0046] As used herein, the terms “reversible,” “reversibly,” “degradable,” or “degradable” relating to the binding of the first part to the second part means that the binding of the first part to the second part is cleavable under physiological conditions, which is an aqueous buffer at pH 7.4 and 37°C, with a half-life ranging from 1 hour to 3 months, preferably 1 hour to 2 months, and more preferably 1 hour to 1 month. The cleavage may be enzymatic or non-enzymatic, preferably non-enzymatic. Thus, the terms “stable” or “permanent” relating to the binding of the first part to the second part means that the binding of the first part to the second part is cleavable under physiological conditions with a half-life longer than 3 months.

[0047] As used herein, the term “modified portion” preferably refers to a substituent or polymer portion.

[0048] As used herein, the term “disulfide crosslinking” refers to the insertion of a portion between two sulfur atoms of a disulfide crosslink. This is achieved by using a reagent having the portion between two thiol-reactive functional groups and reacting each thiol-reactive functional group with one of the sulfur atoms of the disulfide crosslink, thereby inserting the portion between the sulfur atoms after prior reduction of the disulfide bond. If a peptide or protein has more than one disulfide crosslink, the disulfide crosslinks may be inserted between the sulfur atoms of one disulfide crosslink or between the sulfur atoms of different disulfide crosslinks. Such disulfide crosslinks may be naturally present in the peptide or protein, or they may be introduced artificially, for example, by replacing an existing amino acid portion with a cysteine ​​portion, or by adding a cysteine ​​portion to the peptide or protein.

[0049] As used herein, the term “reagent” means a chemical compound containing at least one functional group for reaction with the functional group of another chemical compound or drug. A drug containing a functional group (e.g., a primary or secondary amine, or a hydroxyl functional group) is also understood to be a reagent.

[0050] As used herein, the term “part” means a portion of a molecule that lacks one or more atoms compared to the corresponding reagent. For example, when a reagent of the formula “HXH” reacts with another reagent to become part of a reaction product, the corresponding part of the reaction product has the structure “HX-” or “-X-”, where each “-” indicates a bond to another part. Thus, the bioactive part is released as a drug from a reversible bond.

[0051] If an arrangement or chemical structure of atoms bonded to or interspersed between two parts is provided, it is understood that such arrangement or chemical structure can bond to these two parts in either direction, unless otherwise explicitly stated. For example, part "-C(O)N(R 1 )-" is "-C(O)N(R 1 )-" or "-N(R 1 It can be joined to two parts as either C(O)- or inserted into a part. Similarly, part [ka] teeth, [ka] or [ka] It can be joined to two parts as either, or it can be inserted into a part.

[0052] As used herein, the term “substituted” means that one or more -H atoms in a molecule or part are replaced by different atoms or groups of atoms called “substituents.”

[0053] As used herein, the term "substituent" preferably means halogen, -CN, or -COOR x1 , -OR x1 , -C(O)R x1, -C(O)N(R x1 R x1a ), -S(O)2N(R x1 R x1a ), -S(O)N(R x1 R x1a ), -S(O)2R x1 , -S(O)R x1 , -N(R x1 )S(O)2N(R x1a R x1b ), -SR x1 , -N(R x1 R x1a ), -NO2, -OC(O)R x1 , -N(R x1 )C(O)R x1a , -N(R x1 )S(O)2R x1a , -N(R x1 )S(O)R x1a , -N(R x1 )C(O)OR x1a , -N(R x1 )C(O)N(R x1a R x1b ), -OC(O)N(R x1 R x1a ), -T 0 , C 1-50 alkyl, C[[ID=六一]] 2-50 alkenyl, and C 2-50 alkynyl selected from the group consisting of, where -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl are optionally substituted by one or more of the same or different -R x2 , and C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl are -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 ), -S(O)2N(R x3 ), -S(O)N(R x3 ), -S(O)2-, -S(O)-, -N(R x3 )S(O)2N(R x3a ), -S-, -N(R x3)-, -OC(OR x3 )(R x3a )-, -N(R x3 )C(O)N(R x3a )-, and -OC(O)N(R x3 )- optionally interrupted by one or more groups selected from the group consisting of, -R x1 , -R x1a , -R x1b is independently selected from the group consisting of -H, -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl, where -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally substituted by one or more of the same or different -R x2 and C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 )-, -S(O)2N(R x3 )-, -S(O)N(R x3 )-; -S(O)2-, -S(O)-, -N(R x3 )S(O)2N(R x3a )-, -S-, -N(R x3 )-, -OC(OR x3 )(R x3a )-, -N(R x3 )C(O)N(R x3a )-, and -OC(O)N(R x3 )- optionally interrupted by one or more groups selected from the group consisting of, each T 0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-member heterocyclyl, and 8- to 11-member heterobicyclic, and each T 0 is the same or different one or more -R x2It is independently and arbitrarily replaced by, Each -R x2 These are halogen, -CN, oxo (=O), and -COOR. x4 , -OR x4 , -C(O)R x4 ,-C(O)N(R x4 R x4a ), -S(O)2N(R x4 R x4a ), -S(O)N(R x4 R x4a ), -S(O)2R x4 ,-S(O)R x4 , -N(R x4 )S(O)2N(R x4a R x4b ), -SR x4 , -N(R x4 R x4a ), -NO2, -OC(O)R x4 , -N(R x4 )C(O)R x4a , -N(R x4 )S(O)2R x4a , -N(R x4 )S(O)R x4a , -N(R x4 )C(O)OR x4a , -N(R x4 )C(O)N(R x4a R x4b ), -OC(O)N(R x4 R x4a ), and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 Alkyl is optionally substituted with one or more halogens, one or more of the same or different halogens. Each -R x3 , -R x3a , -R x4 , -R x4a , -R x4b -H and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 Alkyl is optionally substituted with one or more halogens, either the same or different.

[0054] Preferably, up to six -H atoms in the optionally substituted molecule are independently replaced by substituents, for example, five -H atoms are independently replaced by substituents, four -H atoms are independently replaced by substituents, three -H atoms are independently replaced by substituents, two -H atoms are independently replaced by substituents, or one -H atom is independently replaced by a substituent.

[0055] As used herein, the term “fatty acid” refers to a saturated or unsaturated monocarboxylic acid having an aliphatic tail, which may contain 4 to 28 carbon atoms. Fatty acids may be saturated or unsaturated, linear or branched. The term “fatty acid variant” refers to a modified fatty acid, which may be substituted, and in which case certain carbon atoms may be replaced by other atoms or groups of atoms.

[0056] As used herein, the term “peptide” refers to a chain of at least two and no more than 50 amino acid monomer moieties linked by peptide (amide) bonds. The term “peptide” also includes peptide mimetics, such as D-peptides, peptoids, or beta-peptides, and encompasses such peptide mimetic chains having no more than 50 monomer moieties.

[0057] As used herein, the term “protein” means a chain of more than 50 amino acid monomer moieties (which may also be called “amino acid residues”) linked by peptide bonds, preferably 12,000 or fewer amino acid monomers, for example, 10,000 or fewer amino acid monomer moieties, 8,000 or fewer amino acid monomer moieties, 5,000 or fewer amino acid monomer moieties, or 2,000 or fewer amino acid monomer moieties linked by peptide bonds.

[0058] In this specification, the term “about” as used in combination with a number is used to indicate a range (inclusive) of a number plus or minus 25% or less of the number, more preferably 20% or less of the number, and most preferably 10% or less of the number. For example, the phrase “about 200” is used to indicate a range (inclusive) of 200+ / -25%, i.e., the range from 150 to 250; preferably, a range (inclusive) of 200+ / -20%, i.e., the range from 160 to 240; and even more preferably, a range (inclusive) of 200+ / -10%, i.e., the range from 180 to 220. A percentage indicated as “about 50%” is understood to mean “50%+ / -25%,” i.e., the range from 25% to 75%, i.e., the range from 37.5% to 62.5%, i.e., the range (inclusive) of plus or minus 25% of the number 50.

[0059] As used herein, the term “polymer” means a molecule comprising repeating structural units, i.e., monomers, linked by chemical bonds in a linear, cyclic, branched, cross-linked, or dendrimer manner, or in combination thereof, which may be of synthetic origin, bio-origin, or a combination of both. A polymer is understood to also include one or more other chemical groups and / or parts(s), such as one or more functional groups. Similarly, a peptide or protein is also understood to be a polymer, although the side chains of the individual amino acid residues may differ. Preferably, a soluble polymer has a molecular weight of at least 0.5 kDa, for example, at least 1 kDa, at least 2 kDa, at least 3 kDa, or at least 5 kDa. If the polymer is soluble, it preferably has a molecular weight of up to 1000 kDa, for example, up to 750 kDa, for example, up to 500 kDa, for example, up to 300 kDa, for example, up to 200 kDa, for example, up to 100 kDa. For insoluble polymers such as hydrogels, it is understood that a meaningful molecular weight range cannot be provided.

[0060] As used herein, the term “polymeric” means a reagent or part comprising one or more polymers or polymer parts / parts. A polymer reagent or part may optionally also contain one or more other parts / parts, which are preferably selected from the group consisting of: ·C 1-50 Alkyl, C 2-50 Alkenil, C 2-50 Alkinyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl, and • Joins selected from the following groups: [ka] (In the formula, The dashed line indicates binding to the portion or the remainder of the reagent. -R and -R a (These are independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl, and hexyl).

[0061] Those skilled in the art will understand that polymerization products obtained from polymerization reactions do not all have the same molecular weight, but rather exhibit a molecular weight distribution. Therefore, as used herein, molecular weight range, molecular weight, monomer number range in a polymer, and monomer number in a polymer refer to the number-average molecular weight and monomer number average, i.e., the arithmetic mean of the molecular weight of the polymer or polymer portion, and the arithmetic mean of the monomer number of the polymer or polymer portion.

[0062] Therefore, in a polymer portion containing "x" monomer units, any integer substituted for "x" corresponds to the arithmetic mean of the monomers. Any range of integers substituted for "x" provides an integer range in which the arithmetic mean of the monomers resides. An integer "x" expressed as "approximately x" means that the arithmetic mean of the monomers resides within the integer range of x+ / -25%, preferably x+ / -20%, and more preferably x+ / -10%.

[0063] As used herein, the term “number-mean molecular weight” means the usual arithmetic mean of the molecular weights of individual polymers.

[0064] As used herein, the term “PEG-based” with respect to a portion or reagent means that the portion or reagent contains PEG. Preferably, the PEG-based portion or reagent contains at least 10% (w / w) PEG, e.g., at least 20% (w / w) PEG, e.g., at least 30% (w / w) PEG, e.g., at least 40% (w / w) PEG, e.g., at least 50% (w / w), e.g., at least 60% (w / w) PEG, e.g., at least 70% (w / w) PEG, e.g., at least 80% (w / w) PEG, e.g., at least 90% (w / w) PEG, e.g., at least 95%. The remaining weight percentage of the PEG-based portion or reagent is other portions preferably selected from the following portions and conjugates: ·C 1-50 Alkyl, C 2-50 Alkenil, C 2-50 Alkinyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl, and • Joins selected from the group including the following: [ka] (In the formula, The dashed line indicates binding to the portion or the remainder of the reagent. -R and -R a (These are independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl, and hexyl).

[0065] The term "hyaluronic acid-based" is used accordingly.

[0066] As used herein, the term “PEG system containing at least X% PEG” means that the part or reagent contains at least X% (w / w) ethylene glycol units (-CH2CH2O-), which may be arranged alternately in blockwise or randomly distributed within the part or reagent, preferably all of the ethylene glycol units in the part or reagent are in one block, and the remaining weight percentage of the PEG system part or reagent is other parts preferably selected from the following parts and combinations: ·C 1-50 Alkyl, C 2-50 Alkenil, C 2-50 Alkinyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl, and • Joins selected from the following groups: [ka] (In the formula, The dashed line indicates binding to the portion or the remainder of the reagent. -R and -R a (These are independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl, and hexyl).

[0067] The term "hyaluronic acid-based product containing at least X% hyaluronic acid" is used accordingly.

[0068] As used herein, the term "hydrogel" means a hydrophilic or amphiphilic polymer network composed of homopolymers or copolymers that is insoluble due to the presence of hydrophobic interactions, hydrogen bonds, ionic interactions, and / or covalent chemical crosslinks. Crosslinks provide the network structure and physical integrity.

[0069] The term "inserted" means that a portion is inserted between two carbon atoms, or, if the insertion is at one of the ends of the portion, between a carbon or heteroatom and a hydrogen atom, preferably between a carbon and a hydrogen atom.

[0070] As used herein, the term "C 1-4 "Alkyl" refers to a linear or branched alkyl moiety, either alone or in combination, having 1 to 4 carbon atoms. When present at the end of a molecule, it refers to a linear or branched C 1-4 Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Two parts of the molecule are C 1-4 If bonded by alkyl, such C 1-4 Examples of alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, and -C(CH3)2-. 1-4 Each hydrogen atom of the alkyl carbon may be optionally replaced by a substituent as defined above. Optionally, C 1-4 The alkyl group may be interrupted by one or more parts as defined below.

[0071] As used herein, the term "C 1-6 "Alkyl" refers to a linear or branched alkyl moiety, either alone or in combination, having 1 to 6 carbon atoms. When present at the end of a molecule, it refers to linear and branched C 1-6 Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl. Two parts of the molecule are C 1-6 If bonded by an alkyl group, such C 1-6Examples of alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, and -C(CH3)2-. 1-6 Each hydrogen atom of carbon may be optionally replaced by a substituent as defined above. 1-6 The alkyl group may be interrupted by one or more parts as defined below.

[0072] Therefore, "C 1-10 Alkyl," "C 1-20 "Alkyl" or "C 1-50 "Alkyl" refers to an alkyl chain having 1 to 10, 1 to 20, or 1 to 50 carbon atoms, respectively. 1-10 , C 1-20 or C 1-50 Each hydrogen atom of carbon may be optionally replaced by a substituent as defined above. 1-10 or C 1-50 The alkyl group may be interrupted by one or more parts as defined below.

[0073] As used herein, the term "C 2-6 An "alkenyl" refers to a straight-chain or branched hydrocarbon moiety, either alone or in combination, containing 2 to 6 carbon atoms and at least one carbon-carbon double bond. When present at the ends of a molecule, examples include -CH=CH2, -CH=CH-CH3, -CH2-CH=CH2, -CH=CHCH2-CH3, and -CH=CH-CH=CH2. Two parts of a molecule are C 2-6 When bonded by an alkenyl group, such C 2-6 An example of an alkenil is -CH=CH-. 2-6 Each hydrogen atom in the alkenyl moiety may be optionally replaced by a substituent as defined above. Optionally, C 2-6 An alkenil may be interrupted by one or more parts as defined below.

[0074] Therefore, the term "C 2-10 Alkenil, "C 2-20"Alkenil" or "C 2-50 "Alkenyl" means a linear or branched hydrocarbon moiety, either alone or in combination, having 2 to 10, 2 to 20, or 2 to 50 carbon atoms and containing at least one carbon-carbon double bond. 2-10 Alkenil, C 2-20 Alkenyl or C 2-50 Each hydrogen atom of the alkenyl group may be optionally replaced by a substituent as defined above. Optionally, C 2-10 Alkenil, C 2-20 Alkenyl or C 2-50 An alkenil may be interrupted by one or more parts as defined below.

[0075] As used herein, the term "C 2-6 "Alkynyl" refers to a linear or branched hydrocarbon moiety, either alone or in combination, containing 2 to 6 carbon atoms and at least one carbon-carbon triple bond. When present at the end of a molecule, examples include -C≡CH, -CH2-C≡CH, CH2-CH2-C≡CH, and CH2-C≡C-CH3. When two parts of a molecule are linked by an alkynyl group, an example is -C≡C-. 2-6 Each hydrogen atom of the alkynyl group may be optionally replaced by a substituent as defined above. Optionally, one or more double bonds may be present. Optionally, C 2-6 Alkinil may be interrupted by one or more parts as defined below.

[0076] Therefore, as used herein, the term "C 2-10 Alkinyl, C 2-20 "Alkinyl" and "C 2-50 "Alkynyl" means a linear or branched hydrocarbon moiety, either alone or in combination, containing at least one carbon-carbon triple bond, each having 2 to 10, 2 to 20, or 2 to 50 carbon atoms, respectively. 2-10 Alkinyl, C 2-20 Alkinyl or C 2-50Each hydrogen atom of the alkynyl group may be optionally replaced by a substituent as defined above. Optionally, one or more double bonds may be present. Optionally, C 2-10 Alkinyl, C 2-20 Alkinyl or C 2-50 Alkinil may be interrupted by one or more parts as defined below.

[0077] As mentioned above, C 1-4 Alkyl, C 1-6 Alkyl, C 1-10 Alkyl, C 1-20 Alkyl, C 1-50 Alkyl, C 2-6 Alkenil, C 2-10 Alkenil, C 2-20 Alkenil, C 2-50 Alkenil, C 2-6 Alkinyl, C 2-10 Alkinyl, C 2-20 Alkenyl or C 2-50 The alkynyl may optionally be interrupted by one or more parts, the one or more parts preferably [ka] (In the formula, The dashed line indicates binding to the portion or the remainder of the reagent. -R and -R a (These are independently selected from the group consisting of -H, and methyl, ethyl, propyl, butyl, pentyl, and hexyl.) It is selected from the group consisting of the following.

[0078] As used herein, the term "C 3-10 "Cycloalkyl" means a cyclic alkyl chain having 3 to 10 carbon atoms, which may be saturated or unsaturated, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl. 3-10Each hydrogen atom of a cycloalkyl carbon may be replaced by a substituent as defined above. 3-10 "Cycloalkyl" also includes cross-linked birings such as norbornane or norbornene.

[0079] The term "8-30 membered carbopolycyclil" or "8-30 membered carbon polycyclic" refers to a cyclic portion of two or more rings having 8 to 30 ring atoms, where two adjacent rings share at least one ring atom, and which may contain up to the maximum number of double bonds (fully saturated, partially saturated, or unsaturated, aromatic or non-aromatic rings). Preferably, an 8-30 membered carbopolycyclil refers to a cyclic portion of 2, 3, 4, or 5 rings, more preferably 2, 3, or 4 rings.

[0080] As used herein, the terms “3- to 10-membered heterocyclyl” or “3- to 10-membered heterocycle” mean a ring (fully saturated, partially saturated, or unsaturated, aromatic or non-aromatic ring) having 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms and potentially containing up to a maximum number of double bonds, wherein at least one ring atom and up to four ring atoms are replaced by heteroatoms selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen, and nitrogen (including =N(O)-), and the ring is bonded to the remainder of the molecule by carbon or nitrogen atoms. Examples of 3- to 10-membered heterocycles include, but are not limited to, aziridine, oxirane, thiirane, azirine, oxilen, thiirane, azetidine, oxetane, thietan, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazoline, isothiazoline, thiadiazole, thiadiazole, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazoline, isothiazoline, thiadiazole, thiadiazole, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazoline, sulforane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyridine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidin, diazepane, azepine, and homopiperazine. Each hydrogen atom of a 3- to 10-membered heterocyclyl or 3- to 10-membered heterocyclic group may be replaced by a substituent as defined below.

[0081] As used herein, the terms “8-11 membered heterobicyryl” or “8-11 membered heterobicycle” mean a two-ring heterocyclic portion (a fully saturated, partially saturated, or unsaturated aromatic or non-aromatic ring) having 8 to 11 ring atoms and potentially containing up to a maximum number of double bonds, wherein at least one ring atom is shared by both rings, and at least one ring atom, up to a maximum of six ring atoms, is replaced by heteroatoms selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen, and nitrogen (including =N(O)-), and the ring is bonded to the remainder of the molecule by carbon or nitrogen atoms. Examples of 8- to 11-membered heterobicyclic rings include indole, indoline, benzofuran, benzothiophene, benzoxazole, benzoisoxazole, benzothiazole, benzoisothiazole, benzimidazole, benzimidazolin, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine, and pteridine. The term 8- to 11-membered heterobicyclic ring also includes bicyclic spiro structures such as 1,4-dioxa-8-azaspiro[4.5]decane, or bridging heterocyclic rings such as 8-aza-bicyclo[3.2.1]octane. Each hydrogen atom of an 8- to 11-membered heterobicyclyl or 8- to 11-membered heterobicyclic carbon may be replaced by a substituent as defined below.

[0082] Similarly, the term “8-30 membered heteropolycyclil” or “8-30 membered heteropolycycle” means a heterocyclic moiety (fully saturated, partially saturated, or unsaturated, aromatic or non-aromatic ring) having 8 to 30 ring atoms and potentially containing up to a maximum number of double bonds, comprising two or more rings, preferably 3, 4, or 5 rings, wherein two adjacent rings share at least one ring atom, and at least one ring atom, up to a maximum of 10 ring atoms, is replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen, and nitrogen (including =N(O)-), and the ring is bonded to the remainder of the molecule by carbon or nitrogen atoms.

[0083] structure: [ka] Regarding the part, "Pair R x / R y Together with the atoms to which they are bonded, C 3-10 The phrase "forms a cycloalkyl or 3- to 10-membered heterocycline" is understood to mean that Rx and Ry form the following structures: [ka] (In the formula, R is C 3-10 (They are cycloalkyl or 3-10 membered heterocyclines.)

[0084] structure: [ka] Regarding the part, "Pair R x / R y The phrase "they, together with the atoms to which they are bonded, form ring A" is R x and R y However, it is also understood that this means forming the following structure: [ka]

[0085] As used herein, "halogen" means fluoro, chloro, bromo, or iodine. The halogen is generally preferred to be fluoro or chloro.

[0086] As used herein, the term “functional group” means a group of atoms that can react with other groups of atoms. Exemplary functional groups include, for example, carboxylic acids (-(C=O)OH), primary or secondary amines (-NH2, -NH-), maleimides, thiols (-SH), sulfonic acids (-(O=S=O)OH), carbonates, carbamates (-O(C=O)N<), hydroxyls (-OH), aldehydes (-(C=O)H), ketones (-(C=O)-), hydrazines (>NN<), isocyanates, isothiocyanates, phosphoric acids (-O(P=O)OHOH), phosphonic acids (-O(P=O)OHH), haloacetyls, alkyl halides, acryloyls, aryl fluorides, hydroxylamines, disulfides, sulfonamides, sulfuric acids, vinyl sulfones, vinyl ketones, diazoalkanes, oxiranes, and aziridines.

[0087] If the IL-2 conjugate of the present invention contains one or more acidic or basic groups, the present invention also includes the corresponding pharmaceutically or toxicologically acceptable salt thereof, in particular the pharmaceutically usable salt thereof. Thus, the IL-2 conjugate of the present invention containing an acidic group can be used according to the present invention, for example, as an alkali metal salt, an alkaline earth metal salt, or an ammonium salt. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts, or salts with ammonia or organic amines, such as ethylamine, ethanolamine, triethanolamine, or amino acids. The IL-2 conjugate of the present invention containing one or more basic groups, i.e., protonable groups, can exist and be used according to the present invention in the form of its addition salt with an inorganic or organic acid. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. Further methods for converting basic groups to cations are known to those skilled in the art, such as alkylation of amine groups that yield suitable counterions for positively charged ammonium groups and salts. When the IL-2 conjugate of the present invention contains both an acidic and a basic group, the present invention also includes intramolecular salts or betaines (zwitterionic) in addition to the salt forms mentioned. Each salt can be obtained by conventional methods known to those skilled in the art, for example, by contacting these prodrugs with organic or inorganic acids or bases in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of the IL-2 conjugate of the present invention, which, due to their low physiological compatibility, are not directly suitable for use in pharmaceuticals, but can be used, for example, as intermediates in chemical reactions or to prepare pharmaceutically acceptable salts.

[0088] The term "pharmaceutically acceptable" means a substance that does not cause harm when administered to a patient and is approved by a regulatory authority, such as the EMA (Europe) and / or the FDA (US) and / or any other national regulatory authority, preferably for use in animals and preferably for use in humans.

[0089] As used herein, the term “excipient” refers to a diluent, adjuvant, or medium with which a therapeutic agent, such as a drug or prodrug, is administered. Such pharmaceutically excipients may be sterile solutions, such as water, and oils, such as those of petroleum, animal, plant, or synthetic origin, such as, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred excipient when a pharmaceutical composition is administered orally. Saline solution and aqueous dextrose are preferred excipients when a pharmaceutical composition is administered intravenously. Saline solution and aqueous dextrose and glycerol solution are preferably used as liquid excipients for injectable solutions. Suitable pharmaceutically excipients include starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If desired, the pharmaceutical composition may also contain trace amounts of wetting agents or emulsifiers, pH buffers, etc., such as acetates, succinates, tris, carbonates, phosphates, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), or surfactants, such as Tween, poloxamer, poloxamine, CHAPS, Igepal, or amino acids, such as glycine, lysine, or histidine. These pharmaceutical compositions may take the form of liquids, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The pharmaceutical compositions can be formulated as suppositories with common binders and excipients, such as triglycerides. Oral formulations may contain standard excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions contain a therapeutically effective amount of the drug or bioactive portion, along with an appropriate amount of excipients, to provide a form suitable for administration to a patient. The formulation should be adapted to the mode of administration.

[0090] Generally, the terms "comprise" or "comprising" also encompass "consist of" or "consisting of."

[0091] ratio バイアスIL-2 ratio アルデスロイキン The ratio to is greater than 1, preferably greater than 2, preferably greater than 3, preferably greater than 4, and more preferably greater than 5. In certain embodiments, the ratio バイアスIL-2 ratio アルデスロイキン The ratio to is greater than 10, greater than 20, greater than 50, greater than 70, greater than 100, or greater than 150.

[0092] Bias IL-2 portion-D is IL-2 portion, preferably (a) Modifying parts M that may be the same or different mod at least one stable bond, or (b) at least one amino acid mutation, or (c) at least one deletion, Any combination of (a), (b), and (c) It is an aldesleukin that contains [unclear].

[0093] At least one of the modifications described in (a), (b), and (c) is present in -D and is proportional in the bias IL-2 portion. バイアスIL-2 is アルデスロイキン To ensure it is higher. However, the IL-2 part is also compared when viewed individually. バイアスIL-2 is アルデスロイキン It is understood that this may include one or more modifications described in (a), (b), and (c), which do not guarantee a higher result. Such additional modifications may, for example, provide improvements in the production or storage properties of -D.

[0094] In one embodiment, the bias IL-2 portion is the modified portion M modIn another embodiment, the biased IL-2 moiety includes at least one stable binding to the IL-2 moiety. In another embodiment, the biased IL-2 moiety includes at least one amino acid mutation in the IL-2 moiety. In another embodiment, the biased IL-2 moiety includes at least one deletion in the IL-2 moiety. In another embodiment, the biased IL-2 moiety includes modified moiety M mod The modified portion M includes at least one stable binding to the IL-2 moiety and at least one amino acid mutation in the IL-2 moiety. In another embodiment, the biased IL-2 moiety is modified moiety M mod In another embodiment, the biased IL-2 moiety includes at least one stable binding to the IL-2 moiety and at least one deletion in the IL-2 moiety. In yet another embodiment, the biased IL-2 moiety includes at least one amino acid mutation and at least one deletion in the IL-2 moiety. mod This includes at least one stable binding to the IL-2 moiety, at least one amino acid mutation, and at least one deletion in the IL-2 moiety.

[0095] The bias IL-2 portion is the modified portion M mod It may include at least one stable bond to the IL-2 portion. mod The binding may be to the N-terminus, C-terminus, amino acid side chain, or internal site of the IL-2 moiety. mod This may preferably be a substituent or polymer moiety. In a particular embodiment, M mod The bond is at the N-terminus of the IL-2 portion. In a particular embodiment, M mod The bond is at the C-terminus of the IL-2 portion. In a particular embodiment, M mod The bond is to the amino acid side chain of the IL-2 moiety. In certain embodiments, M mod The bond is an internal part of the IL-2 portion. mod When binding occurs to the IL-2 moiety, the binding can occur at any combination of binding sites selected from the group consisting of the N-terminus, C-terminus, amino acid residue side chains, and internal regions.

[0096] In one embodiment, Mmod is a substituent. Preferably, such substituent has a molecular weight in the range of 15 Da to 1 kDa.

[0097] Such part M mod In one embodiment, it may be introduced in the form of a disulfide crosslinking. Preferably, such a disulfide crosslinking is formed between the thiol groups of two cysteine ​​residues. Such a disulfide crosslinking is an example of linking the modified moiety at an internal site. In one embodiment, these cysteine ​​residues may be naturally occurring cysteine ​​residues. In another embodiment, one or both of the cysteine ​​residues are not naturally occurring but are added to or inserted into the IL-2 moiety, preferably the IL-2 moiety of SEQ ID NO: 2, or replaced with a naturally occurring amino acid residue in the IL-2 moiety, preferably the IL-2 moiety of SEQ ID NO: 2.

[0098] A preferred method for obtaining such disulfide crosslinking is disclosed in Jones et al. (J. Am. Chem. Soc., 2012, 134(3), pp 1847-1852), International Publication Nos. 2011 / 018611, 2011 / 018612, and 2011 / 018613.

[0099] Preferably, such disulfide crosslinking occurs at a site related to binding to IL-2Rα. Therefore, preferably, disulfide crosslinking results in a reduction of the affinity of the biased IL-2 moiety to IL-2Rαβ compared to aldesleukine.

[0100] In one embodiment, disulfide crosslinking is formed between C57 and C104 when the IL-2 portion has the sequence of SEQ ID NO: 2.

[0101] In another embodiment, M modThe polymer portion is a polymer. Such polymer portions may include linear, branched, or multi-armed polymers. In one embodiment, the polymer is a linear polymer. In another embodiment, the polymer is a branched polymer. Such a branched polymer preferably has 1, 2, 3, 4, or 5 branching points. From each branching point, preferably 2, 3, or 4 polymer arms extend. In another embodiment, the polymer is a multi-armed polymer. Such a multi-armed polymer preferably has 3, 4, 5, 6, 7, or 8 polymer arms.

[0102] M modIf the polymer portion is a polymer portion, such a polymer portion preferably has a molecular weight in the range of 0.5 kDa to 1000 kDa, for example 1 kDa to 1000 kDa, more preferably 2 kDa to 500 kDa, even more preferably 3 kDa to 200 kDa, most preferably 5 kDa to 120 kDa, or in the range of 7 to 40 kDa. In one embodiment, such a polymer has a molecular weight of about 0.5 kDa. In one embodiment, such a polymer has a molecular weight of about 1 kDa. In one embodiment, such a polymer has a molecular weight of about 2 kDa. In one embodiment, such a polymer has a molecular weight of about 3 kDa. In one embodiment, such a polymer has a molecular weight of about 4 kDa. In one embodiment, such a polymer has a molecular weight of about 5 kDa. In one embodiment, such a polymer has a molecular weight of about 7.5 kDa. In another embodiment, such a polymer portion has a molecular weight of about 10 kDa. In another embodiment, such a polymer portion has a molecular weight of about 15 kDa. In another embodiment, such a polymer portion has a molecular weight of about 20 kDa. In another embodiment, such a polymer portion has a molecular weight of about 30 kDa. In another embodiment, such a polymer portion has a molecular weight of about 40 kDa. In another embodiment, such a polymer portion has a molecular weight of about 50 kDa. In another embodiment, such a polymer portion has a molecular weight of about 70 kDa. In another embodiment, such a polymer portion has a molecular weight of about 80 kDa. In another embodiment, such a polymer portion has a molecular weight of about 90 kDa. In another embodiment, such a polymer portion has a molecular weight of about 100 kDa. In one embodiment, such a polymer has a molecular weight of 0.5 kDa. In one embodiment, such a polymer has a molecular weight of 1 kDa. In one embodiment, such a polymer has a molecular weight of 2 kDa. In one embodiment, such a polymer has a molecular weight of 3 kDa. In one embodiment, such a polymer has a molecular weight of 4 kDa. In one embodiment, such a polymer has a molecular weight of 5 kDa. In one embodiment, such a polymer has a molecular weight of 7.5 kDa.In another embodiment, such a polymer portion has a molecular weight of 10 kDa. In another embodiment, such a polymer portion has a molecular weight of 15 kDa. In another embodiment, such a polymer portion has a molecular weight of 20 kDa. In another embodiment, such a polymer portion has a molecular weight of 30 kDa. In another embodiment, such a polymer portion has a molecular weight of 40 kDa. In another embodiment, such a polymer portion has a molecular weight of 50 kDa. In another embodiment, such a polymer portion has a molecular weight of 70 kDa. In another embodiment, such a polymer portion has a molecular weight of 80 kDa. In another embodiment, such a polymer portion has a molecular weight of 90 kDa. In another embodiment, such a polymer portion has a molecular weight of 100 kDa.

[0103] M odIf the polymer portion is a polymer, such polymer portion is preferably 2-methacryloyloxyethylphosphoryl(phosphoyl)choline, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amideamine), poly(amino acid), poly(acid anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl methacrylamide) The polymers include polymers selected from the group consisting of poly(hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethylcellulose, hydroxypropylmethylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and its derivatives, functionalized hyaluronic acid, alginate, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch, and other carbohydrate-based polymers, xylan, and copolymers thereof.

[0104] In one embodiment, M mod It is a PEG-based polymer.

[0105] In another embodiment, M modIt is a hyaluronic acid-based polymer.

[0106] In another embodiment, M mod This includes a peptide or protein moiety. In one embodiment, such a peptide or protein moiety M mod This may be a translational fusion with the IL-2 moiety contained in bias IL-2. In another embodiment, such a peptide or protein moiety M mod This can be chemically conjugated to the IL-2 portion contained in bias IL-2. Preferably, this peptide or protein portion M mod It is not a fragment or part of IL-2.

[0107] M in the form of a peptide or protein portion mod These may be synthetic or natural protein moieties, parts thereof, or variants thereof. Exemplary non-IL-2 moieties include albumin, antibody domains, such as the Fc domain or antigen-binding domain of immunoglobulin, CTP, and CD25, each in its naturally occurring form or as a variant or fragment thereof.

[0108] Peptide or protein portion M fused to the IL-2 moiety mod It may be attached to the N-terminus or C-terminus, or inserted into the internal position of the IL-2 moiety. One or more peptide or protein moieties M mod It is understood that the IL-2 portion may be translated, fused, chemically conjugated, and / or inserted. The one or more peptide or protein portions M mod These may have the same or different sequences. For example, the biased IL-2 moiety may have a first peptide or protein moiety M that is translated-fused or chemically conjugated to the N-terminus of the IL-2 moiety. mod , and a second peptide or protein moiety M translated fusion or chemically conjugated to the C-terminus of the IL-2 moiety mod It may have a biased IL-2 moiety, which is translated-fused or chemically conjugated to the N-terminus of the IL-2 moiety, or a first peptide or protein moiety M. mod, and a second peptide or protein moiety M that is translated or chemically conjugated to the internal position of the IL-2 moiety. mod This may include: In another example, the biased IL-2 moiety may include a first peptide or protein moiety M that is translated-fused or chemically conjugated to the C-terminus of the IL-2 moiety. mod , and a second peptide or protein moiety M that is translated or chemically conjugated to the internal position of the IL-2 moiety. mod This may include. In further examples, the biased IL-2 moiety may include a first peptide or protein moiety M that is translated-fused or chemically conjugated to the N-terminus of the IL-2 moiety. mod , a second peptide or protein moiety M that is translated or chemically conjugated to the C-terminus of the IL-2 moiety mod , and a third peptide or protein moiety M that is translated or chemically conjugated at the internal position of the IL-2 moiety. mod It may include.

[0109] M od The binding can be a proteogenic or non-proteogenic amino acid residue of the IL-2 moiety. In certain embodiments, M mod The binding occurs at a proteogenic amino acid. Such proteogenic amino acid residues are preferably selected from the group consisting of cysteine, methionine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, glutamine, and arginine. In certain embodiments, M mod The binding occurs with non-proteinogenic amino acids. When the modified portion binds to a non-proteinogenic amino acid residue, it is understood that such a non-proteinogenic amino acid residue is artificially introduced into the IL-2 moiety. Such a non-proteinogenic amino acid residue is M modThis can be any non-proteinogenic amino acid residue having a functional group available for conjugation to the IL-2 moiety. In certain embodiments, such a non-proteinogenic amino acid has a functional group in its side chain selected from the group consisting of carbonyl, carbonyl derivatives, e.g., carbonyl-like groups, labeled carbonyl groups and protected carbonyl groups, azides, oximes, and hydroxylamines.

[0110] In certain embodiments, such non-proteinogenic amino acids are non-proteinogenic amino acids described in International Publication No. 2006 / 069246A2 (these non-proteinogenic amino acids are incorporated herein by reference). In certain embodiments, the non-proteinogenic amino acid is the structure described in formulas (I)

[0265] to

[0283] , formula (XXX)

[0284] , formula (XXX-A)

[0285] , formula (XXX-B)

[0286] , formula (XXXI)

[0287] , formula (XXXI)

[0288] , formula (XXXI-A)

[0289] , formula (XXXI-B)

[0290] , formula (XXXII)

[0291] , formula (XXXII-A)

[0292] , formula (XXXII-B)

[0293] , and formula (XXXX)

[0294] in International Publication No. 2006 / 069246A2 (the non-proteinogenic amino acid is incorporated herein by reference). The structure of XI), the incorrectly displayed paragraph

[0100] , i.e., the structure of formula (XXXXII) in the paragraph between

[0294] and

[0295] , the structure of formula (XXXXIII) in

[0295] and

[0296] , the structure of formula (XIV) in

[0302] to

[0305] , the structure of formula (XV) in

[0306] and

[0307] , the structure of formula (XI) in

[0310] to

[0312] It has the structure of (XII) in

[0313] , the structure of the formulas (XII) in

[0314] and

[0315] , the structure of the formula (XIV) in

[0316] , the structure of the formula (XVI) in

[0317] , the structure of the formulas (XVI) in

[0318] and

[0319] , the structure of the formulas (XVIII) in

[0320] and

[0321] , or the structure of the formula (XXIX) in

[0530] .

[0111] In one embodiment, Mmod Binding occurs at lysine residues of the IL-2 moiety, for example, lysine residues selected from the group consisting of K7, K8, K31, K34, K42, K47, K48, K53, K63, K75, and K96 based on SEQ ID NO: 2, or at the corresponding positions of the homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional lysine residues compared to SEQ ID NO: 2, binding may also occur at such additional lysine residues. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 lysine residues of SEQ ID NO: 2, M mod It is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and contains more lysine residues than the IL-2 portion of SEQ ID NO: 2, then 11 more lysine residues can be used for binding. mod It may be used for binding, i.e., up to the maximum number of lysine residues present in such homologs or variants of SEQ ID NO: 2 mod It may be used for joining. In one embodiment, one part M mod However, it binds to one lysine residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two lysine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, three parts M which may be the same or different mod However, it binds to three lysine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, four parts M which may be the same or different mod However, it binds to four lysine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, five parts M which may be the same or different mod However, it binds to five lysine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, six parts M which may be the same or different mod However, it binds to six lysine residues of SEQ ID NO: 2 or its homolog or variant.

[0112] In another embodiment, M modBinding occurs at the threonine residues of the IL-2 moiety, for example, threonine residues selected from the group consisting of T2, T6, T9, T36, T40, T50, T100, T101, T110, T112, T122, T130, and T132 based on SEQ ID NO: 2, or at the corresponding positions of the homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional threonine residues compared to SEQ ID NO: 2, binding may also occur at such additional threonine residues. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 threonine residues of SEQ ID NO: 2, M mod It is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and contains more threonine residues than the IL-2 portion of SEQ ID NO: 2, then 13 more threonine residues can be used. mod It may be used for binding, that is, up to the maximum number of threonine residues present in such homolog or variant, M mod It may be used for joining. In one embodiment, one part M mod However, it binds to one threonine residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two threonine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, three parts M which may be the same or different mod However, it binds to three threonine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, four parts M which may be the same or different mod However, it binds to four threonine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, five parts M which may be the same or different mod However, it binds to five threonine residues of SEQ ID NO: 1 or its homolog or variant. In another embodiment, six parts M which may be the same or different mod However, it binds to six threonine residues of SEQ ID NO: 2 or its homolog or variant.

[0113] In another embodiment, M modBinding occurs at serine residues of the IL-2 moiety, for example, serine residues selected from the group consisting of S3, S4, S5, S74, S86, S98, S124, S126, and S129 based on SEQ ID NO: 2, or at the corresponding positions of the homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional serine residues compared to SEQ ID NO: 2, binding may also occur at such additional serine residues. The first, second, third, fourth, fifth, sixth, seventh, eighth, or ninth serine residue of SEQ ID NO: 2 can be linked to M mod It is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and contains more serine residues than the IL-2 portion of SEQ ID NO: 2, then 9 more serine residues than M mod It may be used for binding, that is, up to the maximum number of serine residues present in such homolog or variant, M mod It may be used for joining. In one embodiment, one part M mod However, it binds to one serine residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two serine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, three parts M which may be the same or different mod However, it binds to three serine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, four parts M which may be the same or different mod However, it binds to four serine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, five parts M which may be the same or different mod However, it binds to five serine residues of SEQ ID NO: 1 or its homolog or variant. In another embodiment, six parts M which may be the same or different mod However, it binds to six serine residues of SEQ ID NO: 2 or its homolog or variant.

[0114] In another embodiment, M modBinding occurs at tyrosine residues of the IL-2 moiety, for example, tyrosine residues selected from the group consisting of Y30, Y44, and Y106 based on SEQ ID NO: 2, or at the corresponding positions of the homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional tyrosine residues compared to SEQ ID NO: 2, binding may also occur at such additional tyrosine residues. One, two, or three tyrosine residues of SEQ ID NO: 2, M mod It is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and contains more tyrosine residues than the IL-2 portion of SEQ ID NO: 2, then 3 more tyrosine residues can be used. mod It may be used for binding, that is, up to the maximum number of tyrosine residues present in such homolog or variant, M mod It may be used for joining. In one embodiment, one part M mod However, it binds to one tyrosine residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two tyrosine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, three parts M which may be the same or different mod However, it binds to three tyrosine residues of SEQ ID NO: 2 or its homolog or variant.

[0115] In another embodiment, M mod Binding occurs at the histidine residues of the IL-2 moiety, for example, histidine residues selected from the group consisting of H15, H54, and H78 based on SEQ ID NO: 2, or at the corresponding positions of the homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional histidine residues compared to SEQ ID NO: 2, binding may also occur at such additional histidine residues. 1, 2, or 3 histidine residues of SEQ ID NO: 2, M mod It is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and contains more histidine residues than the IL-2 portion of SEQ ID NO: 2, then 3 more histidine residues can be used.mod It may be used for binding, that is, up to the maximum number of histidine residues present in such homolog or variant, M mod It may be used for joining. In one embodiment, one part M mod However, it binds to one histidine residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two histidine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, three parts M which may be the same or different mod However, it binds to three histidine residues of SEQ ID NO: 2 or its homolog or variant.

[0116] In another embodiment, M mod Binding occurs at the tryptophan residue of the IL-2 moiety, for example, the tryptophan residue at position W120 based on SEQ ID NO: 2, or at the corresponding position of the homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional tryptophan residues compared to SEQ ID NO: 2, binding may also occur at such additional tryptophan residues. If the IL-2 moiety is a homolog or variant of SEQ ID NO: 2 and contains more tryptophan residues than the IL-2 moiety of SEQ ID NO: 2, then one or more tryptophan residues may be used for binding. mod It may be used for binding, that is, up to the maximum number of tryptophan residues present in such homolog or variant, M mod It may be used for joining. In one embodiment, one part M mod However, it binds to one tryptophan residue of SEQ ID NO: 2 or its homolog or variant.

[0117] In another embodiment, M modBinding occurs at the aspartic acid residues of the IL-2 moiety, for example, aspartic acid residues selected from the group consisting of D19, D83, and D108 based on SEQ ID NO: 2, or at the corresponding positions of the homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional aspartic acid residues compared to SEQ ID NO: 2, binding may also occur at such additional aspartic acid residues. One, two, or three aspartic acid residues of SEQ ID NO: 2, M mod It is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and contains more aspartic acid residues than the IL-2 portion of SEQ ID NO: 2, then 3 more aspartic acid residues can be used for binding. mod It may be used for binding, that is, up to the maximum number of aspartic acid residues present in such homolog or variant, M mod It may be used for joining. In one embodiment, one part M mod However, it binds to one aspartic acid residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two aspartic acid residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, three parts M which may be the same or different mod However, it binds to three aspartic acid residues of SEQ ID NO: 2 or its homolog or variant.

[0118] In one embodiment, M mod Binding occurs at glutamic acid residues of the IL-2 moiety, for example, glutamic acid residues selected from the group consisting of E14, E51, E59, E60, E61, E66, E67, E94, E99, E105, E109, and E115 based on SEQ ID NO: 2, or at the corresponding positions of homologs or variants of SEQ ID NO: 2. If a homolog or variant of IL-2 contains one or more additional glutamic acid residues compared to SEQ ID NO: 2, binding may also occur at such additional glutamic acid residues. Glutamic acid residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of SEQ ID NO: 2, M modIt is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and contains more glutamate residues than the IL-2 portion of SEQ ID NO: 2, then 12 more glutamate residues can be used. mod It may be used for binding, that is, up to the maximum number of glutamic acid residues present in such homologs or variants of SEQ ID NO: 2 may be used for binding one or more modified moieties. In one embodiment, one moiety M mod However, it binds to one glutamic acid residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two glutamic acid residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, three parts M which may be the same or different mod However, it binds to three glutamic acid residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, four parts M which may be the same or different mod However, it binds to four glutamic acid residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, five parts M which may be the same or different mod However, it binds to five glutamic acid residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, six parts M which may be the same or different mod However, it binds to six glutamic acid residues of SEQ ID NO: 2 or its homolog or variant.

[0119] In another embodiment, M mod Binding occurs at arginine residues of the IL-2 moiety, for example, arginine residues selected from the group consisting of R37, R80, R82, and R119 based on SEQ ID NO: 2, or at the corresponding positions of homologs or variants of SEQ ID NO: 2. If homologs or variants of IL-2 contain one or more additional arginine residues compared to SEQ ID NO: 2, binding may also occur at such additional arginine residues. 1, 2, 3, or 4 arginine residues of SEQ ID NO: 2, M modIt is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and contains more arginine residues than the IL-2 portion of SEQ ID NO: 2, then 4 more arginine residues can be used. mod It may be used for binding, that is, up to the maximum number of arginine residues present in such homolog or variant, M mod It may be used for joining. In one embodiment, one part M mod However, it binds to one arginine residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two arginine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, three parts M which may be the same or different mod However, it binds to three arginine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, four parts M which may be the same or different mod However, it binds to four arginine residues of SEQ ID NO: 2 or its homolog or variant.

[0120] In another embodiment, M mod Binding occurs at a cysteine ​​residue in the IL-2 moiety, for example, a cysteine ​​residue selected from the group consisting of C57 and C104 based on SEQ ID NO: 2, or at the corresponding position in the homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional cysteine ​​residues compared to SEQ ID NO: 2, binding may also occur at such additional cysteine ​​residues. One or two cysteine ​​residues of SEQ ID NO: 2, M mod It is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and contains more cysteine ​​residues than the IL-2 portion of SEQ ID NO: 2, then two more cysteine ​​residues can be used for binding. mod It may be used for binding, that is, up to the maximum number of cysteine ​​residues present in such homolog or variant, M mod It may be used for joining. In one embodiment, one part M modHowever, it binds to one cysteine ​​residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two cysteine ​​residues of SEQ ID NO: 2 or its homolog or variant.

[0121] In another embodiment, M mod Binding occurs at methionine residues of the IL-2 moiety, for example, methionine residues selected from the group consisting of M22, M38, M45, and M103 based on SEQ ID NO: 2, or at the corresponding positions of the homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional methionine residues compared to SEQ ID NO: 2, binding may also occur at such additional methionine residues. It is understood that one, two, three, or four methionine residues of SEQ ID NO: 2 may be used for the binding of one or more modification moieties. If the IL-2 moiety is a homolog or variant of SEQ ID NO: 2 and contains more methionine residues than the IL-2 moiety of SEQ ID NO: 2, then four or more methionine residues may be used for the binding of one or more modification moieties. mod It may be used for binding, that is, up to the maximum number of methionine residues present in such homolog or variant, M mod It may be used for joining. In one embodiment, one part M mod However, it binds to one methionine residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two methionine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, three parts M which may be the same or different mod However, it binds to three methionine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, four parts M which may be the same or different mod However, it binds to four methionine residues of SEQ ID NO: 2 or its homolog or variant.

[0122] In another embodiment, M modBinding occurs at glutamine residues of the IL-2 moiety, for example, glutamine residues selected from the group consisting of Q10, Q12, Q21, Q56, Q73, and Q125 based on SEQ ID NO: 2, or at the corresponding positions of homologs or variants of SEQ ID NO: 2. If homologs or variants of IL-2 contain one or more additional glutamine residues compared to SEQ ID NO: 2, binding may also occur at such additional glutamine residues. Glutaamine residues 1, 2, 3, 4, 5, 6, 7, 8, or 9 of SEQ ID NO: 2, M mod It is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and contains more glutamine residues than the IL-2 portion of SEQ ID NO: 2, then 6 more glutamine residues can be used. mod It may be used for binding, that is, up to the maximum number of glutamine residues present in such homolog or variant, M mod It may be used for joining. In one embodiment, one part M mod However, it binds to one glutamine residue of SEQ ID NO: 2 or its homolog or variant. In another embodiment, two parts M which may be the same or different mod However, it binds to two glutamine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, three parts M which may be the same or different mod However, it binds to three glutamine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, four parts M which may be the same or different mod However, it binds to four glutamine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, five parts M which may be the same or different mod However, it binds to five glutamine residues of SEQ ID NO: 2 or its homolog or variant. In another embodiment, six parts M which may be the same or different mod However, it binds to six glutamine residues of SEQ ID NO: 2 or its homolog or variant.

[0123] In certain embodiments, the bias IL-2 moiety is a moiety M bound to one or more types of amino acid residues, such as cysteine ​​and lysine.mod It is understood that it may have

[0124] M od The bond to the IL-2 portion is via a stable covalent bond. In a particular embodiment, the IL-2 portion and portion M mod The bond between them is via an amide. In certain embodiments, the IL-2 portion and the M portion mod The connection between the two is partial [ka] It is done through.

[0125] Preferably, at least one part M mod The binding occurs at the amino acid position of the IL-2 moiety known to be involved in binding to IL-2Rα. Thus, preferably at least one moiety M mod The binding of this compound results in a substantial reduction in the affinity of the IL-2 moiety, preferably the variant of the IL-2 moiety of SEQ ID NO: 2, to IL-2Rαβ compared to aldesleukin, i.e., a biased IL-2 moiety is introduced.

[0126] Preferably, M mod The binding occurs at an amino acid position selected from the group consisting of K34, R37, M38, T40, F41, K42, F43, Y44, E61, and L71 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. More preferably, M mod The binding occurs at an amino acid position selected from the group consisting of F41, Y44, E61, and L71 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. All of these amino acid positions are M mod It does not contain functional groups that enable direct conjugation, and M mod It is understood that certain steps may be necessary before joining at these amino acid positions, such as replacing naturally occurring amino acids with different amino acids or performing certain chemical modifications. Therefore, M modThe binding may occur at those positions with either naturally occurring amino acids or amino acids that have been replaced by naturally occurring amino acids at those specific positions, and the binding sites may be proteinogenic or non-proteinogenic amino acids, both embodiments of which are described above. In certain embodiments, M mod The binding occurs at amino acid position K34 based on SEQ ID NO: 2 or at the corresponding position of its homolog or variant. In certain embodiments, M mod The binding occurs at amino acid position R37 based on SEQ ID NO: 2 or at the corresponding position of its homolog or variant. In certain embodiments, M mod The binding occurs at amino acid position M38 based on SEQ ID NO: 2 or at the corresponding position of its homolog or variant. In certain embodiments, M mod The binding occurs at amino acid position T40 based on SEQ ID NO: 2 or at the corresponding position of its homolog or variant. In certain embodiments, M mod The binding occurs at amino acid position F41 based on SEQ ID NO: 2 or at the corresponding position of its homolog or variant. In certain embodiments, M mod The binding occurs at amino acid position K42 based on SEQ ID NO: 2 or at the corresponding position of its homolog or variant. In certain embodiments, M mod The binding occurs at amino acid position F43 based on SEQ ID NO: 2 or at the corresponding position of its homolog or variant. In certain embodiments, M mod The binding occurs at amino acid position Y44 based on SEQ ID NO: 2 or at the corresponding position of its homolog or variant. In certain embodiments, M mod The binding occurs at amino acid position E61 based on SEQ ID NO: 2 or at the corresponding position of its homolog or variant. In certain embodiments, M mod The binding occurs at amino acid position L71 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant.

[0127] In a particular embodiment, M mod is equation (A-1) [ka] (In the formula, -FG- is a bond, -SP- is the spacer part. -POL is a polymer.) It belongs to them.

[0128] In a particular embodiment, -FG- in formula (A-1) is [ka] And, In the formula, dashed lines marked with an asterisk indicate bonds to sulfur in the IL-2 portion, while unmarked dashed lines indicate bonds to -SP-. The sulfur may be provided by the side chain of cysteine.

[0129] In a particular embodiment, -FG- in formula (A-1) is [ka] (In the formula, dashed lines marked with an asterisk indicate bonding to nitrogen in the IL-2 portion, while unmarked dashed lines indicate bonding to -SP-.) The nitrogen may be the nitrogen of the N-terminal amine of the IL-2 moiety or the nitrogen of the lysine side chain. In certain embodiments, the nitrogen is the nitrogen of the N-terminal amine of the IL-2 moiety. In certain embodiments, such nitrogen is the nitrogen of the lysine side chain of the IL-2 moiety.

[0130] In a particular embodiment, -FG- in formula (A-1) is [ka] (In the formula, dashed lines marked with an asterisk indicate the bond of nitrogen to sulfur in the IL-2 portion, and dashed lines without an asterisk indicate the bond to -SP-. a1 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.) The sulfur may be sulfur provided by the cysteine ​​side chain, and the nitrogen may be nitrogen from the N-terminal amine of the IL-2 moiety or nitrogen from the lysine side chain. In certain embodiments, the asterisked dashed line indicates a bond to sulfur, and the sulfur is provided by the cysteine ​​side chain. In certain embodiments, a1 is an integer in the range of 1 to 8. In certain embodiments, a1 is an integer in the range of 1 to 6. In certain embodiments, a1 is an integer in the range of 1 to 4. In certain embodiments, a1 is 1. In certain embodiments, a1 is 2. In certain embodiments, a1 is 3. In certain embodiments, a1 is 4. In certain embodiments, a1 is 5. In certain embodiments, a1 is 6.

[0131] In a particular embodiment, -FG- in formula (A-1) is [ka] (In the formula, the dashed lines marked with an asterisk indicate the bond of nitrogen to sulfur in the IL-2 portion, the dashed lines without an asterisk indicate the bond to -SP-, and a2 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.) In certain embodiments, a2 is an integer in the range of 1 to 8. In certain embodiments, a2 is an integer in the range of 1 to 6. In certain embodiments, a2 is an integer in the range of 1 to 4. In certain embodiments, a2 is 1. In certain embodiments, a2 is 2. In certain embodiments, a2 is 3. In certain embodiments, a2 is 4. In certain embodiments, a2 is 5. In certain embodiments, a2 is 6. The sulfur may be sulfur provided by the cysteine ​​side chain, and the nitrogen may be nitrogen from the N-terminal amine of the IL-2 moiety or nitrogen from the lysine side chain. In certain embodiments, the asterisked dashed line indicates a bond to sulfur, and the sulfur is provided by the cysteine ​​side chain.

[0132] In a particular embodiment, -SP- in formula (A-1) is C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Selected from the group consisting of alkynnyls, where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is the same or different one or more R 9 Replaced by optional means, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) 10 )-,-S(O)2N(R 10 )-,-S(O)N(R 10 )-, -S(O)2-, -S(O)-, -N(R 10 )S(O)2N(R 10a )-, -S-, -N(R 10 )-, -OC(OR 10 )(R 10a )-,-N(R 10 )C(O)N(R 10a )-, and -OC(O)N(R 10 Interrupted by one or more elements selected from the group consisting of )-, Each T stands for phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 A molecule is independently selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl, and each T is the same or different R 9 It is independently and arbitrarily replaced by, Each -R 9 These are halogen, -CN, oxo (=O), and -COOR. 11 , -OR 11 , -C(O)R 11 ,-C(O)N(R 11 R 11a ), -S(O)2N(R 11 R 11a ), -S(O)N(R 11 R 11a), -S(O)2R 11 ,-S(O)R 11 , -N(R 11 )S(O)2N(R 11a R 11b ), -SR 11 , -N(R 11 R 11a ), -NO2, -OC(O)R 11 , -N(R 11 )C(O)R 11a , -N(R 11 )S(O)2R 11a , -N(R 11 )S(O)R 11a , -N(R 11 )C(O)OR 11a , -N(R 11 )C(O)N(R 11a R 11b ), -OC(O)N(R 11 R 11a ), and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 Alkyl is optionally substituted with one or more halogens, one or more of the same or different halogens. Each -R 10 , -R 10a , -R 11 , -R 11a , and -R 11b -H and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 Alkyl is optionally substituted with one or more halogens, either the same or different.

[0133] In a particular embodiment, -SP- in formula (A-1) is C 1-20 It is alkyl, C 1-20 Alkyl is one or more -R 9 Replaced by optional means, C 1-20 Alkyl is -O-, -C(O)N(R 10 )-, -S(O)2-, -S(O)-, -S-, -N(R 10 )-, -OC(OR 10 )(R 10a )-,-N(R 10 )C(O)N(R 10a)-, and -OC(O)N(R 10 )- is optionally interrupted by one or more elements selected from the group consisting of -R, where each -R 9 C 1-6 Selected from the group consisting of alkyl groups, each -R 10 and -R 10a -H and C 1-6 It is independently selected from the group consisting of alkyl groups.

[0134] In a particular embodiment, -SP- in formula (A-1) is C 1-10 It is alkyl, C 1-10 Alkyl is one or more -R 9 Replaced by optional means, C 1-10 Alkyl is -O-, -C(O)N(R 10 )-, -S(O)2-, -S(O)-, -S-, -N(R 10 )-, -OC(OR 10 )(R 10a )-,-N(R 10 )C(O)N(R 10a )-, and -OC(O)N(R 10 )- is optionally interrupted by one or more elements selected from the group consisting of -R, where each -R 9 C 1-6 Selected from the group consisting of alkyl groups, each -R 10 and -R 10a -H and C 1-6 It is independently selected from the group consisting of alkyl groups.

[0135] In certain embodiments, -POL in formula (A-1) is a PEG-based polymer. In certain embodiments, -POL is of formula (A-1i). [ka] (In the formula, The dashed line indicates a connection to -SP-. m is either 0 or 1. p is an integer between 12 and 22700. q is selected from the group consisting of 1, 2, 3, 4, 5, and 6).

[0136] In a particular embodiment, m in equation (A-1i) is 0. In a particular embodiment, m in equation (A-1i) is 1.

[0137] In certain embodiments, p in formula (A-1i) is an integer in the range of 23 to 227000, for example, 45 to 11300, or 69 to 4540, or 114 to 2700. In certain embodiments, p in formula (A-1i) is approximately 12. In certain embodiments, p in formula (A-1i) is approximately 23. In certain embodiments, p in formula (A-1i) is approximately 46. In certain embodiments, p in formula (A-1i) is approximately 68. In certain embodiments, p in formula (A-1i) is approximately 90. In certain embodiments, p in formula (A-1i) is approximately 112. In certain embodiments, p in formula (A-1i) is approximately 170. In certain embodiments, p in formula (A-1i) is approximately 227. In certain embodiments, p in formula (A-1i) is approximately 340. In certain embodiments, p in formula (A-1i) is approximately 450. In a particular embodiment, p in formula (A-1i) is approximately 680. In a particular embodiment, p in formula (A-1i) is approximately 900. In a particular embodiment, p in formula (A-1i) is approximately 1130. In a particular embodiment, p in formula (A-1i) is approximately 1350. In a particular embodiment, p in formula (A-1i) is approximately 1590. In a particular embodiment, p in formula (A-1i) is approximately 1800. In a particular embodiment, p in formula (A-1i) is approximately 2045. In a particular embodiment, p in formula (A-1i) is approximately 2275.

[0138] In a particular embodiment, q in formula (A-1i) is 1. In a particular embodiment, q in formula (A-1i) is 2. In a particular embodiment, q in formula (A-1i) is 3. In a particular embodiment, q in formula (A-1i) is 4. In a particular embodiment, q in formula (A-1i) is 5. In a particular embodiment, q in formula (A-1i) is 6.

[0139] In a particular embodiment, -POL in formula (A-1) is equivalent to formula (A-1ii) [ka] (In the formula, The dashed line indicates a connection to -SP-. FG is a functional group, m is either 0 or 1. p is an integer in the range of 12 to 22700. (q is selected from the group consisting of 1, 2, 3, 4, 5, and 6.) It belongs to them.

[0140] Part M of equation (A-1) mod However, further parts, for example, one or more parts - L 1 -L 2 When conjugated with -Z, it is advantageous if the -POL part ends in a functional group. If -POL is of formula (A-1ii), then such a compound is a reagent, and one or more such parts, for example, one or more parts -L 1 -L 2 After conjugation of the reagent Z to the functional group, FG is no longer present, but it is understood to form a bond with the appropriate functional group present in one or more further parts of the reagent form.

[0141] M mod The part that is conjugated, for example, part -L 1 -L 2 It is also understood that other bonding sites for -Z may be possible.

[0142] In a particular embodiment, m in formula (A-1ii) is 0. In a particular embodiment, m in formula (A-1ii) is 1.

[0143] In certain embodiments, p in formula (A-1ii) is an integer in the range of 23 to 227000, for example, 45 to 11300, or 69 to 4540, or 114 to 2700. In certain embodiments, p in formula (A-1ii) is approximately 12. In certain embodiments, p in formula (A-1ii) is approximately 23. In certain embodiments, p in formula (A-1ii) is approximately 46. In certain embodiments, p in formula (A-1ii) is approximately 68. In certain embodiments, p in formula (A-1ii) is approximately 90. In certain embodiments, p in formula (A-1ii) is approximately 112. In certain embodiments, p in formula (A-1ii) is approximately 170. In certain embodiments, p in formula (A-1ii) is approximately 227. In certain embodiments, p in formula (A-1ii) is approximately 340. In certain embodiments, p in formula (A-1ii) is approximately 450. In a particular embodiment, p in formula (A-1ii) is approximately 680. In a particular embodiment, p in formula (A-1ii) is approximately 900. In a particular embodiment, p in formula (A-1ii) is approximately 1130. In a particular embodiment, p in formula (A-1ii) is approximately 1350. In a particular embodiment, p in formula (A-1ii) is approximately 1590. In a particular embodiment, p in formula (A-1ii) is approximately 1800. In a particular embodiment, p in formula (A-1ii) is approximately 2045. In a particular embodiment, p in formula (A-1ii) is approximately 2275.

[0144] In a particular embodiment, q in formula (A-1ii) is 1. In a particular embodiment, q in formula (A-1ii) is 2. In a particular embodiment, q in formula (A-1ii) is 3. In a particular embodiment, q in formula (A-1ii) is 4. In a particular embodiment, q in formula (A-1ii) is 5. In a particular embodiment, q in formula (A-1ii) is 6.

[0145] Further parts, for example, part-L 1 -L 2 -Z is M via the part of equation (A-1) -POL modWhen conjugated, the part-POL is in formula (A-1iii), (A-1iv), (A-1v), or (A-1vi) [ka] (In the formula, The dashed lines marked with an asterisk indicate further subdivisions, such as subdivision L. 1 -L 2 Shows the bond to -Z, Unmarked dashed lines indicate connections to -SP-. m, p, and q are used as defined in equation (A-1i). It could be.

[0146] In a particular embodiment, further parts may be added, for example, part-L 1 -L 2 -Z is M via the part of equation (A-1) -POL mod It is conjugated to give rise to the -POL portion of equation (A-1iii). In certain embodiments, further portions may be added, for example, the -L portion. 1 -L 2 -Z is M via the part of equation (A-1) -POL mod It is conjugated to yield the -POL portion of equation (A-1iv). In certain embodiments, further portions may be added, for example, the -L portion. 1 -L 2 -Z is M via the part of equation (A-1) -POL mod It is conjugated to yield the -POL portion of equation (A-1v). In certain embodiments, further portions may be added, for example, the -L portion. 1 -L 2 -Z is M via the part of equation (A-1) -POL mod It is conjugated to yield the -POL part of equation (A-1vi).

[0147] In certain embodiments, -POL in formula (A-1) is a hyaluronic acid-based polymer.

[0148] In a particular embodiment, M mod is equation (A-1a) [ka] (In the formula, The dashed lines marked with asterisks indicate the binding of the side chain of the amino acid residue in the IL-2 moiety to the sulfur. b1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. b2 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. b3 is an integer in the range of 12 to 22700. It belongs to them.

[0149] In a particular embodiment, b1 in formula (A-1a) is an integer in the range of 1 to 8. In a particular embodiment, b1 in formula (A-1a) is an integer in the range of 1 to 6. In a particular embodiment, b1 in formula (A-1a) is an integer in the range of 1 to 4. In a particular embodiment, b1 in formula (A-1a) is 1. In a particular embodiment, b1 in formula (A-1a) is 2. In a particular embodiment, b1 in formula (A-1a) is 3. In a particular embodiment, b1 in formula (A-1a) is 4. In a particular embodiment, b1 in formula (A-1a) is 5. In a particular embodiment, b1 in formula (A-1a) is 6.

[0150] In a particular embodiment, b2 in formula (A-1a) is an integer in the range of 1 to 8. In a particular embodiment, b2 in formula (A-1a) is an integer in the range of 1 to 6. In a particular embodiment, b2 in formula (A-1a) is an integer in the range of 1 to 4. In a particular embodiment, b2 in formula (A-1a) is 1. In a particular embodiment, b2 in formula (A-1a) is 2. In a particular embodiment, b2 in formula (A-1a) is 3. In a particular embodiment, b2 in formula (A-1a) is 4. In a particular embodiment, b2 in formula (A-1a) is 5. In a particular embodiment, b2 in formula (A-1a) is 6.

[0151] In certain embodiments, b3 in formula (A-1a) is an integer in the range of 23 to 227000, for example, 45 to 11300, or 69 to 4540, or 114 to 2700. In certain embodiments, b3 in formula (A-1a) is about 12. In certain embodiments, b3 in formula (A-1a) is about 23. In certain embodiments, b3 in formula (A-1a) is about 46. In certain embodiments, b3 in formula (A-1a) is about 68. In certain embodiments, b3 in formula (A-1a) is about 90. In certain embodiments, b3 in formula (A-1a) is about 112. In certain embodiments, b3 in formula (A-1a) is about 170. In certain embodiments, b3 in formula (A-1a) is about 227. In a particular embodiment, b3 of formula (A-1a) is approximately 340. In a particular embodiment, b3 of formula (A-1a) is approximately 450. In a particular embodiment, b3 of formula (A-1a) is approximately 680. In a particular embodiment, b3 of formula (A-1a) is approximately 900. In a particular embodiment, b3 of formula (A-1a) is approximately 1130. In a particular embodiment, b3 of formula (A-1a) is approximately 1350. In a particular embodiment, b3 of formula (A-1a) is approximately 1590. In a particular embodiment, b3 of formula (A-1a) is approximately 1800. In a particular embodiment, b3 of formula (A-1a) is approximately 2045. In a particular embodiment, b3 of formula (A-1a) is approximately 2275.

[0152] In a particular embodiment, b1 in formula (A-1a) is 2, b2 in formula (A-1a) is 3, and b3 in formula (A-1a) is approximately 12. In a particular embodiment, b1 in formula (A-1a) is 2, b2 in formula (A-1a) is 3, and b3 in formula (A-1a) is approximately 23. In a particular embodiment, b1 in formula (A-1a) is 2, b2 in formula (A-1a) is 3, and b3 in formula (A-1a) is approximately 46. In a particular embodiment, b1 in formula (A-1a) is 2, b2 in formula (A-1a) is 3, and b3 in formula (A-1a) is approximately 68. In a particular embodiment, b1 in formula (A-1a) is 2, b2 in formula (A-1a) is 3, and b3 in formula (A-1a) is approximately 90. In a particular embodiment, b1 in formula (A-1a) is 2, b2 in formula (A-1a) is 3, and b3 in formula (A-1a) is approximately 112. In a particular embodiment, b1 in formula (A-1a) is 2, b2 in formula (A-1a) is 3, and b3 in formula (A-1a) is approximately 170. In a particular embodiment, b1 in formula (A-1a) is 2, b2 in formula (A-1a) is 3, and b3 in formula (A-1a) is approximately 227. In a particular embodiment, b1 in formula (A-1a) is 2, b2 in formula (A-1a) is 3, and b3 in formula (A-1a) is approximately 340. In a particular embodiment, b1 in formula (A-1a) is 2, b2 in formula (A-1a) is 3, and b3 in formula (A-1a) is approximately 450.

[0153] In a particular embodiment, M mod is equation (A-1b) [ka] (In the formula, The dashed lines marked with asterisks indicate the binding of the side chain of the amino acid residue in the IL-2 moiety to the sulfur. c1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. c2 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. c3 is an integer in the range of 12 to 22700. It belongs to them.

[0154] In a particular embodiment, c1 in formula (A-1b) is an integer in the range of 1 to 8. In a particular embodiment, c1 in formula (A-1b) is an integer in the range of 1 to 6. In a particular embodiment, c1 in formula (A-1b) is an integer in the range of 1 to 4. In a particular embodiment, c1 in formula (A-1b) is 1. In a particular embodiment, c1 in formula (A-1b) is 2. In a particular embodiment, c1 in formula (A-1b) is 3. In a particular embodiment, c1 in formula (A-1b) is 4. In a particular embodiment, c1 in formula (A-1b) is 5. In a particular embodiment, c1 in formula (A-1b) is 6.

[0155] In a particular embodiment, c2 in formula (A-1b) is an integer in the range of 1 to 8. In a particular embodiment, c2 in formula (A-1b) is an integer in the range of 1 to 6. In a particular embodiment, c2 in formula (A-1b) is an integer in the range of 1 to 4. In a particular embodiment, c2 in formula (A-1b) is 1. In a particular embodiment, c2 in formula (A-1b) is 2. In a particular embodiment, c2 in formula (A-1b) is 3. In a particular embodiment, c2 in formula (A-1b) is 4. In a particular embodiment, c2 in formula (A-1b) is 5. In a particular embodiment, c2 in formula (A-1b) is 6.

[0156] In certain embodiments, c3 in formula (A-1b) is an integer in the range of 23 to 227000, for example, 45 to 11300, or 69 to 4540, or 114 to 2700. In certain embodiments, c3 in formula (A-1b) is approximately 12. In certain embodiments, c3 in formula (A-1b) is approximately 23. In certain embodiments, c3 in formula (A-1b) is approximately 46. In certain embodiments, c3 in formula (A-1b) is approximately 68. In certain embodiments, c3 in formula (A-1b) is approximately 90. In certain embodiments, c3 in formula (A-1b) is approximately 112. In certain embodiments, c3 in formula (A-1b) is approximately 170. In certain embodiments, c3 in formula (A-1b) is approximately 227. In certain embodiments, c3 in formula (A-1b) is approximately 340. In a particular embodiment, c3 in formula (A-1b) is approximately 450. In a particular embodiment, c3 in formula (A-1b) is approximately 680. In a particular embodiment, c3 in formula (A-1b) is approximately 900. In a particular embodiment, c3 in formula (A-1b) is approximately 1130. In a particular embodiment, c3 in formula (A-1b) is approximately 1350. In a particular embodiment, c3 in formula (A-1b) is approximately 1590. In a particular embodiment, c3 in formula (A-1b) is approximately 1800. In a particular embodiment, c3 in formula (A-1b) is approximately 2045. In a particular embodiment, c3 in formula (A-1b) is approximately 2275.

[0157] In a particular embodiment, c1 in formula (A-1b) is 2, c2 in formula (A-1b) is 3, and c3 in formula (A-1b) is approximately 12. In a particular embodiment, c1 in formula (A-1b) is 2, c2 in formula (A-1b) is 3, and c3 in formula (A-1b) is approximately 23. In a particular embodiment, c1 in formula (A-1b) is 2, c2 in formula (A-1b) is 3, and c3 in formula (A-1b) is approximately 46. In a particular embodiment, c1 in formula (A-1b) is 2, c2 in formula (A-1b) is 3, and c3 in formula (A-1b) is approximately 68. In a particular embodiment, c1 in formula (A-1b) is 2, c2 in formula (A-1b) is 3, and c3 in formula (A-1b) is approximately 90. In a particular embodiment, c1 in formula (A-1b) is 2, c2 in formula (A-1b) is 3, and c3 in formula (A-1b) is approximately 112. In a particular embodiment, c1 in formula (A-1b) is 2, c2 in formula (A-1b) is 3, and c3 in formula (A-1b) is approximately 170. In a particular embodiment, c1 in formula (A-1b) is 2, c2 in formula (A-1b) is 3, and c3 in formula (A-1b) is approximately 227. In a particular embodiment, c1 in formula (A-1b) is 2, c2 in formula (A-1b) is 3, and c3 in formula (A-1b) is approximately 340. In a particular embodiment, c1 in formula (A-1b) is 2, c2 in formula (A-1b) is 3, and c3 in formula (A-1b) is approximately 450.

[0158] In a particular embodiment, M mod This is equation (A-1c) [ka] (In the formula, The dashed lines marked with asterisks indicate the binding of the side chain of the amino acid residue in the IL-2 moiety to the sulfur. d1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. d2 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. d3 is an integer in the range of 12 to 22700. It belongs to them.

[0159] In a particular embodiment, d1 in equation (A-1c) is an integer in the range of 1 to 8. In a particular embodiment, d1 in equation (A-1c) is an integer in the range of 1 to 6. In a particular embodiment, d1 in equation (A-1c) is an integer in the range of 1 to 4. In a particular embodiment, d1 in equation (A-1c) is 1. In a particular embodiment, d1 in equation (A-1c) is 2. In a particular embodiment, d1 in equation (A-1c) is 3. In a particular embodiment, d1 in equation (A-1c) is 4. In a particular embodiment, d1 in equation (A-1c) is 5. In a particular embodiment, d1 in equation (A-1c) is 6.

[0160] In a particular embodiment, d2 in equation (A-1c) is an integer in the range of 1 to 8. In a particular embodiment, d2 in equation (A-1c) is an integer in the range of 1 to 6. In a particular embodiment, d2 in equation (A-1c) is an integer in the range of 1 to 4. In a particular embodiment, d2 in equation (A-1c) is 1. In a particular embodiment, d2 in equation (A-1c) is 2. In a particular embodiment, d2 in equation (A-1c) is 3. In a particular embodiment, d2 in equation (A-1c) is 4. In a particular embodiment, d2 in equation (A-1c) is 5. In a particular embodiment, d2 in equation (A-1c) is 6.

[0161] In certain embodiments, d3 in formula (A-1c) is an integer in the range of 23 to 227000, for example, 45 to 11300, or 69 to 4540, 114 to 2700, or 160 to 900. In certain embodiments, d3 in formula (A-1c) is approximately 12. In certain embodiments, d3 in formula (A-1c) is approximately 23. In certain embodiments, d3 in formula (A-1c) is approximately 46. In certain embodiments, d3 in formula (A-1c) is approximately 68. In certain embodiments, d3 in formula (A-1c) is approximately 90. In certain embodiments, d3 in formula (A-1c) is approximately 112. In certain embodiments, d3 in formula (A-1c) is approximately 170. In certain embodiments, d3 in formula (A-1c) is approximately 227. In certain embodiments, d3 in formula (A-1c) is approximately 340. In a particular embodiment, d3 in formula (A-1c) is approximately 450. In a particular embodiment, d3 in formula (A-1c) is approximately 680. In a particular embodiment, d3 in formula (A-1c) is approximately 900. In a particular embodiment, d3 in formula (A-1c) is approximately 1130. In a particular embodiment, d3 in formula (A-1c) is approximately 1350. In a particular embodiment, d3 in formula (A-1c) is approximately 1590. In a particular embodiment, d3 in formula (A-1c) is approximately 1800. In a particular embodiment, d3 in formula (A-1c) is approximately 2045. In a particular embodiment, d3 in formula (A-1c) is approximately 2275.

[0162] In a particular embodiment, d1 in formula (A-1c) is 2, d2 in formula (A-1c) is 3, and d3 in formula (A-1cd) is approximately 12. In a particular embodiment, d1 in formula (A-1c) is 2, d2 in formula (A-1c) is 3, and d3 in formula (A-1c) is approximately 23. In a particular embodiment, d1 in formula (A-1c) is 2, d2 in formula (A-1c) is 3, and d3 in formula (A-1c) is approximately 46. In a particular embodiment, d1 in formula (A-1c) is 2, d2 in formula (A-1c) is 3, and d3 in formula (A-1c) is approximately 68. In a particular embodiment, d1 in formula (A-1c) is 2, d2 in formula (A-1c) is 3, and d3 in formula (A-1c) is approximately 90. In a particular embodiment, d1 in formula (A-1c) is 2, d2 in formula (A-1c) is 3, and d3 in formula (A-1c) is approximately 112. In a particular embodiment, d1 in formula (A-1c) is 2, d2 in formula (A-1c) is 3, and d3 in formula (A-1c) is approximately 170. In a particular embodiment, d1 in formula (A-1c) is 2, d2 in formula (A-1c) is 3, and d3 in formula (A-1c) is approximately 227. In a particular embodiment, d1 in formula (A-1c) is 2, d2 in formula (A-1c) is 3, and d3 in formula (A-1c) is approximately 340. In a particular embodiment, d1 in formula (A-1c) is 2, d2 in formula (A-1c) is 3, and d3 in formula (A-1c) is approximately 450.

[0163] The biased IL-2 moiety may include an IL-2 moiety containing at least one amino acid mutation, for example, one, two, three, four, five, six, seven, eight, nine, or ten amino acid mutations. Such amino acid mutations preferably occur at positions involved in binding to IL-2Rα. Therefore, preferably, at least the amino acid mutations result in a reduction in the affinity of the biased IL-2 moiety to IL-2Rαβ compared to aldesleukin.

[0164] Preferably, at least one amino acid mutation occurs at an amino acid position selected from the group consisting of K34, R37, M38, T40, F41, K42, F43, Y44, E61, and L71 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. More preferably, at least one amino acid mutation occurs at an amino acid position selected from the group consisting of F41, Y44, E61, and L71 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. In certain embodiments, at least one amino acid mutation occurs at amino acid position K34 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. In certain embodiments, at least one amino acid mutation occurs at amino acid position R37 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. In certain embodiments, at least one amino acid mutation occurs at amino acid position M38 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. In certain embodiments, at least one amino acid mutation occurs at amino acid position T40 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. In certain embodiments, at least one amino acid mutation occurs at amino acid position F41 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. In certain embodiments, at least one amino acid mutation occurs at amino acid position K42 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. In certain embodiments, at least one amino acid mutation occurs at amino acid position F43 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. In certain embodiments, at least one amino acid mutation occurs at amino acid position Y44 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. In certain embodiments, at least one amino acid mutation occurs at amino acid position E61 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant. In certain embodiments, at least one amino acid mutation occurs at amino acid position L71 based on SEQ ID NO: 2, or at the corresponding position of its homolog or variant.

[0165] In certain embodiments, such a mutation is the substitution of a naturally occurring amino acid with an amino acid residue selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, lysine, serine, threonine, tryptophan, and tyrosine. In certain embodiments, the naturally occurring amino acid is replaced with alanine. In certain embodiments, the naturally occurring amino acid is replaced with arginine. In certain embodiments, the naturally occurring amino acid is replaced with asparagine. In certain embodiments, the naturally occurring amino acid is replaced with aspartic acid. In certain embodiments, the naturally occurring amino acid is replaced with cysteine. In certain embodiments, the naturally occurring amino acid is replaced with glutamine. In certain embodiments, the naturally occurring amino acid is replaced with glutamic acid. In certain embodiments, the naturally occurring amino acid is replaced with glycine. In certain embodiments, the naturally occurring amino acid is replaced with histidine. In certain embodiments, the naturally occurring amino acid is replaced with lysine. In certain embodiments, the naturally occurring amino acid is replaced with serine. In certain embodiments, the naturally occurring amino acid is replaced with threonine. In certain embodiments, the naturally occurring amino acid is replaced with tryptophan. In certain embodiments, the naturally occurring amino acid is replaced with tyrosine. In certain embodiments, such a mutation is the replacement of the naturally occurring amino acid with an amino acid residue selected from the group consisting of arginine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, serine, threonine, tryptophan, and tyrosine. In certain embodiments, such a mutation is the replacement of the naturally occurring amino acid with an amino acid residue selected from the group consisting of cysteine, glutamic acid, lysine, serine, threonine, and tyrosine. In certain embodiments, the naturally occurring amino acid is replaced with a non-proteinogenic amino acid. Embodiments of such non-proteinogenic amino acids are as described above.

[0166] In one embodiment, the IL-2 of the biased IL-2 portion includes an amino acid mutation selected from the group consisting of K34A, K34C, K34G, K34S, K34T, K34Q, K34E, K34N, K34D, K34H, K34W, K34Y, and K34R or their homologs or variants at the corresponding position, based on SEQ ID NO: 2. In a particular embodiment, the IL-2 portion includes the K34A mutation. In a particular embodiment, the IL-2 portion includes the K34C mutation. In a particular embodiment, the IL-2 portion includes the K34G mutation. In a particular embodiment, the IL-2 portion includes the K34S mutation. In a particular embodiment, the IL-2 portion includes the K34T mutation. In a particular embodiment, the IL-2 portion includes the K34Q mutation. In a particular embodiment, the IL-2 portion includes the K34E mutation. In a particular embodiment, the IL-2 portion includes the K34D mutation. In a particular embodiment, the IL-2 portion includes the K34H mutation. In certain embodiments, the IL-2 portion includes the K34W mutation. In certain embodiments, the IL-2 portion includes the K34Y mutation. In certain embodiments, the IL-2 portion includes the K34R mutation.

[0167] In one embodiment, the IL-2 portion of bias IL-2 includes an amino acid mutation selected from the group consisting of R37A, R37C, R37G, R37S, R37T, R37Q, R37E, R37N, R37D, R37H, R37W, R37Y, and R37K or the corresponding position of their homologs or variants, based on SEQ ID NO: 2. In a particular embodiment, the IL-2 portion includes the R37A mutation. In a particular embodiment, the IL-2 portion includes the R37C mutation. In a particular embodiment, the IL-2 portion includes the R37G mutation. In a particular embodiment, the IL-2 portion includes the R37S mutation. In a particular embodiment, the IL-2 portion includes the R37T mutation. In a particular embodiment, the IL-2 portion includes the R37Q mutation. In a particular embodiment, the IL-2 portion includes the R37E mutation. In a particular embodiment, the IL-2 portion includes the R37N mutation. In a particular embodiment, the IL-2 portion includes the R37D mutation. In certain embodiments, the IL-2 portion includes the R37H mutation. In certain embodiments, the IL-2 portion includes the R37K mutation. In certain embodiments, the IL-2 portion includes the R37W mutation. In certain embodiments, the IL-2 portion includes the R37Y mutation. In certain embodiments, the IL-2 portion includes the R37K mutation.

[0168] In one embodiment, at least one amino acid mutation is selected from the group consisting of F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R, F41K, Y44A, Y44C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R, Y44K, L71C, L71G, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R, and L71K or the corresponding positions of their homologs or variants, based on SEQ ID NO: 2.

[0169] In certain embodiments, the IL-2 portion of bias IL-2 includes an amino acid mutation selected from the group consisting of F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R, F41H, F41W, F41Y, and F41K or their homologs or variants at corresponding positions, based on SEQ ID NO: 2. In certain embodiments, the IL-2 portion includes an amino acid mutation selected from the group consisting of F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R, and F41K or their homologs or variants at corresponding positions, based on SEQ ID NO: 2. In one embodiment, the IL-2 portion includes the F41A mutation. In another embodiment, the IL-2 portion includes the F41C mutation. In another embodiment, the IL-2 portion includes the F41G mutation. In another embodiment, the IL-2 portion includes the F41S mutation. In another embodiment, the IL-2 portion includes the F41T mutation. In another embodiment, the IL-2 portion includes the F41Q mutation. In another embodiment, the IL-2 portion includes the F41E mutation. In another embodiment, the IL-2 portion includes the F41N mutation. In another embodiment, the IL-2 portion includes the F41D mutation. In another embodiment, the IL-2 portion includes the F41R mutation. In another embodiment, the IL-2 portion includes the F41H mutation. In another embodiment, the IL-2 portion includes the F41W mutation. In another embodiment, the IL-2 portion includes the F41Y mutation. In another embodiment, the IL-2 portion includes the F41K mutation.

[0170] In certain embodiments, the IL-2 portion includes an amino acid mutation selected from the group consisting of Y44A, Y44C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R, Y44H, Y44W, and Y44K or their homologs or variants at corresponding positions, based on SEQ ID NO: 2. In certain embodiments, the IL-2 portion includes an amino acid mutation selected from the group consisting of Y44A, Y44C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R, and Y44K or their homologs or variants at corresponding positions, based on SEQ ID NO: 2. In one embodiment, the IL-2 portion includes a Y44A mutation. In another embodiment, the IL-2 portion includes a Y44C mutation. In another embodiment, the IL-2 portion includes a Y44G mutation. In another embodiment, the IL-2 portion includes a Y44S mutation. In another embodiment, the IL-2 portion includes the Y44T mutation. In another embodiment, the IL-2 portion includes the Y44Q mutation. In another embodiment, the IL-2 portion includes the Y44E mutation. In another embodiment, the IL-2 portion includes the Y44N mutation. In another embodiment, the IL-2 portion includes the Y44D mutation. In another embodiment, the IL-2 portion includes the Y44R mutation. In another embodiment, the IL-2 portion includes the Y44H mutation. In another embodiment, the IL-2 portion includes the Y44W mutation. In another embodiment, the IL-2 portion includes the Y44K mutation.

[0171] In certain embodiments, the IL-2 moiety includes an amino acid mutation selected from the group consisting of L71G, L71C, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R, L71H, L71W, L71Y, and L71K or their homologs or variants at corresponding positions, based on SEQ ID NO: 2. In certain embodiments, the IL-2 moiety includes an amino acid mutation selected from the group consisting of L71G, L71C, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R, and L71K or their homologs or variants at corresponding positions, based on SEQ ID NO: 2. In one embodiment, the IL-2 moiety includes an L72G mutation. In another embodiment, the IL-2 moiety includes an L72C mutation. In another embodiment, the IL-2 moiety includes an L72A mutation. In another embodiment, the IL-2 moiety includes an L72S mutation. In another embodiment, the IL-2 portion includes the L72T mutation. In another embodiment, the IL-2 portion includes the L72Q mutation. In another embodiment, the IL-2 portion includes the L72E mutation. In another embodiment, the IL-2 portion includes the L72N mutation. In another embodiment, the IL-2 portion includes the L72D mutation. In another embodiment, the IL-2 portion includes the L72R mutation. In another embodiment, the IL-2 portion includes the L72H mutation. In another embodiment, the IL-2 portion includes the L72W mutation. In another embodiment, the IL-2 portion includes the L72Y mutation. In another embodiment, the IL-2 portion includes the L72K mutation.

[0172] In another embodiment, the IL-2 moiety includes amino acid mutations selected from the group consisting of F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R, and F41K or their homologs or variants at corresponding positions, based on SEQ ID NO: 2, and further amino acid mutations selected from the group consisting of Y44A, Y44C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R, and Y44K. In one embodiment, the IL-2 moiety includes F41A and Y44A mutations. In another embodiment, the IL-2 moiety includes F41C and Y44A mutations. In yet another embodiment, the IL-2 moiety includes F41A and Y44C mutations.

[0173] In another embodiment, the IL-2 moiety includes amino acid mutations selected from the group consisting of F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R, and F41K or their homologs or variants at corresponding positions, based on SEQ ID NO: 2, and further amino acid mutations selected from the group consisting of L71G, L71C, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R, and L71K. In one embodiment, the IL-2 moiety includes F42A and L72G mutations. In another embodiment, the IL-2 moiety includes F42C and L72G mutations. In yet another embodiment, the IL-2 moiety includes F42A and L72C mutations.

[0174] In another embodiment, the IL-2 moiety includes amino acid mutations selected from the group consisting of Y44A, Y44C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R, and Y44K or their homologs or variants at corresponding positions, based on SEQ ID NO: 2, and further amino acid mutations selected from the group consisting of L71G, L71C, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R, and L71K. In one embodiment, the IL-2 moiety includes Y45A and L72G mutations. In another embodiment, the IL-2 moiety includes Y45C and L72G mutations. In yet another embodiment, the IL-2 moiety includes Y45A and L72C mutations.

[0175] In another embodiment, the IL-2 moiety includes amino acid mutations selected from the group consisting of F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R, and F41K or their homologs or variants at corresponding positions, further amino acid mutations selected from the group consisting of Y44A, Y41C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R, and Y44K, and further amino acid mutations selected from the group consisting of L71G, L71C, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R, and L71K. In one embodiment, the IL-2 moiety includes F41A, Y44A, and L71G mutations. In another embodiment, the IL-2 moiety includes the F41C, Y44A, and L71G mutations. In another embodiment, the IL-2 moiety includes the F41A, Y44C, and L71G mutations. In another embodiment, the IL-2 moiety includes the F41A, Y44C, and L71C mutations.

[0176] The IL-2 moiety may further or optionally include amino acid mutations that remove an O-glycosylation site. Preferably, such amino acid mutations are at the position corresponding to residue 2 of aldethleukin, and more preferably, such amino acid mutations are selected from the group consisting of T2A, T2G, T2Q, T2E, T2N, T2D, T2R, T2K, and T2P or their homologs or variants at the corresponding position, based on SEQ ID NO: 2, and most preferably T2A.

[0177] The IL-2 moiety may further or or otherwise include one or more additional amino acid mutations that may provide additional benefits, such as increased expression or stability. For example, the methionine at position 103 of aldesleukin may be replaced with a neutral amino acid, such as alanine, as described in U.S. Patent No. 5,206,344.

[0178] In a particular embodiment, the biased IL-2 moiety includes at least one amino acid mutation and modification moiety M mod at least one stable bond, for example, one amino acid mutation and one partial Mmod , one amino acid mutation and two partial M mod , two amino acid mutations and one partial M mod , 3 amino acid mutations and 1 partial M mod , or one amino acid mutation and three partial M mod This is the IL-2 portion containing amino acid mutations and partial M mod The number of can be selected independently of each other and can be selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In certain embodiments, amino acid mutations and partial M mod The number is independently selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8. In a particular embodiment, the number of amino acid mutations is 1. In a particular embodiment, the number of amino acid mutations is 2. In a particular embodiment, the number of amino acid mutations is 3. In a particular embodiment, the number of amino acid mutations is 4. In a particular embodiment, the number of amino acid mutations is 5. In a particular embodiment, the number of amino acid mutations is 6. In a particular embodiment, partial M mod The number is 1. In a particular embodiment, part M mod The number is 2. In a particular embodiment, part M mod The number is 3. In a particular embodiment, part M mod The number is 4. In a particular embodiment, part M mod The number is 5. In a particular embodiment, part M mod The number is 6. Part M mod The mutation site / mutation embodiment is as described above.

[0179] In a particular embodiment, the bias IL-2 moiety is at least one amino acid mutation, and at least one moiety M bound to such mutated amino acid. mod This is the IL-2 portion containing amino acid mutations and partial M mod The number of parts does not have to be the same, and further subdivisions M mod The IL-2 moiety may be conjugated with a non-mutated amino acid residue, and at least one moiety M modAs long as it is conjugated to one mutant amino acid residue, not all mutant amino acid residues are necessarily partial M mod It is understood that it does not need to be conjugated. Therefore, amino acid mutations and partial M mod The number of may be selected independently of each other, or may be selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In certain embodiments, amino acid mutations and partial M mod The number is independently selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8. In a particular embodiment, the number of amino acid mutations is 1. In a particular embodiment, the number of amino acid mutations is 2. In a particular embodiment, the number of amino acid mutations is 3. In a particular embodiment, the number of amino acid mutations is 4. In a particular embodiment, the number of amino acid mutations is 5. In a particular embodiment, the number of amino acid mutations is 6. In a particular embodiment, partial M mod The number is 1. In a particular embodiment, part M mod The number is 2. In a particular embodiment, part M mod The number is 3. In a particular embodiment, part M mod The number is 4. In a particular embodiment, part M mod The number is 5. In a particular embodiment, part M mod The number is 6. Part M mod The mutation site / mutation embodiment is as described above.

[0180] In a particular embodiment, the biased IL-2 moiety is an IL-2 moiety comprising at least one amino acid mutation at a position selected from the group consisting of K34, R37, M38, T40, F41, K42, F43, Y44, E61, and L71, wherein a naturally occurring amino acid is replaced with cysteine, resulting in mutations K34C, R37C, M38C, T40C, F41C, K42C, F43C, Y44C, E61C, and L71C, and at least one moiety M modHowever, the IL-2 moiety is conjugated to the sulfur of such cysteine. In certain embodiments, the biased IL-2 moiety is a K34C mutation, and further a cysteine ​​sulfur-conjugated moiety M is replaced by lysine at position 34. mod This includes. In certain embodiments, the bias IL-2 moiety is an R37C mutation, and further a moiety M conjugated to the sulfur of cysteine ​​replaced by arginine at position 37. mod This includes. In certain embodiments, the bias IL-2 moiety is the M38C mutation, and further the moiety M conjugated to the sulfur of cysteine ​​replaced by methionine at position 38. mod This includes. In certain embodiments, the bias IL-2 moiety is a T40C mutation, and further a moiety M conjugated to the sulfur of cysteine ​​replaced by threonine at position 40. mod This includes. In certain embodiments, the bias IL-2 moiety is an F41C mutation, and further a moiety M conjugated to the sulfur of cysteine ​​replaced by phenylalanine at position 41. mod This includes. In certain embodiments, the bias IL-2 moiety is a K42C mutation, and further a moiety M conjugated to the sulfur of cysteine ​​replaced by lysine at position 42. mod This includes. In certain embodiments, the bias IL-2 moiety is an F43C mutation, and further a moiety M conjugated to the sulfur of cysteine ​​replaced by phenylalanine at position 43. mod This includes. In certain embodiments, the bias IL-2 moiety is a Y44C mutation, and further a moiety M conjugated to the sulfur of cysteine ​​replaced by tyrosine at position 44. mod This includes. In certain embodiments, the bias IL-2 moiety is an E61C mutation, and further a moiety M conjugated to the sulfur of cysteine ​​replaced by glutamic acid at position 61. mod Includes. In certain embodiments, the bias IL-2 portion includes the L71C mutation, and Furthermore, the sulfur-conjugated portion of cysteine ​​that replaced lysine at position 71 is M modIncludes. In a particular embodiment, the bias IL-2 portion includes one mutation selected from the group consisting of K34C, R37C, M38C, T40C, F41C, K42C, F43C, Y44C, E61C, and L71C, and one portion M mod It is conjugated to a naturally occurring amino acid, namely, in the case of the R37C mutation, the sulfur of the cysteine ​​that replaces arginine at position 37.

[0181] The IL-2 moiety may contain at least one deletion (see point (c)). Such a deletion is understood to preferably refer to the sequence of SEQ ID NO: 2. Such a deletion may be in the form of a naturally occurring deletion, e.g., a splice variant, or it may be an artificially introduced deletion. One such naturally occurring splice variant is the IL-2δ2 mutant, in which exon 2 is excluded, resulting in a deletion of amino acid residues N30-K49 based on the sequence of SEQ ID NO: 1, corresponding to a deletion of amino acid residues N29-K48 based on the sequence of SEQ ID NO: 2 (see Tsytsikov et al., JBC 1996, 271(38): 23055-23060).

[0182] Preferably, such deletions occur at sites related to binding to IL-2Rα. Therefore, preferably, at least one deletion results in a reduction in the affinity of the biased IL-2 moiety to IL-2Rαβ compared to aldesleukin.

[0183] In one embodiment, -D includes at least one mutation introducing a cysteine ​​residue, wherein the modified portion M is bound to the cysteine ​​mutation. modThe cysteine ​​mutation further includes an IL-2 moiety, preferably an IL-2 moiety having the sequence of Sequence ID No. 2. Preferably, the cysteine ​​mutation is selected from the group including K34C, R37C, M38C, T40C, F41C, K42C, F43C, Y44C, E61C, and L71C. In certain embodiments, the cysteine ​​mutation is the K34C mutation. In certain embodiments, the cysteine ​​mutation is the R37C mutation. In certain embodiments, the cysteine ​​mutation is the M38C mutation. In certain embodiments, the cysteine ​​mutation is the T40C mutation. In certain embodiments, the cysteine ​​mutation is the F41C mutation. In certain embodiments, the cysteine ​​mutation is the K42C mutation. In certain embodiments, the cysteine ​​mutation is the F43C mutation. In certain embodiments, the cysteine ​​mutation is the Y44C mutation. In certain embodiments, the cysteine ​​mutation is the E61C mutation. In certain embodiments, the cysteine ​​mutation is the L71C mutation.

[0184] In one embodiment, the IL-2 moiety is a superkine as described by Levin et al. (Nature, 2012, 484: 529-535). Preferably, in addition to or instead of the above modifications, such an IL-2 moiety includes one or more mutations of amino acid residues Q73, L79, R80, L84, I85, and I91, and in particular includes one or more mutations selected from the group consisting of Q73H, L79F, R80D, L84V, I85V, and I91F. In one embodiment, the IL-2 moiety includes the Q73H, L79F, R80D, L84V, I85V, and I91F mutations in addition to or instead of the above modifications. In another embodiment, the IL-2 moiety includes the L79F, R80D, L84V, I85V, and I91F mutations in addition to or instead of the above modifications. If the IL-2 portion is based on superkine, the corresponding biased IL-2 has a higher affinity for IL-2Rβ than aldethleukin.

[0185] The IL-2 conjugate of the present invention comprises a polymer moiety and / or substituted fatty acid moiety-Z that are reversibly bonded by at least one covalent bond.

[0186] The addition of such a polymer moiety and / or substituted fatty acid moiety, which is reversibly bonded by at least one covalent bond, can further extend the cyclic half-life of the biased IL-2 moiety, and it was surprisingly found that the reversible bond ensures sufficient affinity for IL-2Rβ after cleavage of the polymer moiety or substituted fatty acid moiety, which is reversibly bonded by at least one covalent bond.

[0187] In one embodiment, the IL-2 conjugate of the present invention comprises a single moiety-Z which is either a substituted fatty acid or a polymer moiety. In one embodiment, -Z is a substituted fatty acid. In another embodiment, -Z is a polymer moiety.

[0188] In another embodiment, the IL-2 conjugate of the present invention comprises two parts-Z which may be the same or different. In one embodiment, both parts-Z are substituted fatty acids which may be the same or different. In another embodiment, both parts-Z are polymer parts which may be the same or different. In yet another embodiment, one part-Z is a substituted fatty acid and the other part-Z is a polymer part.

[0189] In another embodiment, the IL-2 conjugate of the present invention comprises three parts-Z which may be the same or different. In one embodiment, all three parts-Z are substituted fatty acids which may be the same or different. In another embodiment, all three parts-Z are polymer parts which may be the same or different. In yet another embodiment, one or two parts-Z are substituted fatty acids and the remaining parts / parts-Z are polymer parts.

[0190] In another embodiment, the IL-2 conjugate of the present invention comprises four parts-Z which may be the same or different. In one embodiment, all four parts-Z are substituted fatty acids which may be the same or different. In another embodiment, all four parts-Z are polymer parts which may be the same or different. In yet another embodiment, one, two, or three parts-Z are substituted fatty acids and the remaining parts / parts-Z are polymer parts.

[0191] If -Z is a substituted fatty acid moiety, it is preferably a substituted fatty acid moiety disclosed in International Publication 2005 / 027978A2 and International Publication 2014 / 060512A1, which are incorporated herein by reference.

[0192] If -Z is a polymer portion, such a polymer portion preferably has a molecular weight in the range of 1 kDa to 1000 kDa, more preferably 2 kDa to 500 kDa, even more preferably 3 kDa to 200 kDa, even more preferably 5 kDa to 120 kDa, even more preferably 10 kDa to 100 kDa, and most preferably 15 kDa to 80 kDa. In one embodiment, -Z is a polymer portion having a molecular weight of about 2 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of about 5 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of about 10 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of about 15 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of about 20 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of about 30 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of about 40 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of approximately 50 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of approximately 60 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of approximately 70 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of approximately 80 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of approximately 90 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of approximately 100 kDa. In one embodiment, -Z is a polymer portion having a molecular weight of 2 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of 5 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of 10 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of 15 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of 20 kDa. In another embodiment, -Z is a polymer portion having a molecular weight of 30 kDa. In another embodiment, -Z is a polymer moiety having a molecular weight of 40 kDa. In another embodiment, -Z is a polymer moiety having a molecular weight of 50 kDa. In another embodiment, -Z is a polymer moiety having a molecular weight of 60 kDa. In another embodiment, -Z is a polymer moiety having a molecular weight of 70 kDa.In another embodiment, -Z is a polymer moiety having a molecular weight of 80 kDa. In another embodiment, -Z is a polymer moiety having a molecular weight of 90 kDa. In another embodiment, -Z is a polymer moiety having a molecular weight of 100 kDa.

[0193] If -Z is a polymer moiety, such polymer moieties are preferably 2-methacryloyloxyethylphosphoryl(phosphoyl)choline, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amideamine), poly(amino acid), poly(acid anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl methacrylamide) The polymers include polymers selected from the group consisting of poly(hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethylcellulose, hydroxypropylmethylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and its derivatives, functionalized hyaluronic acid, alginate, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch, and other carbohydrate-based polymers, xylan, and copolymers thereof.

[0194] In one embodiment, -Z is a peptide or protein moiety. Preferably, such a peptide or protein moiety is not an IL-2 moiety or fragment thereof. Such a peptide or protein moiety -Z is -L1 -L 2 - may be chemically conjugated to -D via - or the reversible linker portion -L 1 - may be translated and fused to -D via -L 1 - is a peptide or protein portion, -L 2 - is preferably a chemical bond. In one embodiment, such a peptide or protein portion -Z is -L 1 -L 2 - is chemically conjugated to -D via -. In another embodiment, such peptide or protein moiety -Z is reversible linker moiety -L 1 - is translated and fused to -D via -L 1 - is a peptide or protein, -L 2 - is preferably a chemical bond. Such a peptide or protein reversible linker moiety -L 1 -It is understood that - may be degradable enzymatically or non-enzymatically. To promote enzymatic degradation, -L 1 - may include a protease recognition site.

[0195] If -Z is a peptide or protein moiety, it is preferably selected from the group consisting of a carboxyl-terminal peptide moiety of a chorionic gonadotropin described in U.S. Patent Application Publication No. 2012 / 0035101A1, incorporated herein by reference, an albumin moiety, a random coil protein moiety, and an Fc fusion protein moiety.

[0196] In one embodiment, -Z comprises a random coil peptide or protein moiety.

[0197] Preferably, such a random coil protein portion contains at least 25 amino acid residues and at most 2000 amino acids. More preferably, such a random coil peptide or protein portion contains at least 30 amino acid residues and at most 1500 amino acid residues. Even more preferably, such a random coil peptide or protein portion contains at least 50 amino acid residues and at most 500 amino acid residues.

[0198] In a preferred embodiment, -Z comprises a random coil protein moiety in which at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99% of the total number of amino acids forming the random coil protein moiety are selected from alanine and proline. More preferably, at least 10%, but less than 75%, preferably less than 65%, of the total number of amino acid residues in such a random coil protein moiety are proline residues. Preferably, such a random coil protein moiety is one described in WO2011 / 144756A1 (the whole thereof is incorporated herein by reference). More preferably, -Z includes at least one portion selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 51, and 61 as disclosed in WO2011 / 144756 (incorporated herein by reference). Such a portion containing alanine and proline, comprising a random coil protein, is referred to as "PA" or "PA portion".

[0199] Therefore, in one embodiment, -Z includes the PA portion.

[0200] In another embodiment, -Z comprises a random coil protein moiety in which at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99% of the total number of amino acids forming the random coil protein moiety are selected from alanine, serine, and proline. More preferably, at least 4%, but less than 40%, of the total number of amino acid residues in such a random coil protein moiety are proline residues. Preferably, such a random coil protein moiety is as described in WO2008 / 155134A1 (the whole thereof is incorporated herein by reference). More preferably, -Z includes at least one portion selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 40, 42, 44, 46, 50, 52, 54, and 56 as disclosed in WO2008 / 155134A1 (incorporated herein by reference). A portion including such a random coil protein portion containing alanine, serine, and proline is referred to as "PAS" or "PAS portion".

[0201] Therefore, in one embodiment, -Z includes the PAS portion.

[0202] In another embodiment, -Z comprises a random coil protein moiety in which at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99% of the total number of amino acids forming the random coil protein moiety are selected from alanine, glycine, serine, threonine, glutamic acid, and proline. Preferably, such a random coil protein moiety is as described in WO2010 / 091122A1 (incorporated herein by reference). More preferably, -Z refers to the sequences 182, 183, 184; 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214 disclosed in WO2010 / 091122A1 (incorporated herein by reference). , comprising at least one part selected from the group consisting of SEQ ID NOs: 215, 216, 217, 218, 219, 220, 221, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 1715, 1716, 1718, 1719, 1720, 1721, and 1722.Such a random coil protein moiety containing alanine, glycine, serine, threonine, glutamic acid, and proline is referred to as "XTEN" or "XTEN moiety" according to the designation in WO2010 / 091122A1.

[0203] Therefore, in one embodiment, -Z includes the XTEN portion.

[0204] In another embodiment, -Z is a hyaluronic acid-based polymer.

[0205] In another embodiment, -Z is a PEG system portion, such as a linear, branched, or multi-arm PEG system portion. In one embodiment, -Z is a branched PEG system portion. Preferably, such a branched PEG system portion -Z is a branched PEG system portion having 1, 2, 3, 4, 5, or 6 branch points. Preferably, -Z is a branched PEG system portion having 1, 2, or 3 branch points. In one embodiment, -Z is a branched PEG system portion having 1 branch point. In another embodiment, -Z is a branched PEG system portion having 2 branch points. In another embodiment, -Z is a branched PEG system portion having 3 branch points.

[0206] Each branch point is preferably independently selected from the group consisting of -N<, -CH<, and >C<.

[0207] In a particular embodiment, -Z is in equation (A) [ka] (In the formula, -BP 1 <, -BP 2 <, -BP 3 <But -N< and -C(R 8 Selected independently from the group consisting of )<, R 8 However, H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Selected from the group consisting of alkynnyls, -P 1, -P 2 , -P 3 , -P 4 However, each is independently a PEG-based chain containing at least 40% PEG and having a molecular weight in the range of 3 to 40 kDa. -C 1 -, -C 2 -but, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Selected independently from the group consisting of alkynnyls, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl, one or more R, either the same or different 9 Replaced by optional means, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) 10 )-,-S(O)2N(R 10 )-,-S(O)N(R 10 )-, -S(O)2-, -S(O)-, -N(R 10 )S(O)2N(R 10a )-, -S-, -N(R 10 )-, -OC(OR 10 )(R 10a )-,-N(R 10 )C(O)N(R 10a )-, and -OC(O)N(R 10 Interrupted by one or more elements selected from the group consisting of )-, Each T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 A molecule is independently selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl, and each T is the same or different R 9 It is independently and arbitrarily replaced by, Each R 9 However, halogen, -CN, oxo (=O), -COOR 11 , -OR 11 , -C(O)R11 ,-C(O)N(R 11 R 11a ), -S(O)2N(R 11 R 11a ), -S(O)N(R 11 R 11a ), -S(O)2R 11 ,-S(O)R 11 , -N(R 11 )S(O)2N(R 11a R 11b ), -SR 11 , -N(R 11 R 11a ), -NO2, -OC(O)R 11 , -N(R 11 )C(O)R 11a , -N(R 11 )S(O)2R 11a , -N(R 11 )S(O)R 11a , -N(R 11 )C(O)OR 11a , -N(R 11 )C(O)N(R 11a R 11b ), -OC(O)N(R 11 R 11a ), and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 The alkyl group is optionally substituted with one or more halogens, either the same or different. Each R 10 , R 10a , R 11 , R 11a , and R 11b However, -H and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 (The alkyl group is optionally substituted with one or more halogens, either the same or different.) This includes the part.

[0208] In a particular embodiment, -P 1 , -P 2 , -P 3 , -P 4These are PEG-based chains that independently contain at least 50% PEG and have a molecular weight in the range of 3 to 40 kDa. In certain embodiments, -P 1 , -P 2 , -P 3 , -P 4 These are PEG-based chains that independently contain at least 60% PEG and have a molecular weight in the range of 3 to 40 kDa. In certain embodiments, -P 1 , -P 2 , -P 3 , -P 4 These are PEG-based chains that independently contain at least 70% PEG and have a molecular weight in the range of 3 to 40 kDa. In certain embodiments, -P 1 , -P 2 , -P 3 , -P 4 These are PEG-based chains that independently contain at least 80% PEG and have a molecular weight in the range of 3 to 40 kDa.

[0209] In a particular embodiment, part P of formula (A) 1 , P 2 , P 3 , and P 4 The molecular weights are independently in the range of 5 to 30 kDa, for example, 5 to 25 kDa, or 8 to 20 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight can be approximately 5 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight can be approximately 7 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight may be approximately 10 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight can be approximately 12 kDa. In certain embodiments, partial P 1 , P2 , P 3 , or P 4 The molecular weight can be approximately 15 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight can be approximately 20 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight can be approximately 25 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight may be approximately 30 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight may be 7 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight may be 10 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight may be 12 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight may be 15 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight may be 20 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 The molecular weight may be 25 kDa. In certain embodiments, partial P 1 , P 2 , P 3 , or P 4 Its molecular weight may be 30 kDa.

[0210] In a particular embodiment, P of formula (A) 1 , P 2 , P 3 , and P 4 They have the same structure.

[0211] In a particular embodiment, the BP of formula (A) 1 -N < .

[0212] In a particular embodiment, the BP of formula (A) 2 and BP 3 They have the same structure. In a particular embodiment, the BP of formula (A) 2 and BP 3 All of these are -CH<.

[0213] In a particular embodiment, C of formula (A) 1 and C 2 They have the same structure. In a particular embodiment, C of formula (A) 1 and C 2 is -O-, -C(O)N(R 10 )-, and C is interrupted by one or more groups selected from the group consisting of 3- to 10-membered heterocyclines. 1-50 It is alkyl, where a 3- to 10-membered heterocycline is substituted by at least one oxo (=O).

[0214] In a particular embodiment, C of formula (A) 1 and C 2 is equation (Aa) [ka] (In the formula, The dashed line with a star indicates BP 1 This shows a binding to, The dashed lines that are not marked are BP 2 or BP 3 This shows a binding to, q1 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8. q2 is selected from the group consisting of 1, 2, 3, 4, and 5. q3 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8. (q4 is selected from the group consisting of 1, 2, and 3.) It belongs to them.

[0215] In a particular embodiment, q1 in formula (Aa) is selected from the group consisting of 4, 5, 6, 7, and 8. In a particular embodiment, q1 in formula (Aa) is selected from the group consisting of 5, 6, and 7. In a particular embodiment, q1 in formula (Aa) is 1. In a particular embodiment, q1 in formula (Aa) is 2. In a particular embodiment, q1 in formula (Aa) is 3. In a particular embodiment, q1 in formula (Aa) is 4. In a particular embodiment, q1 in formula (Aa) is 5. In a particular embodiment, q1 in formula (Aa) is 6. In a particular embodiment, q1 in formula (Aa) is 7. In a particular embodiment, q1 in formula (Aa) is 8.

[0216] In a particular embodiment, q2 in formula (Aa) is selected from the group consisting of 1, 2, and 3. In a particular embodiment, q2 in formula (Aa) is 1. In a particular embodiment, q2 in formula (Aa) is 2. In a particular embodiment, q2 in formula (Aa) is 3. In a particular embodiment, q2 in formula (Aa) is 4. In a particular embodiment, q2 in formula (Aa) is 5.

[0217] In a particular embodiment, q3 of formula (Aa) is selected from the group consisting of 2, 3, 4, and 5. In a particular embodiment, q3 of formula (Aa) is selected from the group consisting of 2, 3, and 4. In a particular embodiment, q3 of formula (Aa) is 1. In a particular embodiment, q3 of formula (Aa) is 2. In a particular embodiment, q3 of formula (Aa) is 3. In a particular embodiment, q3 of formula (Aa) is 4. In a particular embodiment, q3 of formula (Aa) is 5. In a particular embodiment, q3 of formula (Aa) is 6. In a particular embodiment, q3 of formula (Aa) is 7. In a particular embodiment, q3 of formula (Aa) is 8.

[0218] In a particular embodiment, q4 in equation (Aa) is 1. In a particular embodiment, q4 in equation (Aa) is 2. In a particular embodiment, q4 in equation (Aa) is 3.

[0219] In a particular embodiment, P of formula (A) 1 , P 2 , P 3 , and P 4 These are independent of each other and form equation (Ab) [ka] (In the formula, The dashed line indicates the connection to the remainder of -Z. m is either 0 or 1. p is an integer in the range of 70 to 900. (q is selected from the group consisting of 1, 2, 3, 4, 5, and 6.) It belongs to them.

[0220] In a particular embodiment, m in equation (Ab) is 0. In a particular embodiment, m in equation (Ab) is 1.

[0221] In a particular embodiment, p in formula (Ab) is an integer in the range of 115 to 680. In a particular embodiment, p in formula (Ab) is an integer in the range of 115 to 560. In a particular embodiment, p in formula (Ab) is an integer in the range of 185 to 450. In a particular embodiment, p in formula (Ab) is approximately 115. In a particular embodiment, p in formula (Ab) is approximately 160. In a particular embodiment, p in formula (Ab) is approximately 225. In a particular embodiment, p in formula (Ab) is approximately 270. In a particular embodiment, p in formula (Ab) is approximately 340. In a particular embodiment, p in formula (Ab) is approximately 450. In a particular embodiment, p in formula (Ab) is approximately 560.

[0222] In a particular embodiment, q in equation (Ab) is 1. In a particular embodiment, q in equation (Ab) is 2. In a particular embodiment, q in equation (Ab) is 3. In a particular embodiment, q in equation (Ab) is 4. In a particular embodiment, q in equation (Ab) is 5. In a particular embodiment, q in equation (Ab) is 6.

[0223] In a particular embodiment, -Z is in the formula (Ac) [ka] (In the formula, p1, p2, p3, and p4 are independent integers in the range of 70 to 900. This includes the part.

[0224] In certain embodiments, p1 in formula (Ac) is an integer in the range of 115 to 680. In certain embodiments, p1 in formula (Ac) is an integer in the range of 115 to 560. In certain embodiments, p1 in formula (Ac) is an integer in the range of 185 to 450. In certain embodiments, p1 in formula (Ac) is an integer in the range of 220 to 240. In certain embodiments, p1 in formula (Ac) is approximately 115. In certain embodiments, p1 in formula (Ac) is approximately 160. In certain embodiments, p1 in formula (Ac) is approximately 225. In certain embodiments, p1 in formula (Ac) is approximately 270. In certain embodiments, p1 in formula (Ac) is approximately 340. In certain embodiments, p1 in formula (Ac) is approximately 450. In certain embodiments, p1 in formula (Ac) is approximately 560.

[0225] In certain embodiments, p2 in formula (Ac) is an integer in the range of 115 to 680. In certain embodiments, p2 in formula (Ac) is an integer in the range of 115 to 560. In certain embodiments, p2 in formula (Ac) is an integer in the range of 185 to 450. In certain embodiments, p2 in formula (Ac) is an integer in the range of 220 to 240. In certain embodiments, p2 in formula (Ac) is approximately 115. In certain embodiments, p2 in formula (Ac) is approximately 160. In certain embodiments, p2 in formula (Ac) is approximately 225. In certain embodiments, p2 in formula (Ac) is approximately 270. In certain embodiments, p2 in formula (Ac) is approximately 340. In certain embodiments, p2 in formula (Ac) is approximately 450. In certain embodiments, p2 in formula (Ac) is approximately 560.

[0226] In certain embodiments, p3 in formula (Ac) is an integer in the range of 115 to 680. In certain embodiments, p3 in formula (Ac) is an integer in the range of 115 to 560. In certain embodiments, p3 in formula (Ac) is an integer in the range of 185 to 450. In certain embodiments, p3 in formula (Ac) is an integer in the range of 220 to 240. In certain embodiments, p3 in formula (Ac) is approximately 115. In certain embodiments, p3 in formula (Ac) is approximately 160. In certain embodiments, p3 in formula (Ac) is approximately 225. In certain embodiments, p3 in formula (Ac) is approximately 270. In certain embodiments, p3 in formula (Ac) is approximately 340. In certain embodiments, p3 in formula (Ac) is approximately 450. In certain embodiments, p3 in formula (Ac) is approximately 560.

[0227] In certain embodiments, p4 in formula (Ac) is an integer in the range of 115 to 680. In certain embodiments, p4 in formula (Ac) is an integer in the range of 115 to 560. In certain embodiments, p4 in formula (Ac) is an integer in the range of 185 to 450. In certain embodiments, p4 in formula (Ac) is an integer in the range of 220 to 240. In certain embodiments, p4 in formula (Ac) is approximately 115. In certain embodiments, p4 in formula (Ac) is approximately 160. In certain embodiments, p4 in formula (Ac) is approximately 225. In certain embodiments, p4 in formula (Ac) is approximately 270. In certain embodiments, p4 in formula (Ac) is approximately 340. In certain embodiments, p4 in formula (Ac) is approximately 450. In certain embodiments, p4 in formula (Ac) is approximately 560.

[0228] In certain embodiments, p1, p2, p3, and p4 in formula (Ac) are identical. In certain embodiments, p1, p2, p3, and p4 are in the range of 220 to 240.

[0229] In one embodiment, -Z is a portion disclosed in International Publication No. 2012 / 02047A1, which is incorporated herein by reference.

[0230] In another embodiment, -Z is a portion disclosed in International Publication No. 2013 / 024048A1, which is incorporated herein by reference.

[0231] In certain embodiments, -Z is water-insoluble. In certain embodiments, -Z is a hydrogel.

[0232] In certain embodiments, such hydrogels include 2-methacryloyloxyethyl phosphorylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amideamine), poly(amino acid), poly(acid anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(alkylene glycol), for example, poly(ethylene glycol) and poly(propylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(Hy) The polymers include polymers selected from the group consisting of droxypropyl methacrylate, poly(hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethylcellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and its derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch, and other carbohydrate-based polymers, xylan, and its copolymers.

[0233] In certain embodiments, -Z is a poly(alkylene glycol) or hyaluronic acid hydrogel.

[0234] In a particular embodiment, -Z is a poly(propylene glycol)-based hydrogel.

[0235] In a particular embodiment, -Z is a PEG-based hydrogel.

[0236] In certain embodiments, -Z is a PEG-based hydrogel disclosed in International Publication No. 2011 / 012715A1 or International Publication No. 2014 / 056926A1, which are incorporated herein by reference.

[0237] In a particular embodiment, -Z is a hyaluronic acid-based hydrogel.

[0238] In certain embodiments, -Z is a hyaluronic acid-based hydrogel disclosed in International Publication No. 2018 / 175788A1, which is incorporated herein by reference.

[0239] Part-L 1 - is through the IL-2 moiety, particularly through the amino acid residues of the IL-2 moiety, or through the modified moiety M present in -D. mod It may be coupled to -D through. In one embodiment, -L 1 - binds to -D through the IL-2 moiety, particularly through the amino acid residues of the IL-2 moiety. In another embodiment, -L 1 - is the modifying part M present in -D mod Joins to -D through one or more parts -L 1 - is part M mod It is understood that it may be combined with [this].

[0240] In one embodiment, all parts of the IL-2 conjugate of the present invention -L 1 - binds to the amino acid residue of -D.

[0241] -L 1 When - binds to an amino acid residue of the IL-2 moiety, such amino acid residues can be proteinogenic or non-proteinogenic amino acid residues of -D. In one embodiment, -L 1- binds to a non-proteinogenic amino acid residue, preferably the non-proteinogenic amino acid mentioned above. In another embodiment, -L 1 The binding of - is to a protogenic amino acid residue. When the binding occurs at a protogenic amino acid residue, in certain embodiments, the protogenic amino acid residue is selected from the group consisting of cysteine, methionine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, glutamine, and arginine. In certain embodiments, such a protogenic amino acid residue is selected from the group consisting of cysteine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, and arginine.

[0242] In one embodiment, -L 1 - binds to a cysteine ​​residue of -D, for example, a cysteine ​​residue selected from the group consisting of C57 and C104 based on SEQ ID NO: 2, or to the corresponding position of a homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional cysteine ​​residues compared to SEQ ID NO: 2, binding may also occur with such cysteine ​​residues that may be naturally occurring or that may be the result of addition, insertion, or mutation. It is understood that one or two cysteine ​​residues of SEQ ID NO: 2 may each be used for the binding of one or two partial -Z to -D. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and has more cysteine ​​residues than the IL-2 portion of SEQ ID NO: 2, two or more cysteine ​​residues may be used for the binding of -Z, i.e., up to the maximum number of cysteine ​​residues present in such homolog or variant may be used for the binding of -Z. In one embodiment, one partial -Z binds to one cysteine ​​residue. In another embodiment, two partial -Zs in total bind to two cysteine ​​residues.

[0243] In another embodiment, -L 1- binds to a histidine residue of -D, for example, a histidine residue selected from the group consisting of H15, H54, and H78 based on SEQ ID NO: 2, or to the corresponding position of the homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional histidine residues compared to SEQ ID NO: 2, binding may also occur with such histidine residues that may be naturally occurring or that may be the result of addition, insertion, or mutation. One, two, or three histidine residues of SEQ ID NO: 2 are bound to one, two, or three partial -L 1 It is understood that - may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and has more histidine residues than the IL-2 portion of SEQ ID NO: 2, then three or more histidine residues may be used for -D to -L 1 -It may be used for binding, that is, up to the maximum number of histidine residues present in such homolog or variant, -L 1 -May be used for joining. In one embodiment, one part -L 1 - binds to one histidine residue. In another embodiment, there are two parts in total -L 1 - binds to two histidine residues. In another embodiment, there are three parts in total -L 1 - binds to three histidine residues.

[0244] In another embodiment, -L 1 - binds to lysine residues, for example, lysine residues selected from the group consisting of K7, K8, K31, K34, K42, K47, K48, K53, K63, K75, or K96 based on SEQ ID NO: 2, or to the corresponding position of a homolog or variant of SEQ ID NO: 2. If a homolog or variant of IL-2 contains one or more additional lysine residues compared to SEQ ID NO: 2, binding may also occur with such lysine residues that may be naturally occurring or that may be the result of addition, insertion, or mutation. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 lysine residues of SEQ ID NO: 2 are bound to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 partial-L residues, respectively. 1It is understood that - may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and has more lysine residues than the IL-2 portion of SEQ ID NO: 2, then 11 or more lysine residues may be used for -L 1 - may be used for binding, that is, up to the maximum number of lysine residues present in such homolog or variant, -L 1 -May be used for joining. In one embodiment, one part -L 1 - binds to one lysine residue. In another embodiment, there are two parts in total -L 1 - binds to two lysine residues. In another embodiment, there are three parts in total -L 1 - binds to three lysine residues. In another embodiment, there are four parts in total -L 1 - binds to four lysine residues. In another embodiment, there are five parts in total -L 1 - binds to five lysine residues. In another embodiment, there are six parts in total -L 1 - binds to six lysine residues.

[0245] In another embodiment, -L 1 - binds to a tryptophan residue, for example, the tryptophan residue at position W120 based on SEQ ID NO: 2, or the corresponding position of a homolog or variant of SEQ ID NO: 2. If the homolog or variant of IL-2 contains one or more additional tryptophan residues compared to SEQ ID NO: 2, binding can also occur with such tryptophan residues, whether naturally occurring or resulting from addition, insertion, or mutation. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and has more tryptophan residues than the IL-2 portion of SEQ ID NO: 2, then one or more tryptophan residues are bound to -L 1 -It may be used for binding, that is, up to the maximum number of tryptophan residues present in such homolog or variant, -L 1 -May be used for joining. In one embodiment, one part -L 1 - binds to one tryptophan residue.

[0246] In another embodiment, -L 1 - binds to serine residues, for example, serine residues selected from the group consisting of S3, S4, S5, S74, S86, S98, S124, S126, and S129 based on SEQ ID NO: 2, or to the corresponding position of a homolog or variant of SEQ ID NO: 2. If a homolog or variant of IL-2 contains one or more additional serine residues compared to SEQ ID NO: 2, binding may also occur with such serine residues that may be naturally occurring or that may be the result of addition, insertion, or mutation. - binds to serine residues 1, 2, 3, 4, 5, 6, 7, 8, or 9 of SEQ ID NO: 2, -L 1 It is understood that - may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and has more serine residues than the IL-2 portion of SEQ ID NO: 2, then 9 more serine residues may be used for -L to -D. 1 - May be used for binding, that is, up to the maximum number of serine residues present in such homolog or variant, -L 1 -May be used for joining. In one embodiment, one part -L 1 - binds to one serine residue. In another embodiment, there are two parts in total -L 1 - binds to two serine residues. In another embodiment, there are three parts in total -L 1 - binds to three serine residues. In another embodiment, there are four parts in total -L 1 - binds to four serine residues. In another embodiment, there are five parts in total -L 1 - binds to five serine residues. In another embodiment, there are six parts in total -L 1 - binds to six serine residues.

[0247] In another embodiment, -L 1- binds to threonine residues, for example, threonine residues selected from the group consisting of T2, T6, T9, T36, T40, T50, T100, T102, T110, T112, T122, T130, and T132 based on SEQ ID NO: 2, or to the corresponding position of a homolog or variant of SEQ ID NO: 2. If a homolog or variant of IL-2 contains one or more additional threonine residues compared to SEQ ID NO: 2, the binding may also occur with such threonine residues that may be naturally occurring or that may be the result of addition, insertion, or mutation. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 threonine residues of SEQ ID NO: 2 are bound to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 partial-L 1 It is understood that - may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and has more threonine residues than the IL-2 portion of SEQ ID NO: 2, then 13 more threonine residues may be used for -L to -D. 1 - May be used for binding, that is, up to the maximum number of threonine residues present in such homolog or variant, -L 1 -May be used for joining. In one embodiment, one part -L 1 - binds to one threonine residue. In another embodiment, there are two parts in total -L 1 - binds to two threonine residues. In another embodiment, there are three parts in total -L 1 - binds to three threonine residues. In another embodiment, there are four parts in total -L 1 - binds to four threonine residues. In another embodiment, there are five parts in total -L 1 - binds to five threonine residues. In another embodiment, there are six parts in total -L 1 - binds to six threonine residues.

[0248] In another embodiment, -L 1- binds to a tyrosine residue, for example, a tyrosine residue selected from the group consisting of Y30, Y44, and Y106 based on SEQ ID NO: 2, or to the corresponding position of a homolog or variant of SEQ ID NO: 2. If a homolog or variant of IL-2 contains one or more additional tyrosine residues compared to SEQ ID NO: 2, binding may also occur with such tyrosine residues, whether naturally occurring or resulting from addition, insertion, or mutation. One, two, or three tyrosine residues of SEQ ID NO: 2 are bound to -D, or to one, two, or three partial -L 1 It is understood that - may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and has more tyrosine residues than the IL-2 portion of SEQ ID NO: 2, then three or more tyrosine residues may be used for -L 1 - may be used for binding, that is, up to the maximum number of tyrosine residues present in such homolog or variant, -L for -D 1 -May be used for joining. In one embodiment, one part -L 1 - binds to one tyrosine residue. In another embodiment, there are two parts in total -L 1 - binds to two tyrosine residues. In another embodiment, there are three parts in total -L 1 - binds to three tyrosine residues.

[0249] In another embodiment, -L 1 - binds to an aspartic acid residue, for example, an aspartic acid residue selected from the group consisting of D19, D83, and D108 based on SEQ ID NO: 2, or to the corresponding position of a homolog or variant of SEQ ID NO: 2. If a homolog or variant of IL-2 contains one or more additional aspartic acid residues compared to SEQ ID NO: 2, binding may also occur with such aspartic acid residues that may be naturally occurring or that may be the result of addition, insertion, or mutation. One, two, or three aspartic acid residues of SEQ ID NO: 2 are bound to one, two, or three partial -L 1It is understood that - may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and has more aspartic acid residues than the IL-2 portion of SEQ ID NO: 2, then three or more aspartic acid residues may be used for -L 1 - may be used for binding, that is, up to the maximum number of aspartic acid residues present in such homolog or variant, -L to -D 1 -May be used for joining. In one embodiment, one part -L 1 - is attached to one aspartic acid residue. In another embodiment, there are two parts in total -L 1 - binds to two aspartic acid residues. In another embodiment, there are three parts in total -L 1 - binds to three aspartic acid residues.

[0250] In another embodiment, -L 1 - binds to glutamate residues, for example, glutamate residues selected from the group consisting of E14, E51, E59, E60, E61, E66, E67, E94, E99, E105, E109, and E115 based on SEQ ID NO: 2, or to the corresponding position of a homolog or variant of SEQ ID NO: 2. If a homolog or variant of IL-2 contains one or more additional glutamate residues compared to SEQ ID NO: 2, binding may also occur with such glutamate residues that may be naturally occurring or that may be the result of addition, insertion, or mutation. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 glutamate residues of SEQ ID NO: 2 are bound to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 partial-L 1 -It is understood that it may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and has more glutamic acid residues than the IL-2 portion of SEQ ID NO: 2, then 12 more glutamic acid residues may be used for -L 1 - May be used for binding, that is, up to the maximum number of glutamic acid residues present in such homolog or variant, -L 1 -May be used for joining. In one embodiment, one part -L 1- binds to one glutamate residue. In another embodiment, there are two parts in total -L 1 - binds to two glutamate residues. In another embodiment, there are three parts in total -L 1 - binds to three glutamate residues. In another embodiment, there are four parts in total -L 1 - binds to four glutamate residues. In another embodiment, there are five parts in total -L 1 - binds to five glutamate residues. In another embodiment, there are six parts in total -L 1 - binds to six glutamate residues.

[0251] In another embodiment, -L 1 - binds to an arginine residue, for example, an arginine residue selected from the group consisting of R37, R80, R82, and R119 based on SEQ ID NO: 2, or to the corresponding position of a homolog or variant of SEQ ID NO: 2. If a homolog or variant of IL-2 contains one or more additional arginine residues compared to SEQ ID NO: 2, binding may also occur with such arginine residues that may be naturally occurring or that may be the result of addition, insertion, or mutation. 1, 2, 3, or 4 arginine residues of SEQ ID NO: 2 are bound to 1, 2, 3, or 4 partial-L 1 It is understood that - may be used for binding. If the IL-2 portion is a homolog or variant of SEQ ID NO: 2 and has more arginine residues than the IL-2 portion of SEQ ID NO: 2, then four or more arginine residues may be used for -L 1 - May be used for binding, that is, up to the maximum number of arginine residues present in such homolog or variant, -L 1 -May be used for joining. In one embodiment, one part -L 1 - binds to one arginine residue. In another embodiment, there are two parts in total -L 1 - binds to two arginine residues. In another embodiment, there are three parts in total -L 1 - binds to four arginine residues. In another embodiment, there are four parts in total -L 1 - binds to four arginine residues.

[0252] In another embodiment, at least one part - L 1 - binds to the amino acid residue of -D, and the remaining part / part(s) -L 1 - is attached to the modifier present in -D.

[0253] In another embodiment, all parts of the IL-2 conjugate of the present invention -L 1 - is attached to the modifying part present in -D. In one embodiment, one part -L 1 - is joined to one modifying part of -D. In another embodiment, there are two parts in total -L 1 - joins to one modifying part, i.e., the same modifying part. In another embodiment, there are three parts in total -L 1 - is joined to one modifying part. In another embodiment, there are four parts in total -L 1 - is joined to one modifying part. In another embodiment, there are five parts in total -L 1 - is joined to one modifying part. In another embodiment, there are six parts in total -L 1 - is joined to one modifying part. In another embodiment, there are two parts in total -L 1 - combines with two modifying parts, i.e., one part -L per modifying part. 1 - joins. In another embodiment, there are three parts in total -L 1 - is combined with three modifying parts, i.e., one part -L per modifying part. 1 - joins. In another embodiment, there are four parts in total -L 1 - is combined with four modifying parts, i.e., one part -L per modifying part. 1 - joins. In another embodiment, there are five parts in total -L 1 - is joined to five modifying parts, meaning one part is joined to each modifying part. In another embodiment, there are six parts in total -L 1 - is combined with six modifying parts, meaning one -L per modifying part. 1 They combine.

[0254] In one embodiment, -L 1- has the structure disclosed in WO 2009 / 095479 A2. Thus, in one embodiment, Sub-L 1 - is of formula (II):

Chemical formula

[0255] Preferably, -L of formula (II) 1 - is one part -L 2 Replaced with -Z

[0256] In one embodiment, -L of formula (II) 1 - is not further replaced.

[0257] -R in equation (II) 3 / -R 3a However, when they combine with the nitrogen atom to which they are bonded to form a 3- to 10-membered heterocycle, the atom directly bonded to the nitrogen is SP 3It is understood that only such 3- to 10-membered heterocycles, which are hybrid carbon atoms, can be formed. In other words, -R 3 / -R 3a Such 3- to 10-membered heterocycles, formed by these atoms and the nitrogen atoms to which they are bonded, have the following structure: [ka] (In the formula, The dashed line is -L 1 - indicates the bond to the remainder, The ring contains 3 to 10 atoms, including at least one nitrogen atom, and R # and R ## SP 3 (Represents hybrid carbon atoms).

[0258] It is also understood that 3- to 10-membered complex rings may be further substituted.

[0259] -R in equation (II) 3 / -R 3a A suitable exemplary embodiment of a 3- to 10-membered heterocycle formed by these atoms and the nitrogen atoms to which they are bonded is as follows: [ka] (In the formula, The dashed lines indicate bonding to the rest of the molecule. -R is -H and C 1-6 (Selected from the group consisting of alkyl groups).

[0260] -L in equation (II) 1 - may be further substituted at will. In general, any substituent may be used as long as it does not affect the principle of cleavage, i.e., the asterisked hydrogen in formula (II) is not substituted, and the part of formula (II) [ka] The nitrogen remains as part of the primary, secondary, or tertiary amine, i.e., -R 3 and -R3a These are either -H independently of each other, or SP 3 It is linked to -N< through hybridized carbon atoms.

[0261] In one embodiment, the -R of formula (II) 1 or -R 1a is, -L 2 -Z or -L 2 It is replaced by -Z'. In another embodiment, -R in equation (II) 2 or -R 2a is, -L 2 -Z or -L 2 It is replaced by -Z'. In another embodiment, -R in equation (II) 3 or -R 3a is, -L 2 -Z or -L 2 It is replaced by -Z'. In another embodiment, -R in equation (II) 4 is, -L 2 -Z or -L 2 It is replaced by -Z'. In another embodiment, -R in equation (II) 5 or -R 5a is, -L 2 -Z or -L 2 It is replaced by -Z'. In another embodiment, -R in equation (II) 6 is, -L 2 -Z or -L 2 It is replaced by -Z'. In another embodiment, -R in equation (II) 7 or -R 7a is, -L 2 -Z or -L 2 It is replaced by -Z'. In another embodiment, -R in equation (II) 8 or -R 8a is, -L 2 -Z or -L 2 It is replaced by -Z'. In another embodiment, -R in equation (II) 9 or -R 9a is, -L 2 -Z or -L 2 It is replaced with -Z'.

[0262] In another embodiment, -L 1- has the structure disclosed in International Publication No. 2016 / 020373A1. Therefore, in another embodiment, part -L 1 - is equation (III): [ka] (In the formula, The dashed lines indicate bonding by forming amide or ester bonds to primary or secondary amines or hydroxyls of -D, respectively. -R 1 , -R 1a , -R 2 , -R 2a , -R 3 and -R 3a is -H, -C(R 8 R 8a R 8b ), -C(=O)R 8 -C≡N, -C(=NR) 8 )R 8a ,-CR 8 (=CR 8a R 8b ), -C≡CR 8 and -T are independently selected from the group, -R 4 , -R 5 and -R 5a is -H, -C(R 9 R 9a R 9b ) and -T are independently selected from the group, and a1 and a2 are independently 0 or 1. Each-R 6 , -R 6a , -R 7 , -R 7a , -R 8 , -R 8a , -R 8b , -R 9 , -R 9a , -R 9b -H, HALogen, -CN, -COOR 10 , -OR 10 , -C(O)R 10 ,-C(O)N(R 10 R 10a ), -S(O)2N(R 10 R10a ), -S(O)N(R 10 R 10a ), -S(O)2R 10 ,-S(O)R 10 , -N(R 10 )S(O)2N(R 10a R 10b ), -SR 10 , -N(R 10 R 10a ), -NO2, -OC(O)R 10 , -N(R 10 )C(O)R 10a , -N(R 10 )S(O)2R 10a , -N(R 10 )S(O)R 10a , -N(R 10 )C(O)OR 10a , -N(R 10 )C(O)N(R 10a R 10b ), -OC(O)N(R 10 R 10a ), -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 They are independently selected from a group consisting of alkynnyls, where -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkinyl is the same or different -R of one or more R's. 11 Replaced by optional means, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) 12 )-,-S(O)2N(R 12 )-,-S(O)N(R 12 )-, -S(O)2-, -S(O)-, -N(R 12 )S(O)2N(R 12a )-, -S-, -N(R 12 )-, -OC(OR 12 )(R 12a )-,-N(R 12 )C(O)N(R 12a )-, and -OC(O)N(R 12Interrupted by one or more elements selected from the group consisting of )-, Each-R 10 , -R 10a , -R 10b -H, -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Independently selected from the group consisting of alkynyls, -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkinyl is the same or different -R of one or more R's. 11 Replaced by optional means, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) 12 )-,-S(O)2N(R 12 )-,-S(O)N(R 12 )-, -S(O)2-, -S(O)-, -N(R 12 )S(O)2N(R 12a )-, -S-, -N(R 12 )-, -OC(OR 12 )(R 12a )-,-N(R 12 )C(O)N(R 12a )-, and -OC(O)N(R 12 Interrupted by one or more elements selected from the group consisting of )-, Each T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 The group consists of cycloalkyls, 3-10 membered heterocyclyls, and 8-11 membered heterobicyclyls, and each T is independently selected from the group, and each T is independently one or more of the same or different -R 11 Replaced by choice, Each-R 11 These are halogen, -CN, oxo (=O), and -COOR. 13 , -OR 13 , -C(O)R 13 ,-C(O)N(R 13 R 13a ), -S(O)2N(R 13 R 13a), -S(O)N(R 13 R 13a ), -S(O)2R 13 ,-S(O)R 13 , -N(R 13 )S(O)2N(R 13a R 13b ), -SR 13 , -N(R 13 R 13a ), -NO2, -OC(O)R 13 , -N(R 13 )C(O)R 13a , -N(R 13 )S(O)2R 13a , -N(R 13 )S(O)R 13a , -N(R 13 )C(O)OR 13a , -N(R 13 )C(O)N(R 13a R 13b ), -OC(O)N(R 13 R 13a ), and C 1-6 Selected independently from alkyl groups, C 1-6 Alkyl is optionally substituted with one or more halogens, one or more of the same or different halogens. Each-R 12 , -R 12a , -R 13 , -R 13a , -R 13b -H and C 1-6 Independently selected from the group consisting of alkyls, C 1-6 Alkyl is optionally substituted with one or more halogens, one or more of the same or different halogens. Optional: Pair-R 1 / -R 1a , -R 2 / -R 2a , -R 3 / -R 3a , -R 6 / -R 6a , -R 7 / -R 7a One or more of them, together with the atom they are bonded to, C 3-10 Forming cycloalkyl or 3-10 membered heterocyclines, Optional: Pair-R1 / -R 2 , -R 1 / -R 3 , -R 1 / -R 4 , -R 1 / -R 5 , -R 1 / -R 6 , -R 1 / -R 7 , -R 2 / -R 3 , -R 2 / -R 4 , -R 2 / -R 5 , -R 2 / -R 6 , -R 2 / -R 7 , -R 3 / -R 4 , -R 3 / -R 5 , -R 3 / -R 6 , -R 3 / -R 7 , -R 4 / -R 5 , -R 4 / -R 6 , -R 4 / -R 7 , -R 5 / -R 6 , -R 5 / -R 7 , -R 6 / -R 7 One or more of these, together with the atoms to which they are bonded, form ring A. A is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 (Selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl) -L 1 - is at least one -L 2 -Z is used as a substitute, and -L is optional. 1 - is further replaced.

[0263] -L in equation (III) 1Any further optional substituents are preferably as described above.

[0264] Preferably, -L of formula (III) 1 - is one part -L 2 Replaced with -Z

[0265] In one embodiment, the -L of formula (III) 1 - is not further replaced.

[0266] In another embodiment, -L 1 - has the structure disclosed in European Patent No. 1536334 B1, WO2009 / 009712A1, WO2008 / 034122A1, WO2009 / 143412A2, WO2011 / 082368A2 and U.S. Patent No. 8,618,124 B2, which are incorporated herein by reference.

[0267] In another embodiment, -L 1 - has the structure disclosed in U.S. Patent No. 8,946,405 B2 and U.S. Patent No. 8,754,190 B2, which are incorporated herein by reference. Thus, in another embodiment, -L 1 - is equation (IV): [ka] (In the formula, The dashed lines indicate bonding to -D through a functional group of -D selected from the group consisting of -OH, -SH, and -NH2. m is either 0 or 1. -R 1 and -R 2 At least one or both of are -CN, -NO2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -C(O)R 3 ,-S(O)R 3 -S(O)2R 3 , and -SR 4 Selected independently from a group consisting of, -R1 and -R 2 One or only one of these is selected from the group consisting of -H, optionally substituted alkyl, optionally substituted arylalkyl, and optionally substituted heteroarylalkyl. -R 3 -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 9 and -N(R 9 Selected from the group consisting of )2, -R 4 This is selected from the group consisting of optionally substituted alkyls, optionally substituted aryls, optionally substituted arylalkyls, optionally substituted heteroaryls, and optionally substituted heteroarylalkyls. Each-R 5 It is independently selected from the group consisting of -H, optionally substituted alkyl, optionally substituted alkenylalkyl, optionally substituted alkynylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl. -R 9 It is selected from the group consisting of -H and optionally substituted alkyl groups, -Y- does not exist, -X- is either -O- or -S-, or -Y- is -N(Q)CH2-, and -X- is -O-, Q is selected from the group consisting of optionally substituted alkyls, optionally substituted aryls, optionally substituted arylalkyls, optionally substituted heteroaryls, and optionally substituted heteroarylalkyls. Optionally, -R 1 and -R 2 They may come together to form a 3- to 8-membered ring. Optionally, both -R 9 (These atoms, together with the nitrogen atoms they are bonded to, form a heterocyclic ring.) It is, -L 1 - is, -L 2 -Z is used as a substitute, and -L is optional. 1 - is further replaced.

[0268] The terms used only in relation to equation (IV) have the following meanings:

[0269] The term "alkyl" as used herein includes linear, branched, or cyclic saturated hydrocarbon groups comprising 1 to 8 carbon atoms, or in some embodiments, 1 to 6 or 1 to 4 carbon atoms.

[0270] The term "alkoxy" includes alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and similar groups.

[0271] The term "alkenyl" includes non-aromatic unsaturated hydrocarbons that have a carbon-carbon double bond.

[0272] The term "alkynyl" includes non-aromatic unsaturated hydrocarbons that have a carbon-carbon triple bond.

[0273] The term "aryl" includes aromatic hydrocarbon groups with 6 to 18 carbon atoms, preferably 6 to 10 carbon atoms, including groups such as phenyl, naphthyl, and anthracenyl. The term "heteroaryl" includes aromatic rings with 3 to 15 carbon atoms and at least one N, O, or S atom, preferably 3 to 7 carbon atoms and at least one N, O, or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and similar groups.

[0274] In some cases, the alkenyl, alkynyl, aryl, or heteroaryl moiety may be coupled to the rest of the molecule through an alkylene bond. In these situations, the substituent is referred to as alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl, indicating that the alkylene moiety is located between the alkenyl, alkynyl, aryl, or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl, or heteroaryl is coupled.

[0275] The term "halogen" includes bromo, fluoro, chloro, and iodine.

[0276] The term "heterocyclic ring" refers to a 4-8 member aromatic or aromatic ring containing 3-7 carbon atoms and at least one N, O, or S atom. Examples include piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groups provided above for the term "heteroaryl."

[0277] When the ring system is optionally substituted, suitable substituents are selected from the group consisting of alkyl, alkenyl, alkynyl, or further rings, each of which may be optionally further substituted. Optional substituents for any of the above groups include halo, nitro, cyano, -OR, -SR, -NR2, -OCOR, -NRCOR, -COOR, -CONR2, -SOR, -SO2R, -SONR2, and -SO2NR2, where each R is independently alkyl, alkenyl, alkynyl, aryl, or heteroaryl, or two R groups cooperate with the atom to which they are bonded to form a ring.

[0278] Preferably, -L of formula (IV) 1 - is one part - L 2 It is replaced with -Z.

[0279] In another embodiment, -L 1- has the structure disclosed in WO2013 / 036857A1, which is incorporated herein by reference. Thus, in another embodiment, -L 1 - is equation (V): [ka] (In the formula, The dashed line indicates the bond to -D through the amine functional group of -D. -R 1 These include optionally substituted C1-C6 linear, branched or cyclic alkyl groups, optionally substituted aryl groups, optionally substituted heteroaryl groups, alkoxy groups, and -NR groups. 5 Selected from a group consisting of 2, -R 2 This is selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl. -R 3 This is selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl. -R 4 This is selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl. Each-R 5 When the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl is independently selected from each other or acts in cooperation with two -R 5 (This can be a cycloalkyl or cycloheteroalkyl group.) It is, -L 1 - is, -L 2 -Z is used as a substitute, and -L is optional. 1 - is further replaced.

[0280] The terms used only in relation to equation (V) have the following meanings:

[0281] "Alkyl," "alkenyl," and "alkynyl" are linear, branched, or cyclic hydrocarbon groups comprising 1 to 8, 1 to 6, or 1 to 4 carbon atoms, where alkyl is a saturated hydrocarbon, alkenyl contains one or more carbon-carbon double bonds, and alkynyl contains one or more carbon-carbon triple bonds. Unless otherwise specified, these contain 1 to 6 carbon atoms.

[0282] "Aryl" includes aromatic hydrocarbon groups with 6 to 18 carbon atoms, preferably 6 to 10 carbon atoms, including groups such as phenyl, naphthyl, and anthracenin. "Heteroaryl" includes aromatic rings with 3 to 15 carbon atoms and at least one N, O, or S atom, preferably 3 to 7 carbon atoms and at least one N, O, or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and similar groups.

[0283] The term "substituted" refers to an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group that contains one or more substituents in place of one or more hydrogen atoms. Substituents generally include halogens (including F, Cl, Br, and I), lower alkyls (including linear, branched, and cyclic), lower haloalkyls (including fluoroalkyls, chloroalkyls, bromoalkyls, and iodoalkyls), OH, lower alkoxys (including linear, branched, and cyclic), SH, lower alkylthios (including linear, branched, and cyclic), aminos, alkylaminos, dialkylaminos, silyls (including alkylsilyls, alkoxysilyls, and arylsilyls), nitros, cyanos, carbonyls, carboxylic acids, carboxylic acid esters, carboxylic acid amides, aminocarbonyls, aminoacyls, carbamates, ureas, and thiocarbamates. The following can be selected: thiourea, ketone, sulfone, sulfonamide, aryl (including phenyl, naphthyl, and anthracenyl), and heteroaryl (including five-membered heteroaryls such as pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole, and tetrazole; six-membered heteroaryls such as pyridine, pyrimidine, and pyrazine; and fusion heteroaryls such as benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzoisoxazole, and benzoisothiazole).

[0284] Preferably, -L of formula (V) 1 - is one part - L 2 Replaced with -Z

[0285] In another embodiment, -L 1 - has the structure disclosed in U.S. Patent No. 7,585,837 B2, which is incorporated herein by reference. Thus, in another embodiment, -L 1 - is represented by the following equation (VI): [ka] (In the formula, The dashed line indicates the bond to -D through the amine functional group of -D. R 1 and R 2 Hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkalyl, aralkyl, halogen, nitro, -SO3H, -SO2NHR 5 Independently selected from the group consisting of amino, ammonium, carboxyl, PO3H2 and OPO3H2, R 3 , R 4 and R 5 (The element is independently selected from the group consisting of hydrogen, alkyl, and aryl atoms.) It is, -L 1 - is, -L 2 -Z is replaced with o, and is optional, -L 1 - is further replaced.

[0286] Suitable substituents for formula (VI) are alkyl (e.g., C 1-6 Alkyl), alkenyl (e.g., C 2-6 Alkenyl), Alkinyl (for example, C 2-6 The moiety is an alkynyl, aryl (e.g., phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl (e.g., an aromatic 4-7 membered heterocycle), or halogen moiety.

[0287] The terms used only in relation to equation (VI) have the following meanings:

[0288] The terms "alkyl," "alkoxy," "alkoxyalkyl," "aryl," "alkalil," and "aralkyl" refer to alkyl radicals with 1 to 8 carbon atoms, preferably 1 to 4, such as methyl, ethyl, propyl, isopropyl, and butyl, and aryl radicals with 6 to 10 carbon atoms, such as phenyl and naphthyl. The term "halogen" includes bromo, fluoro, chloro, and iodine.

[0289] Preferably, -L of formula (VI) 1- is one part - L 2 It is replaced with -Z.

[0290] In another embodiment, -L 1 - has the structure disclosed in WO2002 / 089789A1, which is incorporated herein by reference. Thus, in another embodiment, -L 1 - is represented by the following equation (VII): [ka] (In the formula, The dashed line indicates the bond to -D through the amine functional group of -D. L1 is a bifunctional bonding group, Y1 and Y2 are independently O, S, or NR 7 And, R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is hydrogen, C 1-6 Alkyl, C 3-12 Branched alkyl, C 3-8 Cycloalkyl, C 1-6 Substituting alkyl, C 3-8 Substituted cycloalkyl, aryl, substituted aryl, aralkyl, C 1-6 Heteroalkyl, substituted C 1-6 Heteroalkyl, C 1-6 Alkoxy, phenoxy and C 1-6 Independently selected from the group consisting of heteroalkoxys, When Ar is included in formula (VII), it is the part that forms a polysubstituted aromatic hydrocarbon or a polysubstituted heterocyclic group. X is a chemical bond, or a portion that is actively transported to the target cell, a hydrophobic portion, or a combination thereof. (y is either 0 or 1) It is, -L 1 - is, -L 2 -Z is used as a substitute, and -L is optional. 1 - is further replaced.

[0291] The terms used only in relation to equation (VII) have the following meanings:

[0292] The term "alkyl" is used, for example, in alkoxy, C 3-8 Linear, branched, and substituted carbon atoms, including cycloalkyl or substituted cycloalkyl groups. 1-12 It is understood that it contains alkyl groups.

[0293] The term "substituted" shall be understood to include the addition of one or more different atoms, or the replacement of one or more atoms contained in a functional group or compound with one or more different atoms.

[0294] Substituted alkyls include carboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls, and mercaptoalkyls; substituted cycloalkyls include parts such as 4-chlorocyclohexyl; aryls include parts such as naphthyl; substituted aryls include parts such as 3-bromophenyl; aralkyls include parts such as toluyl; heteroalkyls include parts such as ethylthiophene; substituted heteroalkyls include parts such as 3-methoxythiophene; alkoxys include parts such as methoxy; and phenoxys include parts such as 3-nitrophenoxy. Halos are understood to include fluoro, chloro, iodine, and bromo.

[0295] Preferably, the -L of formula (VII) 1 - is one part - L 2 It is replaced with -Z.

[0296] In another embodiment, -L 1 - is a substructure of equation (VIII) [ka] (In the formula, The dashed lines marked with an asterisk indicate the bond of -D to nitrogen due to the formation of an amide bond, while the dashed lines without an asterisk indicate -L 1 (This indicates a bond to the remainder.) Includes, -L 1 - is, -L 2 -Z is used as a substitute, and -L is optional. 1 - is further replaced.

[0297] Preferably, -L of formula (VIII) 1 - is one part -L 2 Replaced with -Z

[0298] In one embodiment, the -L of formula (VIII) 1 - is not further replaced.

[0299] In another embodiment, -L 1 - is a substructure of equation (IX) [ka] (In the formula, The dashed lines marked with an asterisk indicate bonding by carbamate bonding to nitrogen in -D, while the dashed lines without an asterisk indicate bonding by carbamate bonding to nitrogen in -L. 1 (This indicates a bond to the remainder.) Includes, -L 1 - is, -L 2 -Z is used as a substitute, and -L is optional. 1 - is further replaced.

[0300] Preferably, -L of formula (IX) 1 - is one part -L 2 Replaced with -Z

[0301] In one embodiment, the -L of formula (IX) 1 - is not further replaced.

[0302] In one embodiment, -L 1 - is equation (IX-a) [ka] (In the formula, The dashed lines marked with an asterisk indicate the bond to nitrogen in -D, while the dashed lines without an asterisk indicate -L2 Shows the bond to -Z, n is 0, 1, 2, 3, or 4. =Y1 is selected from the group consisting of =O and =S, -Y2- is selected from the group consisting of -O- and -S-, -Y3- is selected from the group consisting of -O- and -S-, -Y4- is -O-, -NR 5 -, and -C(R 6 R 6a Selected from the group consisting of )-, =Y5 is selected from the group consisting of =O and =S, -R 3 , -R 5 , -R 6 , -R 6a These are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl. -R 4 This is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl. -W- is C 3-10 Cycloalkyl, 8-30 membered carbopolycyclyl, 3-10 membered heterocyclyl, -C(O)-, -C(O)N(R) 7 )-, -O-, -S-, and -N(R 7 C is optionally interrupted by one or more elements selected from the group consisting of )- 1-20 Selected from the group consisting of alkyl groups, -Nu is -N(R 7 R 7a ), -N(R 7 OH), -N(R7 )-N(R 7a R 7b ), -S(R 7 ), -COOH, [ka] A nucleophile selected from the group consisting of, -Ar- is, [ka] (In the formula, The dashed line is -L 1 - indicates the bond to the remainder, -Z 1 - is -O-, -S-, and -N(R 7 Selected from the group consisting of )-, -Z 2 - is -N(R 7 )-is) Selected from the group consisting of, -R 7 , -R 7a , -R 7b is -H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 (Selected independently from a group consisting of alkinyls) It is, -L 1 - can be optionally replaced further.

[0303] In one embodiment, the -L of formula (IX-a) 1 - is not further replaced.

[0304] In another embodiment, -L 1 - is equation (IX-b) [ka] (In the formula, The dashed lines marked with an asterisk indicate the bond to nitrogen in -D, while the dashed lines without an asterisk indicate -L 2 Shows the bond to -Z, n is 0, 1, 2, 3, or 4. =Y1 is selected from the group consisting of =O and =S, -Y2- is selected from the group consisting of -O- and -S-, -Y3- is selected from the group consisting of -O- and -S-, -Y4- is -O-, -NR 5 -, and -C(R 6 R 6a Selected from the group consisting of )-, =Y5 is selected from the group consisting of =O and =S, -R 2 , -R 3 , -R 5 , -R 6 , -R 6a These are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl. -R 4 This is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl. -W- is C 3-10 Cycloalkyl, 8-30 membered carbopolycyclyl, 3-10 membered heterocyclyl, -C(O)-, -C(O)N(R) 7 )-, -O-, -S-, and -N(R 7 C is optionally interrupted by one or more groups selected from the group consisting of )- 1-20 Selected from the group consisting of alkyl groups, -Nu is -N(R 7 R 7a ), -N(R 7 OH), -N(R 7 )-N(R 7a R 7b ), -S(R 7), -COOH, [ka] A nucleophile selected from the group consisting of, -Ar- is, [ka] (In the formula, The dashed line is -L 1 - indicates the bond to the remainder, -Z 1 - is -O-, -S-, and -N(R 7 Selected from the group consisting of )-, -Z 2 - is -N(R 7 )-is) Selected from the group consisting of, -R 7 , -R 7a , -R 7b is -H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 (Selected independently from a group consisting of alkinyls) It is, -L 1 - can be optionally replaced further.

[0305] In one embodiment, the -L of formula (IX-b) 1 - is not further replaced.

[0306] In a particular embodiment, equations (IX-a) and (IX-b) = Y 1 The value is O.

[0307] In certain embodiments, formulas (IX-a) and (IX-b)-Y 2 - is -O-

[0308] In certain embodiments, the -Y in formulas (IX-a) and (IX-b) 3 - is -O-

[0309] In certain embodiments, the -Y in formulas (IX-a) and (IX-b) 4 - is -NR 5 - is

[0310] In a particular embodiment, equations (IX-a) and (IX-b) = Y 5 The value is O.

[0311] In certain embodiments, n in formulas (IX-a) and (IX-b) is 0 or 1. In certain embodiments, n in formulas (IX-a) and (IX-b) is 0. In certain embodiments, n in formulas (IX-a) and (IX-b) is 1.

[0312] In a particular embodiment, (IX-b) -R 2 The group is selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, the -R of formula (IX-b) 2 The -R of formula (IX-b) is selected from the group consisting of -H, methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, the -R of formula (IX-b) is selected. 2 is selected from -H, methyl, and ethyl. In certain embodiments, -R of formula (IX-b) 2 is -H.

[0313] In certain embodiments, the -R in formulas (IX-a) and (IX-b) 3 The -R is selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, the -R of formulas (IX-a) and (IX-b) 3 The -R is selected from the group consisting of -H, methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, the -R of formulas (IX-a) and (IX-b) is selected. 3 is selected from -H, methyl, and ethyl. In certain embodiments, -R of formulas (IX-a) and (IX-b) 3 is -H.

[0314] In a preferred embodiment, the -R in each of the formulas (IX-a) and (IX-b) 4 is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, the -R of formulas (IX-a) and (IX-b) 4 The -R of formulas (IX-a) and (IX-b) is selected from the group consisting of methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, the -R of formulas (IX-a) and (IX-b) is selected. 4 It is selected from methyl and ethyl.

[0315] In certain embodiments, the -R in formulas (IX-a) and (IX-b) 5 The -R is selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, the -R of formulas (IX-a) and (IX-b) 5 The -R is selected from the group consisting of -H, methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, the -R of formulas (IX-a) and (IX-b) is selected. 5 The -R of formulas (IX-a) and (IX-b) is selected from methyl and ethyl. In certain embodiments, the -R of formulas (IX-a) and (IX-b) is selected. 5 It is methyl.

[0316] In certain embodiments, the -R in formulas (IX-a) and (IX-b) 6 and -R 6a -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, -R of formulas (IX-a) and (IX-b) 6 and -R 6a -H, methyl, ethyl, n-propyl, and isopropyl are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, and isopropyl. In certain embodiments, -R of formulas (IX-a) and (IX-b) 6 and -R 6aThe -R is independently selected from -H, methyl, and ethyl. In certain embodiments, the -R of formulas (IX-a) and (IX-b) is selected. 6 and -R 6a All of these are -H.

[0317] In certain embodiments, Ar in formulas (IX-a) and (IX-b) is phenyl. In certain embodiments, Ar in formulas (IX-a) and (IX-b) is [ka] (In the equation, the dashed line indicates the combination of parts (IX-a) and (IX-b) to the remainder.) That is the case.

[0318] In a particular embodiment, W in formulas (IX-a) and (IX-b) is C 3-10 Cycloalkyl, -C(O)-, -C(O)N(R) 7 )-, -O-, -S-, and -N(R 7 )- Interrupted by choice by C 1-20 It is alkyl. In certain embodiments, W in formulas (IX-a) and (IX-b) is C 3-10 Cycloalkyl, -C(O)-, -C(O)N(R) 7 )-, -O-, -S-, and -N(R 7 )- Interrupted by choice by C 1-10 It is alkyl. In certain embodiments, W in formulas (IX-a) and (IX-b) is C 3-10 Cycloalkyl, -C(O)-, -C(O)N(R) 7 )-, -O-, -S-, and -N(R 7 )- Interrupted by choice by C 1-6 It is alkyl. In certain embodiments, W in formulas (IX-a) and (IX-b) is [ka] (In the formula, The dashed lines indicate the connection to the remainder of the part of equation (IX-a) or (IX-b), respectively. That is the case.

[0319] In a particular embodiment, -Nu in formulas (IX-a) and (IX-b) is -N(R 7 R 7a )

[0320] In certain embodiments, the -R in formulas (IX-a) and (IX-b) 7 , -R 7a , and -R 7b The -R of formulas (IX-a) and (IX-b) is independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments, the -R of formulas (IX-a) and (IX-b) is independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. 7 , -R 7a , and -R 7b -H, methyl, ethyl, n-propyl, and isopropyl are independently selected from each other. In certain embodiments, -R of formulas (IX-a) and (IX-b) 7 , -R 7a , and -R 7b The -R of formulas (IX-a) and (IX-b) is selected independently from each other. In certain embodiments, the -R 7 , -R 7a , and -R 7b All of them are methyl.

[0321] In a particular embodiment, -L 1 - is equation (IX-c) [ka] (In the formula, The dashed lines marked with an asterisk indicate the bond of -D to nitrogen. Dashed lines without a mark are -L 2 Shows the bond to -Z, s1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. It belongs to them.

[0322] In a particular embodiment, s1 in equation (IX-c) is an integer selected from the group consisting of 1, 2, 3, 4, and 5. In a particular embodiment, s1 in equation (IX-c) is 1. In a particular embodiment, s1 in equation (IX-c) is 2. In a particular embodiment, s1 in equation (IX-c) is 3. In a particular embodiment, s1 in equation (IX-c) is 4. In a particular embodiment, s1 in equation (IX-c) is 5.

[0323] In a particular embodiment, the -L of formula (IX-d) 1 -teeth, [ka] (In the formula, The dashed lines marked with an asterisk indicate the bond of -D to nitrogen. Dashed lines without a mark are -L 2 (This indicates a bond to -Z) That is the case.

[0324] Part-L 1 - can be linked to -D through any type of bond, as long as it is reversible. Preferably, -L 1 - is linked to -D through a bond selected from the group consisting of amides, esters, carbamates, acetals, aminals, imines, oximes, hydrazones, disulfides, and acylguanidines. More preferably, -L 1 - is linked to -D through a bond selected from the group consisting of amides, esters, carbamates, and acylguanidines. These bonds do not have to be reversible themselves, but reversibility is -L 1 It is understood that this could be due to the action of a specific group or part of atoms present in -.

[0325] In one embodiment, -L 1 - is linked to -D through an ester bond.

[0326] In another embodiment, -L 1 - is linked to -D through a carbamate bond.

[0327] In another embodiment, -L 1 - is linked to -D via acylguanidine.

[0328] In a preferred embodiment, -L 1 - is linked to -D through an amide bond.

[0329] In a particular embodiment, -L 1 - is linked to -D via the nitrogen of the amine functional group of the side chain of the lysine residue of -D. In certain embodiments, -L 1 - is linked to -D via the nitrogen of the amine functional group of the side chain of the lysine residue of -D, and -D and -L 1 The bond formed between - and is a carbamate. Embodiments of such lysine residues are as described above.

[0330] In one embodiment, -L 2 - represents a chemical bond.

[0331] In another embodiment, -L 2 - is the spacer part.

[0332] In a particular embodiment, -L 2 - stands for -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-,-S(O)2N(R y1 )-,-S(O)N(R y1 )-, -S(O)2-, -S(O)-, -N(R y1 )S(O)2N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-,-N(R y1 )C(O)N(R y1a )-,-OC(O)N(R y1 )-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Selected from the group consisting of alkynnyls, where -T-, C 1-50Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is the same or different -R of one or more R's. y2 Replaced by optional means, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) y3 )-,-S(O)2N(R y3 )-,-S(O)N(R y3 )-, -S(O)2-, -S(O)-, -N(R y3 )S(O)2N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(R y3a )-,-N(R y3 )C(O)N(R y3a )-, and -OC(O)N(R y3 Interrupted by one or more elements selected from the group consisting of )-, -R y1 and -R y1a -H, -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 They are independently selected from a group consisting of alkynnyls, where -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is the same or different one or more -R y2 Replaced by optional means, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) y4 )-,-S(O)2N(R y4 )-,-S(O)N(R y4 )-, -S(O)2-, -S(O)-, -N(R y4 )S(O)2N(R y4a )-, -S-, -N(R y4 )-, -OC(OR y4 )(R y4a )-,-N(R y4)C(O)N(R y4a )-, and -OC(O)N(R y4 )- Interrupted by one or more elements selected from the group consisting of, Each T stands for phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 A molecule is independently selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl, and each T is the same or different -R y2 It is independently and arbitrarily replaced by, Each -R y2 These are halogen, -CN, oxo (=O), and -COOR. y5 , -OR y5 , -C(O)R y5 ,-C(O)N(R y5 R y5a ), -S(O)2N(R y5 R y5a ), -S(O)N(R y5 R y5a ), -S(O)2R y5 ,-S(O)R y5 , -N(R y5 )S(O)2N(R y5a R y5b ), -SR y5 , -N(R y5 R y5a ), -NO2, -OC(O)R y5 , -N(R y5 )C(O)R y5a , -N(R y5 )S(O)2R y5a , -N(R y5 )S(O)R y5a , -N(R y5 )C(O)OR y5a , -N(R y5 )C(O)N(R y5a R y5b ), -OC(O)N(R y5 R y5a ), and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 Alkyl is optionally substituted with one or more halogens, one or more of the same or different halogens. Each -R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b are -H and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 Alkyl is optionally substituted with one or more halogens, either the same or different.

[0333] In a particular embodiment, -L 2 - stands for -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-,-S(O)2N(R y1 )-,-S(O)N(R y1 )-, -S(O)2-, -S(O)-, -N(R y1 )S(O)2N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-,-N(R y1 )C(O)N(R y1a )-,-OC(O)N(R y1 )-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Selected from the group consisting of alkynnyls, where -T-, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkinyl is the same or different -R of one or more R's. y2 Replaced by optional means, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) y3 )-,-S(O)2N(R y3 )-,-S(O)N(R y3 )-, -S(O)2-, -S(O)-, -N(R y3 )S(O)2N(R y3a )-, -S-, -N(R y3 )-, -OC(ORy3 )(R y3a )-,-N(R y3 )C(O)N(R y3a )-, and -OC(O)N(R y3 Interrupted by one or more elements selected from the group consisting of )-, -R y1 and -R y1a -H, -T, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 They are independently selected from a group consisting of alkynnyls, where -T, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkinyl is the same or different -R of one or more R's. y2 Replaced by optional means, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) y4 )-,-S(O)2N(R y4 )-,-S(O)N(R y4 )-, -S(O)2-, -S(O)-, -N(R y4 )S(O)2N(R y4a )-, -S-, -N(R y4 )-, -OC(OR y4 )(R y4a )-,-N(R y4 )C(O)N(R y4a )-, and -OC(O)N(R y4 )- Interrupted by one or more elements selected from the group consisting of; Each T stands for phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 A molecule is independently selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl, and each T is the same or different -R y2 It is independently and arbitrarily replaced by, -R y2 These are halogen, -CN, oxo (=O), and -COOR.y5 , -OR y5 , -C(O)R y5 ,-C(O)N(R y5 R y5a ), -S(O)2N(R y5 R y5a ), -S(O)N(R y5 R y5a ), -S(O)2R y5 ,-S(O)R y5 , -N(R y5 )S(O)2N(R y5a R y5b ), -SR y5 , -N(R y5 R y5a ), -NO2, -OC(O)R y5 , -N(R y5 )C(O)R y5a , -N(R y5 )S(O)2R y5a , -N(R y5 )S(O)R y5a , -N(R y5 )C(O)OR y5a , -N(R y5 )C(O)N(R y5a R y5b ), -OC(O)N(R y5 R y5a ), and C 1-6 Selected from the group consisting of alkyl groups, where C 1-6 Alkyl is optionally substituted with one or more halogens, one or more of the same or different halogens. Each -R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b are -H and C 1-6 They are independently selected from the group consisting of alkyls, where C 1-6 Alkyl atoms are optionally substituted with one or more halogens, either the same or different.

[0334] In a particular embodiment, -L 2 - stands for -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1)-,-S(O)2N(R y1 )-,-S(O)N(R y1 )-, -S(O)2-, -S(O)-, -N(R y1 )S(O)2N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-,-N(R y1 )C(O)N(R y1a )-,-OC(O)N(R y1 )-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Selected from the group consisting of alkynnyls, where -T-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is the same or different -R of one or more R's. y2 Replaced by optional means, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) y3 )-,-S(O)2N(R y3 )-,-S(O)N(R y3 )-, -S(O)2-, -S(O)-, -N(R y3 )S(O)2N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(R y3a )-,-N(R y3 )C(O)N(R y3a )-, and -OC(O)N(R y3 Interrupted by one or more elements selected from the group consisting of )-, -R y1 and -R y1a -H, -T, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Independently selected from the group consisting of alkinyls, Each T stands for phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10Independently selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl, Each-R y2 is halogen and C 1-6 Independently selected from the group consisting of alkyls, Each-R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b -H and C 1-6 They are independently selected from the group consisting of alkyls, where C 1-6 Alkyl atoms are optionally substituted with one or more halogens, either the same or different.

[0335] In a particular embodiment, -L 2 - is C 1-20 It is an alkyl chain, and it consists of -O-, -T- and -C(O)N(R) y1 )- is optionally interrupted by one or more elements independently selected from and its C 1-20 The alkyl chain consists of -OH, -T, and -C(O)N(R) y6 R y6a ) is optionally replaced by one or more elements independently selected from, where -R y1 , -R y6 , -R y6a H and C 1-4 Independently selected from the group consisting of alkyls, T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 The group is selected from cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl.

[0336] In a particular embodiment, -L 2 - has a molecular weight in the range of 14 g / mol to 750 g / mol.

[0337] In a particular embodiment, -L2 -teeth, [ka] JPEG0007857984000057.jpg159163 (in the formula, The dashed lines represent -L 1 -, -L 2 - indicates a bond to the remainder of -Z, -R and -R a (These are independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl, and hexyl.) Includes a portion selected from the group consisting of

[0338] In a particular embodiment, -L 2 - is equation (IX-e): [ka] (In the formula, the dashed line marked with an asterisk indicates a connection to -L1-, Unmarked dashed lines indicate connections to -Z. s2 is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. It belongs to them.

[0339] In a particular embodiment, s2 in formula (IX-e) is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In a particular embodiment, s2 in formula (IX-e) is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8. In a particular embodiment, s2 in formula (IX-e) is 1. In a particular embodiment, s2 in formula (IX-e) is 2. In a particular embodiment, s2 in formula (IX-e) is 3. In a particular embodiment, s2 in formula (IX-e) is 4. In a particular embodiment, s2 in formula (IX-e) is 5. In a particular embodiment, s2 in formula (IX-e) is 6. In a particular embodiment, s2 in formula (IX-e) is 7. In a particular embodiment, s2 in formula (IX-e) is 8.

[0340] In a particular embodiment, part-L 1 -L 2 - is equation (IX-f) [ka] (In the formula, the dashed line marked with an asterisk indicates the bond of -D to nitrogen.) Unmarked dashed lines indicate connections to -Z. s1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. s2 is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. It belongs to them.

[0341] Therefore, the molar -L formed in the compound of formula (IX-f) 1 The bond between -D and - is a carbamate.

[0342] In a particular embodiment, s1 in equation (IX-f) is an integer selected from the group consisting of 1, 2, 3, 4, and 5. In a particular embodiment, s1 in equation (IX-f) is 1. In a particular embodiment, s1 in equation (IX-f) is 2. In a particular embodiment, s1 in equation (IX-f) is 3. In a particular embodiment, s1 in equation (IX-f) is 4. In a particular embodiment, s1 in equation (IX-f) is 5.

[0343] In a particular embodiment, s2 in formula (IX-f) is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In a particular embodiment, s2 in formula (IX-f) is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8. In a particular embodiment, s2 in formula (IX-f) is 1. In a particular embodiment, s2 in formula (IX-f) is 2. In a particular embodiment, s2 in formula (IX-f) is 3. In a particular embodiment, s2 in formula (IX-e) is 4. In a particular embodiment, s2 in formula (IX-f) is 5. In a particular embodiment, s2 in formula (IX-e) is 6. In a particular embodiment, s2 in formula (IX-f) is 7. In a particular embodiment, s2 in formula (IX-f) is 8.

[0344] In a particular embodiment, s1 in equation (IX-f) is 3, and s2 in equation (IX-f) is 3.

[0345] In one embodiment, the IL-2 conjugate of the present invention is of formula (Ia). In one embodiment, x is 1. In another embodiment, x is 2. In another embodiment, x is 3. In another embodiment, x is 4.

[0346] In another embodiment, the IL-2 conjugate of the present invention is of formula (Ib). In one embodiment, y is 2. In another embodiment, y is 3. In yet another embodiment, y is 4.

[0347] Another aspect of the present invention is a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention and at least one excipient.

[0348] Preferably, the pharmaceutical composition comprising at least one IL-2 conjugate of the present invention has a pH in the range of pH 3 to pH 8 (including both ends).

[0349] In one embodiment, a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention and at least one excipient is a liquid formulation.

[0350] In another embodiment, a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention and at least one excipient is a dry formulation.

[0351] Such liquid or dry pharmaceutical compositions contain at least one excipient. Excipients used in parenteral formulations may be classified, for example, as buffers, isotonic modifiers, preservatives, stabilizers, anti-adsorption agents, antioxidants, viscosifiers / viscosity enhancing agents, or other adjuvants. However, in some cases, one excipient may have two or three functions. Preferably, the at least one excipient included in the pharmaceutical composition of the present invention is selected from the group consisting of: (i) Buffering agents: Physiologically acceptable buffering agents for maintaining pH within a desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate, chloride, and pyruvate; antacids, such as Mg(OH)2 or ZnCO3, may also be used; (ii) Isotonic modifiers: used to minimize pain that may result from cell damage caused by osmotic pressure differences at injection depots; glycerin and sodium chloride are examples; the effective concentration can be determined by osmotic measurement using the assumed molar osmolality of serum between 285 and 315 mOsmol / kg; (iii) Preservatives and / or antimicrobial agents: Parenteral formulations for multiple doses require the addition of preservatives at sufficient concentrations to minimize the risk of patient infection by injection, and corresponding regulatory requirements have been established; typical preservatives include m-cresol, phenol, methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenylmercury nitrate, thimerosal, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and benzalkonium chloride; (iv) Stabilizers: Stabilization is achieved by enhancing the protein stabilizing power, by destabilizing the denatured state, or by the direct binding of excipients to the protein; stabilizers may be amino acids (e.g., alanine, arginine, aspartic acid, glycine, histidine, lysine, proline), sugars (e.g., glucose, sucrose, trehalose), polyols (e.g., glycerol, mannitol, sorbitol), salts (e.g., potassium phosphate, sodium sulfate), chelating agents (e.g., EDTA, hexaphosphate), ligands (e.g., divalent metal ions (zinc, calcium, etc.)), other salts, or organic molecules (e.g., phenolic derivatives); in addition, oligomers or polymers, such as cyclodextrin, dextran, dendrimers, PEG or PVP, or protamine or HSA may be used; (v) Anti-adsorption agents: Primarily ionic or nonionic surfactants or other proteins or soluble polymers are used to coat the inner surface of the formulation container or to competitively adsorb onto the inner surface; for example, poloxamer (Pluronic F-68), PEG dodecyl ether (Brij 35), polysorbate 20 and 80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA, and gelatin; the concentration and type of excipient selected depends on the action to be avoided, but typically a single layer of surfactant is formed at the interface just above the CMC value; (vi) Antioxidant agents: Antioxidants such as ascorbic acid, ectoin, methionine, glutathione, monothioglycerol, morin, polyethyleneimine (PEI), propyl gallate, and vitamin E; chelating agents such as citric acid, EDTA, hexaphosphate, and thioglycolic acid may also be used; (vii) Thickeners or viscosity enhancers: used to delay the sedimentation of particles in vials and syringes, to facilitate the mixing and resuspension of particles, and to make the suspension easier to inject (i.e., less force on the syringe plunger); suitable thickeners or viscosity enhancers include, for example, carbomer thickeners (e.g., Carbopol 940, Carbopol Ultrez 10), cellulose derivatives (e.g., hydroxypropyl methylcellulose (hypromellose, HPMC) or diethylaminoethylcellulose (DEAE or DEAE-C)), colloidal magnesium silicate (Veegum) or sodium silicate, hydroxyapatite gel, tricalcium phosphate gel, xanthan gum, carrageenan (e.g., Satia gum UTC) 30) Aliphatic poly(hydroxy acids) (e.g., poly(D,L- or L-lactic acid) (PLA) and poly(glycolic acid) (PGA) and their copolymers (PLGA), terpolymers of D,L-lactide, glycolide and caprolactone), poloxamers, hydrophilic poly(oxyethylene) blocks and hydrophobic poly(oxypropylene) blocks for constituting a poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) triblock (e.g., Pluronic®), polyether ester copolymers (e.g., polyethylene glycol terephthalate / polybutylene terephthalate copolymer), sucrose acetate isobutyrate (SAIB), dextran or its derivatives The body is an ABA triblock or AB block copolymer composed of a combination of dextran and PEG, polydimethylsiloxane, collagen, chitosan, vinyl alcohol (PVA) and its derivatives, polyalkylimide, poly(acrylamide-co-diallyldimethylammonium (DADMA)), polyvinylpyrrolidone (PVP), glycosaminoglycans (GAG) (e.g., dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronan), a hydrophobic A block (e.g., polylactide (PLA) or poly(lactide-co-glycolide) (PLGA)) and a hydrophilic B block (e.g., polyethylene glycol (PEG) or polyvinylpyrrolidone); such block copolymers Furthermore, the poloxamers mentioned above may exhibit reverse thermal gelation behavior (a fluid state at room temperature to facilitate administration, and a gel state at temperatures higher than the sol-gel transition temperature at body temperature after injection); (viii) Spreading or diffusing agents: modulate the permeability of connective tissue by hydrolysis of components of the extracellular matrix in the intrafiber space of the tissue (e.g., hyaluronic acid, a polysaccharide found in the intercellular space of connective tissue, though not limited to these); spreading agents, e.g., hyaluronidase, though not limited to these, reduce the viscosity of the extracellular matrix and promote the diffusion of injected drugs; and (ix) Other auxiliary agents: wetting agents, viscosity modifiers, antibiotics, hyaluronidase, etc.; acids and bases such as hydrochloric acid and sodium hydroxide are auxiliary agents necessary for pH adjustment during manufacturing.

[0352] Another aspect of the present invention is a pharmaceutical composition for use as a pharmaceutical, comprising the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or at least one IL-2 conjugate.

[0353] Another aspect of the present invention is a pharmaceutical composition comprising the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or at least one IL-2 conjugate, for use in the treatment of diseases treatable with IL-2.

[0354] Preferably, the disease is cancer. More preferably, the disease is selected from the group consisting of sarcoma, chordoma, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminomas, fetal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, renal cell carcinoma, urothelial carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia, and leukemia. Even more preferable are diseases selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, renal cell carcinoma, urothelial carcinoma, breast cancer, colorectal cancer, gastric cancer, and sarcoma.

[0355] Preferred types of sarcomas include fibrosarcoma, myxosarcoma, leiomyosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endosarcoma, gastrointestinal stromal tumor, lymphangiosarcoma, lymphangioendotheliosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, and rhabdomyosarcoma.

[0356] In one embodiment, the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate, for use in the treatment of a disease treatable by IL-2, is administered to the patient before, concurrently with, or after the administration of one or more further drugs, the one or more further drugs preferably selected from the group consisting of PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, e.g., tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; Toll-like receptor agonists (TLRs), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8, and TLR9; and agonists that stimulate immune-activating receptors, e.g., 41BB (CD137), OX40, ICOS, CD40, CD28, NKG2D, NKp30, NKp44, NKp46, LFA1, CD16, CD64, CD32A, and CD3. In one embodiment, the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate, for use in the treatment of a disease treatable by IL-2, is administered to a patient before, concurrently with, or after the administration of one or more further drugs, the one or more further drugs preferably selected from the group consisting of PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, e.g., tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; and Toll-like receptor agonists (TLRs), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8, and TLR9. The administration of the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate, together with one or more further drugs may be a single event or repeated multiple times.

[0357] In one embodiment, the PD-1 inhibitor is pembrolizumab. In another embodiment, the PD-1 inhibitor is nivolumab.

[0358] In one embodiment, the PD-L1 inhibitor is atezolizumab. In another embodiment, the PD-L1 inhibitor is avelumab. In yet another embodiment, the PD-L1 inhibitor is durvalumab.

[0359] In one embodiment, the CTLA-4 inhibitor is ipilimumab. In another embodiment, the CTLA-4 inhibitor is tremelimumab.

[0360] In another embodiment, the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate, is administered to the patient before, concurrently with, or after CAR-T therapy. Such administration of the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate, in combination with CAR-T therapy may be a single event or repeated multiple times.

[0361] Preferably, the IL-2 conjugate of the present invention for therapeutic use, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate is administered to a mammalian patient, preferably a human patient.

[0362] Another aspect of the present invention is the use of the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate, for the manufacture of a medicament for treating a disease that can be treated by IL-2.

[0363] Preferably, the disease is cancer. More preferably, the disease is selected from the group consisting of sarcoma, chordoma, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminomas, fetal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, renal cell carcinoma, urothelial carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia, and leukemia. Even more preferable are diseases selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, renal cell carcinoma, urothelial carcinoma, breast cancer, colorectal cancer, gastric cancer, and sarcoma.

[0364] Preferred types of sarcomas include fibrosarcoma, myxosarcoma, leiomyosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endosarcoma, gastrointestinal stromal tumor, lymphangiosarcoma, endosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, and rhabdomyosarcoma.

[0365] In one embodiment, the pharmaceutical is administered to the patient before, concurrently with, or after the administration of one or more further drugs, the one or more further drugs preferably selected from the group consisting of PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, e.g., tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; Toll-like receptor agonists (TLRs), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8, and TLR9; and agonists that stimulate immune-activating receptors, e.g., 41BB (CD137), OX40, ICOS, CD40, CD28, NKG2D, NKp30, NKp44, NKp46, LFA1, CD16, CD64, CD32A, and CD3. The administration of the pharmaceutical in this manner with one or more further drugs may be a single event or may be repeated multiple times. In one embodiment, the pharmaceutical is administered to the patient before, concurrently with, or after the administration of one or more further drugs, the one or more further drugs are preferably selected from the group consisting of PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, e.g., tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; and Toll-like receptor agonists (TLRs), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8, and TLR9. The administration of the pharmaceutical in conjunction with one or more further drugs may be a single event or may be repeated multiple times.

[0366] In one embodiment, the PD-1 inhibitor is pembrolizumab. In another embodiment, the PD-1 inhibitor is nivolumab.

[0367] In one embodiment, the PD-L1 inhibitor is atezolizumab. In another embodiment, the PD-L1 inhibitor is avelumab. In yet another embodiment, the PD-L1 inhibitor is durvalumab.

[0368] In one embodiment, the CTLA-4 inhibitor is ipilimumab. In another embodiment, the CTLA-4 inhibitor is tremelimumab.

[0369] In another embodiment, the drug is administered to the patient before, concurrently with, or after CAR-T therapy. Such administration of the drug in combination with CAR-T therapy may be a single event or may be repeated multiple times.

[0370] Preferably, the drug is administered to a mammalian patient, more preferably to a human patient.

[0371] A further aspect of the present invention is a method for treating, controlling, delaying or preventing in a mammalian patient, preferably a human patient, who requires treatment for one or more diseases treatable by IL-2, the method comprising the step of administering to the patient requiring such treatment a therapeutically effective amount of the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the IL-2 conjugate.

[0372] Preferably, one or more diseases that can be treated with IL-2 are cancers. More preferably, the disease is selected from the group consisting of sarcoma, chordoma, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminomas, fetal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, renal cell carcinoma, urothelial carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia, and leukemia. Even more preferable are diseases selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, renal cell carcinoma, urothelial carcinoma, breast cancer, colorectal cancer, gastric cancer, and sarcoma.

[0373] Preferred types of sarcomas include fibrosarcoma, myxosarcoma, leiomyosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endosarcoma, gastrointestinal stromal tumor, lymphangiosarcoma, endosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, and rhabdomyosarcoma.

[0374] In one embodiment, the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the IL-2 conjugate, is administered to a patient before, concurrently with, or after the administration of one or more further drugs, one or more of which are preferably selected from the group consisting of PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, e.g., tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; Toll-like receptor agonists (TLRs), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8, and TLR9; and agonists that stimulate immune-activating receptors, e.g., 41BB (CD137), OX40, ICOS, CD40, CD28, NKG2D, NKp30, NKp44, NKp46, LFA1, CD16, CD64, CD32A, and CD3. Administration of the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing at least one IL-2 conjugate, together with one or more further drugs may be a single event or repeated multiple times. In one embodiment, the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing an IL-2 conjugate, is administered to the patient before, concurrently with, or after the administration of one or more further drugs, the one or more further drugs are preferably selected from the group consisting of PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, e.g., tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; and Toll-like receptor agonists (TLRs), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8, and TLR9. Administration of the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing at least one IL-2 conjugate, together with one or more further drugs, may be a single event or repeated multiple times.

[0375] In one embodiment, the PD-1 inhibitor is pembrolizumab. In another embodiment, the PD-1 inhibitor is nivolumab.

[0376] In one embodiment, the PD-L1 inhibitor is atezolizumab. In another embodiment, the PD-L1 inhibitor is avelumab. In yet another embodiment, the PD-L1 inhibitor is durvalumab.

[0377] In one embodiment, the CTLA-4 inhibitor is ipilimumab. In another embodiment, the CTLA-4 inhibitor is tremelimumab.

[0378] In another embodiment, the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate, is administered to the patient before, concurrently with, or after CAR-T therapy. Such administration of the IL-2 conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate, in combination with CAR-T therapy may be a single event or repeated multiple times.

[0379] An additional aspect of the present invention is a method for administering the IL-2 conjugate, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention, which preferably includes the step of administering the IL-2 conjugate, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention by topical, intra-intestinal, or parenteral administration, by topical application, injection, or infusion, for example, by intra-articular, peri-articular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, intracapsular, intrasacral, intraorbital, intratympanic, intravesical, intracardiac, transtracheal, subepidermal, subcapsular, intraarachnoid, intraspinal, intraventricular, intrasternal injection and infusion, direct delivery to the brain via an implantable device (e.g., an Onmeyer reservoir) that enables delivery of the present invention to brain tissue or cerebral fluid, direct intraventricular injection or infusion, direct intraventricular injection or infusion, injection or infusion into the brain or brain-related region, injection into the subchoroidal space, posterior orbital injection and ophthalmic instillation, etc., via subcutaneous injection.

[0380] In one embodiment, the present invention relates to an IL-2 conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, for use in the treatment of one or more diseases that can be treated by IL-2 via subcutaneous injection. In another embodiment, the present invention relates to an IL-2 conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, for use in the treatment of one or more diseases that can be treated by IL-2 via subcutaneous injection. [Examples]

[0381] material 0.7 kDa PEG maleimide (MeO-dPEG(12)-mal, maleimidyl-N-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)propanamide, CAS 724722-89-8) was obtained from Iris Biotech GmbH, Marktredwitz, Germany. 2kDa PEG-maleimide (Sunbright ME-020-MA, CAS 883993-35-9), 5kDa PEG-maleimide (Sunbright ME-050MA, CAS 883993-35-9), 10kDa PEG-maleimide (Sunbright ME-100MA, CAS 883993-35-9), and 20kDa PEG-maleimide (Sunbright ME-200MA0B, CAS 883993-35-9) were obtained from NOF Europe NV, Grobbendonk, Belgium.

[0382] 10 kDa PEGamine (Sunbright ME-100EA, CAS 80506-64-5) can be obtained from NOF Europe NV, Grobbendonk, Belgium.

[0383] Recombinant human IL-2 (aldesleukin, catalog number AF-200-02) was obtained from Peprotech, Rocky Hill, NJ, USA.

[0384] Microbial transglutaminase (catalog number T001) and MTG inhibitor (catalog number C102) were obtained from Zedira GmbH, Darmstadt, Germany.

[0385] 10kDa Mal-PEG-NH2 (catalog number PHB-943) may be obtained, for example, from Creative PEGWorks, Chapel Hill, NC, USA.

[0386] method [Example 1] Preparation of IL-2 variants The IL-2 variant (mutein) was custom-made, supplied from an external source, and after expressing the protein in Escherichia coli (E. coli), it was purified using standard purification strategies known to those skilled in the art. The following proteins were prepared: JPEG0007857984000060.jpg44166JPEG0007857984000061.jpg49167

[0387] [Example 2] Preparation of 0.7 kDa PEG-IL-2 mutein conjugate 2 Approximately 2 mL of IL-2 mutein 1a, formulated at a concentration of 0.2 mg / mL in 50 mM acetic acid, pH 3, was concentrated via a centrifugation filter to obtain a final volume of 0.47 mL with a concentration of 0.85 mg / mL determined by UV(A280). Conjugation was carried out at approximately pH 7.5 as follows. 0.385 mg of 1a (0.455 mL at 0.85 mg / mL) in 50 mM acetic acid, pH 3 was mixed with 0.36 volume equivalents (164 μL) of 0.5 M sodium phosphate, pH 8, 16 μL of 37 mM acetic acid, 132 mM sodium phosphate, pH 7, and 15.1 mM maleimidyl-N-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)propanamide in 3 molar equivalents (5 μL) of water. The solutions were carefully shaken and allowed to stand at ambient temperature for 15 minutes. The quantitative conversion of 1a to each monoconjugate was confirmed by MS. The conjugate was isolated from the reaction mixture by size exclusion chromatography (SEC) using a GE Healthcare Superdex 200 Increase 10 / 300 GL (24 mL volume) column connected to an Akta system, with a mobile phase of 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, at a flow rate of 0.75 mL / min. One fraction mainly containing the conjugate was concentrated using a centrifugation filter to obtain fraction 2 as a 0.45 mL protein solution with a protein content of 0.22 mg / mL and an approximate purity of 79% as determined by SEC.

[0388] [Example 3] Preparation of 2kDa PEG-IL-2 mutein conjugate 3 Approximately 2 mL of IL-2 mutein 1a, formulated at a concentration of 0.2 mg / mL in 50 mM acetic acid, pH 3, was concentrated via a centrifugation filter to obtain a final volume of 0.34 mL at a concentration of 1.16 mg / mL determined by A280. Conjugation was performed at approximately pH 7 as follows: 0.38 mg of 1a in 50 mM acetic acid, pH 3 (0.33 mL at 1.16 mg / mL) was mixed with 0.36 volume equivalents (118 μL) of 0.5 M sodium phosphate, pH 8, and 24.8 mM 2 kDa PEG-Mal in 20 molar equivalents (20 μL) of water. The solution was carefully shaken and incubated at ambient temperature for 15 minutes. As described in Example 29, page 159 of International Publication No. 2014056923, 0.82 mL of a thiol-functionalized hydrogel suspension with a hydrogel content of 21.8 mg / mL and a thiol content of 3 μmol / mL (corresponding to 5 molar equivalents of thiol per 2 kDa PEG-maleimide) was transferred to a 2 mL PP reactor equipped with PE frit. The reaction mixture was transferred to a reaction vessel and incubated at ambient temperature for 30 minutes with gentle stirring to allow excess PEG-maleimide to bind to the hydrogel. The protein-containing solution was removed, and the protein conjugate was isolated from the conjugation mixture by size exclusion chromatography (SEC) using a GE Healthcare Superdex 75 Increase 10 / 300 GL (volume 24 mL) column connected to an Akta system, with a mobile phase of 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, at a flow rate of 0.75 mL / min. The conjugate-containing fraction was concentrated using a centrifugation filter to obtain 0.37 mL of 2 kDa PEG IL-2 mutein conjugate 3 with a protein concentration of 0.27 mg / mL and a purity of 78% by SEC.

[0389] [Example 4] Preparation of 5kDa PEG-IL-2 mutein conjugate 4 Approximately 2 mL of IL-2 mutein 1a, formulated in 50 mM acetic acid, pH 3, was concentrated via centrifugation to obtain a final volume of 0.36 mL at a concentration of 1.01 mg / mL as determined by A280. Conjugation was performed at approximately pH 7.5 as follows: 0.35 mg of protein (0.35 mL at 1.01 mg / mL) in 50 mM acetic acid, pH 3 was mixed with 0.36 volume equivalents (125 μL) of 0.5 M sodium phosphate, pH 8, and 7.49 mM 5 kDa PEG-maleimide in 3 molar equivalents (9.8 μL, activity-corrected) of water. The solution was carefully shaken and incubated at ambient temperature for 15 minutes. To quench the excess PEG-maleimide, 13.8 mM L-cysteine ​​in 6 molar equivalents (10 μL) of water per 1a was added to the conjugation mixture. The solution was carefully shaken and incubated at ambient temperature for 2.5 hours. The conjugate was isolated from the conjugation mixture by size exclusion chromatography (SEC) using a GE Healthcare Superdex 75 Increase 10 / 300 GL (24 mL volume) column connected to an Akta system, with a mobile phase of 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, at a flow rate of 0.75 mL / min. The fraction mainly containing the conjugate was pooled and concentrated using a centrifugation filter to obtain 0.42 mL of 5 kDa PEG IL-2 mutaine conjugate 4 with a protein concentration of 0.30 mg / mL. The isolated conjugate was analyzed by SEC and RP-HPLC. Quantitative depletion of impurities could not be achieved by the purification step, as shown by RP-HPLC. The approximate purity of conjugate 4 was determined to be 68% by SEC analysis at 215 nm.

[0390] [Example 5] Preparation of 10kDa PEG-IL-2 mutein conjugate 5 Approximately 2 mL of IL-2 mutein 1a formulated at a concentration of 0.2 mg / mL in 50 mM acetic acid, pH 3, was concentrated via a centrifugation filter to obtain a final volume of 0.35 mL at a concentration of 1.03 mg / mL determined by A280. Conjugation was performed at approximately pH 7.5 as follows: 0.34 mg of protein (0.33 mL at 1.03 mg / mL) in 50 mM acetic acid, pH 3 was mixed with 0.36 volume equivalents (120 μL) of 0.5 M sodium phosphate, pH 8, and 13.54 mM 10 kDa PEG-maleimide in 3 molar equivalents (5 μL) of water. The solution was carefully shaken and incubated at ambient temperature for 15 minutes. The conjugate was isolated from the conjugation mixture by size exclusion chromatography (SEC) using a GE Healthcare Superdex 200 Increase 10 / 300 GL (24 mL volume) column connected to an Akta system, with a mobile phase of 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, at a flow rate of 0.75 mL / min. The fraction mainly containing the conjugate was pooled and concentrated using a centrifugation filter to obtain 0.55 mL of 10 kDa PEG IL-2 mutaine conjugate 5 with a protein concentration of 0.22 mg / mL. The isolated conjugate was analyzed by SEC and RP-HPLC. Quantitative depletion of impurities could not be achieved by the purification step. The approximate purity of conjugate 5 was determined to be 57% by SEC analysis at 215 nm, with excess PEG-Mal being the main impurity.

[0391] [Example 6] Preparation of 20kDa PEG-IL-2 mutein conjugate 6 Approximately 2 mL of IL-2 mutein 1a formulated at a concentration of 0.2 mg / mL in 50 mM acetic acid, pH 3, was concentrated via a centrifugation filter to obtain a final volume of 0.33 mL at a concentration of 1.11 mg / mL as determined by A280. Conjugation was carried out at approximately pH 7.5 as follows: 0.35 mg of protein (0.32 mL at 1.11 mg / mL) in 50 mM acetic acid, pH 3 was mixed with 0.36 volume equivalents (115 μL) of 0.5 M sodium phosphate, pH 8, 6 μL of 37 mM acetic acid, 132 mM sodium phosphate, pH 7, and 3 molar equivalents (10 μL) of 6.96 mM 20 kDa PEG-maleimide in water. The solution was carefully shaken and incubated at ambient temperature for 15 minutes. The conjugate was isolated from the conjugation mixture by size exclusion chromatography (SEC) using a GE Healthcare Superdex 200 Increase 10 / 300 GL (24 mL volume) column connected to an Akta system, with a mobile phase of 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, at a flow rate of 0.75 mL / min. The fraction mainly containing the conjugate was pooled and concentrated using a centrifugation filter to obtain 0.67 mL of 20 kDa PEG IL-2 mutaine conjugate 6 with a protein concentration of 0.18 mg / mL. The isolated conjugate was analyzed via SEC and RP-HPLC. Quantitative depletion of impurities could not be achieved by the purification step. The approximate purity of conjugate 6 was determined to be 44% by RP-HPLC analysis at 215 nm, with excess PEG-Mal being the main impurity.

[0392] [Example 7] Preparation of 5kDa PEG-IL-2 mutein conjugate 7 1.1 mL of IL-2 mutein 1c, formulated at a concentration of 0.66 mg / mL in 50 mM acetic acid, pH 3, was mixed with 0.15 volume equivalents (165 μL) of 0.5 M sodium phosphate, pH 8, and 141 μL of 0.6 M hydroxylamine hydrochloride, 0.5 M sodium phosphate, pH 6.4. The resulting reaction mixture was incubated overnight at 25°C. The solution was concentrated using a centrifuge filter to obtain a final volume of 0.67 mL at a concentration of 0.99 mg / mL as determined by A280. Conjugation was performed as follows: 0.65 mg of protein (0.66 mL at 0.99 mg / mL) in 58 mM sodium phosphate, approximately 57 mM hydroxylamine hydrochloride, and 39 mM acetic acid, pH 6.5 was mixed with 81 mM 5 kDa PEG-maleimide in 6 molar equivalents (320 μL) of water. The resulting reaction mixture was incubated at ambient temperature for 2.25 hours. Next, buffer was exchanged using a centrifugation filter to 37 mM acetic acid, 132 mM phosphate, pH 7, to obtain a final volume of 0.75 mL. 1.5 molar equivalents (80 μL) of 81 mM 5 kDa PEG-maleimide in water was added to the reaction solution, and after 3.5 hours, 3 molar equivalents (160 μL) of 81 mM 5 kDa PEG-maleimide in water was added. The resulting reaction mixture was incubated overnight at ambient temperature. The conjugate was isolated from the conjugation mixture by size exclusion chromatography (SEC) using a GE Healthcare Superdex 75 Increase 10 / 300 GL (24 mL) column connected to an Akta system, with a mobile phase of 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, at a flow rate of 0.75 mL / min. The fraction containing the recovered conjugate was concentrated using a centrifugation filter to obtain 0.45 mL of 5 kDa PEG IL-2 mutein 7 at a concentration of 0.25 mg / mL. The isolated conjugate was analyzed by SEC and RP-HPLC. Depletion of impurities could not be quantitatively achieved by the purification step, and due to their presence in the conjugate sample, the approximate purity of conjugate 7 by SEC was 60%.

[0393] [Example 8] Preparation of 10kDa PEG-IL-2 mutein conjugate 8 1.1 mL of IL-2 mutein 1c formulation at 0.66 mg / mL in 50 mM acetic acid, pH 3 was mixed with 0.15 volume equivalents (165 μL) of 0.5 M sodium phosphate, pH 8, and 141 μL of 0.5 M hydroxylamine hydrochloride and 0.05 M sodium phosphate, pH 7. The resulting reaction mixture was incubated overnight at 25°C. The solution was concentrated using a centrifuge filter to obtain a final volume of 0.48 mL at a concentration of 1.42 mg / mL as determined by A280. Conjugation was performed as follows: 0.678 mg of protein (0.48 mL at 1.42 mg / mL) in 64 mM sodium phosphate, 50 mM hydroxylamine hydrochloride, 39 mM acetic acid, pH 6.5-7.0 was mixed with 7.42 mM 10 kDa PEG10-maleimide in 3 molar equivalents (18 μL) of water. The resulting reaction mixture was carefully shaken and incubated at ambient temperature for 15 minutes. An additional 3 molar equivalents (17.9 μL) of 7.42 mM PEG-maleimide in water were added to the conjugation mixture and incubated at ambient temperature for 15 minutes. The conjugate was isolated from the conjugation mixture by size exclusion chromatography (SEC) using a GE Healthcare Superdex 200 Increase 10 / 300 GL (24 mL) column connected to an Akta system, with a mobile phase of 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, at a flow rate of 0.75 mL / min. The recovered fraction was analyzed by RP-HPLC, and the fraction containing the conjugate was pooled and concentrated using a centrifuge filter to obtain 0.57 mL of 10 kDa PEG IL-2 mutein 8 at 0.50 mg / mL. Analysis of the isolated conjugate was performed by SEC and RP-HPLC. Depletion of impurities from the starting material could not be quantitatively achieved by the purification step, and impurities were present in the conjugate sample as shown by RP-HPLC. The approximate purity of conjugate 8 was determined to be 25% by RP-HPLC at 280 nm, with excess PEG-Mal being the main impurity.

[0394] [Example 9] SPR analysis of PEG-IL-2 mutein conjugate The binding kinetics of aldesleukin and bias IL-2 compounds were evaluated using surface plasmon resonance (SPR) spectroscopy with a Biacore instrument (T200, GE Healthcare) using the immobilized extracellular domain of the IL-2 receptor subunit. Briefly, a carboxymethylated dextran biosensor chip (CM5, GE Healthcare) was activated with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions for use. Immobilization of monoclonal mouse anti-human IgG (Fc) antibody was also performed according to the supplier's instructions for use (GE Healthcare, order number BR-1008-39).

[0395] To determine the binding kinetics for IL-2Rα, the following tip preparations were used: Human IL-2 receptor alpha, Fc-Tag (Symansis, New Zealand) was diluted to approximately 0.67 μg / mL in HBS-EP running buffer (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4), fixed at a flow rate of 10 μL / min for 60 seconds, and 80–100 response units (RUs) were achieved.

[0396] To determine the binding kinetics to IL-2Rβ, the following tip preparations were used: Human IL-2 receptor beta, Fc-Tag (Symansis, New Zealand) was diluted to approximately 0.80 μg / mL in HBS-EP running buffer (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4), fixed at a flow rate of 10 μL / min for 60 seconds, and 140–180 response units (RUs) were achieved.

[0397] To determine the binding kinetics to the IL-2Rαβ complex, the following tip preparations were used: human IL-2 receptor alpha, Fc-Tag (Symansis, New Zealand), and human IL-2 receptor beta, Fc-Tag (Symansis, New Zealand) were mixed and diluted to approximately 1.33 μg / mL each in HBS-EP running buffer (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4). The mixtures were fixed at a flow rate of 10 μL / min for 60 seconds, achieving 360–440 response units (RUs).

[0398] For kinetic measurements of all three receptor subunit configurations (setups), aldesleukin or Bias IL-2 compounds were analyzed using multi-cycle kinetics, and therefore injected into HBS-EP running buffer at 25°C at at least five different concentrations (flow rate 30 μL / min, contact time 120 sec, dissociation time 600 sec). Double-referenced data (data subtracted from samples with only the reference flow cell and buffer) were analyzed kinetically via a 1:1 binding model or steady-state analysis, if possible (Biacore T200 Evaluation Software, version 3.1), K D , and if possible, k a and k d The following was determined. Regeneration after each cycle was performed with 3M MgCl2 at a flow rate of 30 μL / min for 30 seconds.

[0399] The obtained data is summarized in Table 1.

[0400] [Table 1]

[0401] [Example 10] Calculation of bias ratio As used herein, the term “bias IL-2” means the K of bias IL-2 relative to IL-2Rβ.D K for the bias IL-2 with respect to IL-2Rαβ D The ratio of aldesleukin to IL-2Rβ is K D K for aldesleukin against IL-2Rαβ D This refers to a modified IL-2 with a ratio greater than the given value. This is described by the following formula:

number

number

[0402] Using the above formula, the following ratios were calculated and summarized in Table 2.

[0403] [Table 2]

[0404] [Example 11] Preparation of 10kDa PEG-IL-2 conjugate 9 Recombinant human IL-2 is buffered to 0.2 M Tris / HCl, pH 7.5 using a HiTrap desalting column connected to an Akta system. Then, IL-2 is enzymatically PEGylated using microbial transglutaminase (MTG) according to the procedure described in Sato et al., Bioconjugate Chem 2001, 12(5), 701-710. 5 μM IL-2 is incubated in 0.2 M Tris / HCl, pH 7.5 at 25°C for 12 hours in the presence of 1.25 mM 10 kDa PEGamine (10 kDa methoxy-PEG-(CH2)2-NH2) and 0.2 units / mL of MTG. The 10kDa PEG IL-2 conjugate (9) was purified from the reaction mixture by size exclusion chromatography using a GE Healthcare Superdex 200 Increase 10 / 300 GL column connected to an Akta system, with a mobile phase of 10mM HEPES, 150mM sodium chloride, 0.05% Tween 20, pH 7.4, at a flow rate of 0.75 mL / min.

[0405] [Example 12] Preparation of 10kDa PEG-IL-2 conjugate 10 Recombinant human IL-2 (2.7 mg / mL in 50 mM acetate, pH 3.0) was mixed with 1 volume equivalent of 140 mM HEPES, 300 mM NaCl, 6 mM sodium EDTA, 0.1% Tween 20, pH 8.2 to obtain 1.35 mg / mL of IL-2 in 70 mM HEPES, 150 mM NaCl, 3 mM sodium EDTA, 0.05% Tween 20, 25 mM AcOH, pH 7.4. IL-2 was enzymatically PEGylated using microbial transglutaminase (MTG). For this purpose, IL-2 at a concentration of 0.1 mg / mL was incubated at 37°C for 4 hours in 70 mM HEPES, 150 mM NaCl, 3 mM sodium EDTA, 0.05% Tween 20, 25 mM AcOH, pH 7.4, in the presence of 1 mM 10 kDa PEGamine (10 kDa methoxy-PEG-(CH2)2-NH2) and 1 U / mL MTG. Subsequently, an MTG inhibitor was added to a final concentration of 0.5 mM, and the reaction mixture was incubated for a further 30 minutes. The 10kDa PEG IL-2 conjugate (10) was purified from the reaction mixture by size exclusion chromatography using a GE Healthcare Superdex 200 Increase 10 / 300 GL column connected to an Akta system, with 10mM HEPES, 150mM sodium chloride, 0.05% Tween 20, pH 7.4 as the mobile phase, at a flow rate of 0.75 mL / min.

[0406] [Example 13] Preparation of Bias IL-2 mutain polymer prodrug 11 PEG IL-2 mutaine is buffered with 100 mM sodium borate pH 9 and concentrated to a concentration of approximately 2.5 mg / mL IL-2 equivalent. A 5-fold molar excess of 40 kDa mPEG-linker reagent (as described in Example 2 of International Patent Publication No. 2016079114) is dissolved in an equal volume of water compared to the protein solution used for the conjugation reaction. The protein solution (2.5 mg / mL IL-2 equivalent) and the 40 kDa mPEG-linker solution (32 g / L) are mixed and incubated at 14-16°C for 2 hours. Biased IL-2 mutaine polymer prodrug 11 is isolated by CIEX and analyzed by SEC.

[0407] [Example 14] Preparation of the novel conjugate 12 Dissolve the 40kDa mPEG-linker reagent (as described in Example 2 of International Patent Publication No. 2016079114) in water to obtain a 32g / L solution. Dissolve 10kDa Mal-PEG-NH2 in 0.1M sodium phosphate, 6mM sodium EDTA, pH 7.4 to a final concentration of 1mM. Mix both solutions in a 1:1 volume ratio and incubate at ambient temperature for 2 hours. Then, add 0.5 volume equivalents (relative to the volume of the reaction mixture of 40kDa mPEG-linker reagent and 10kDa Mal-PEG-NH2) of 2mg / mL IL-2 mutaine 1a solution in 50mM sodium phosphate, 3mM sodium EDTA, pH 7.4 to the reaction mixture and incubate at ambient temperature for 1 hour. The 40+10kDa PEG IL-2 mutaine conjugate 12 is isolated from the reaction mixture by cation exchange chromatography and analyzed by size exclusion chromatography.

[0408] [Table 3] This disclosure includes the following embodiments. [1] Equation (Ia) or (Ib) [1] JPEG0007857984000067.jpg41161 (In the formula, -D is the bias IL-2 portion, and the bias IL-2 portion includes the IL-2 portion, and with respect to the bias IL-2 portion, K of the bias IL-2 with respect to IL-2Rβ D K for the bias IL-2 with respect to IL-2Rαβ D The ratio of aldesleukin to IL-2Rβ is K D K for aldesleukin against IL-2Rαβ D Larger than the ratio, -L 1 - is a linker portion covalently and reversibly bonded to -D. -L 2 - represents a chemical bond or spacer portion. -Z is a polymer portion or a substituted fatty acid portion. x is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. (y is an integer selected from the group consisting of 2, 3, 4, and 5.) IL-2 conjugate or a pharmaceutically acceptable salt thereof. [2] Bias IL-2 portion-D is, (a) Modifying parts M that may be the same or different mod at least one stable bond, or (b) at least one amino acid mutation, or (c) at least one deletion, Any combination of (a), (b), and (c) An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to Embodiment 1, comprising an IL-2 portion containing the IL-2 portion. [3] The IL-2 conjugate or pharmaceutically acceptable salt thereof according to Embodiment 1 or 2, wherein the IL-2 moiety comprises at least one amino acid mutation at position K34, R37, M38, T40, F41, K42, F43, Y44, E61, or L71 based on SEQ ID NO: 2. [4] An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 3, wherein the IL-2 moiety comprises at least one amino acid mutation selected from the group consisting of K34C, R37C, M38C, T40C, F41C, K42C, F43C, Y44C, E61C, and L71C based on SEQ ID NO: 2. [5] An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 4, wherein the IL-2 moiety has the sequence of Sequence ID No. 2 and contains the R37C mutation. [6] IL-2 part is modified part M mod An IL-2 conjugate according to any one of Embodiments 1 to 5, or a pharmaceutically acceptable salt thereof, comprising a combination of at least one stable bond and at least one amino acid mutation. [7] Part M mod An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 2 to 6, wherein is bound to cysteine. [8] The IL-2 portion has the sequence of SEQ ID NO: 2, contains the R37C mutation, and is part M mod The IL-2 conjugate or pharmaceutically acceptable salt thereof according to any of Embodiments 2 to 7, which is conjugated to the sulfur of cysteine ​​at position R37C. [9] The IL-2 conjugate according to any one of Embodiments 1 to 8 or a pharmaceutically acceptable salt thereof, wherein the IL-2 conjugate is of formula (Ia) and x is 1.

[10] M mod An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 2 to 9, wherein the polymer portion is the polymer portion.

[11] M mod However, equation (A-1) [2] JPEG0007857984000068.jpg20168 (In the formula, -FG- is a bond, -SP- is the spacer part. -POL is a polymer.) It belongs to An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of Embodiments 2 to 10.

[12] M mod However, equation (A-1a) [3] JPEG0007857984000069.jpg26152 (In the formula, The dashed lines marked with asterisks indicate the binding of the side chain of the amino acid residue in the IL-2 region to the sulfur. b1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. b2 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. (b3 is an integer in the range of 12 to 22700) An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 2 to 11.

[13] - An IL-2 conjugate or pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 12, wherein Z is a polymer portion.

[14] -Z is given by equation (A) [4] JPEG0007857984000070.jpg47146 (In the formula, -BP 1 <, -BP 2 <, -BP 3 <But -N< and -C(R 8 Selected independently from the group consisting of )<, R 8 However, H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Selected from the group consisting of alkynnyls, -P 1 、-P 2 、-P 3 、-P 4 However, each is independently a PEG-based chain containing at least 40% PEG and having a molecular weight in the range of 3 to 40 kDa. -C 1 -、-C 2 -but, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Selected independently from the group consisting of alkynnyls, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl, one or more R, either the same or different 9 Replaced by optional means, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) 10 )-,-S(O) 2 N(R 10 )-,-S(O)N(R 10 )-,-S(O) 2 -, -S(O)-, -N(R 10 )S(O) 2 N(R 10a )-, -S-, -N(R 10 )-, -OC(OR 10 )(R 10a )-,-N(R 10 )C(O)N(R 10a )-, and -OC(O)N(R 10 Interrupted by one or more elements selected from the group consisting of )-, Each T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 A molecule is independently selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl, and each T is the same or different R 9 It is independently and arbitrarily replaced by, Each R 9 However, halogen, -CN, oxo (=O), -COOR 11 , -OR 11 , -C(O)R 11 ,-C(O)N(R 11 R 11a ), -S(O) 2 N(R 11 R 11a ), -S(O)N(R 11 R 11a ), -S(O) 2 R11 ,-S(O)R 11 , -N(R 11 )S(O) 2 N(R 11a R 11b ), -SR 11 , -N(R 11 R 11a ), -NO 2 ,-OC(O)R 11 , -N(R 11 )C(O)R 11a , -N(R 11 )S(O) 2 R 11a , -N(R 11 )S(O)R 11a , -N(R 11 )C(O)OR 11a , -N(R 11 )C(O)N(R 11a R 11b ), -OC(O)N(R 11 R 11a ), and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 The alkyl group is optionally substituted with one or more halogens, either the same or different. Each R 10 、R 10a 、R 11 、R 11a , and R 11b However, -H and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 (The alkyl group is optionally substituted with one or more halogens, either the same or different.) An IL-2 conjugate or pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 13, including the portion thereof.

[15] C in equation (A) 1 and C 2 However, equation (Aa) [5] JPEG0007857984000071.jpg37146 (In the formula, The dashed line with a star is BP 1 This shows a binding to, Each of the unmarked dashed lines is BP 2 or BP 3 This shows a binding to, q1 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8. q2 is selected from the group consisting of 1, 2, 3, 4, and 5. q3 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8. (q4 is selected from the group consisting of 1, 2, and 3.) It belongs to The IL-2 conjugate described in Embodiment 14 or a pharmaceutically acceptable salt thereof.

[16] P in equation (A) 1 、P 2 、P 3 , and P 4 However, equation (Ab) is independent of each other. [6] JPEG0007857984000072.jpg40123 (In the formula, The dashed line indicates the connection to the remainder of -Z. m is 0 or 1, p is an integer in the range of 70 to 900. (q is selected from the group consisting of 1, 2, 3, 4, 5, and 6), An IL-2 conjugate or a pharmaceutically acceptable salt thereof as described in Embodiment 14 or 15.

[17] BP of equation (A) 1 An IL-2 conjugate or pharmaceutically acceptable salt thereof according to any one of embodiments 14 to 16, wherein -N<

[18] BP of formula (A) 2 and BP 3 However, all of them are -CH<, the IL-2 conjugate or pharmaceutically acceptable salt thereof according to any one of embodiments 14 to 17.

[19] -Z is given by equation (Ac) [7] JPEG0007857984000073.jpg81159 (In the formula, p1, p2, p3, and p4 are independent integers in the range of 70 to 900. An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 18, including the portion thereof.

[20] -L1 - The IL-2 conjugate or pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 19, wherein - is bound to an amino acid residue of the IL-2 moiety.

[21] -L 1 - An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 20, wherein - is bound to an amino acid residue of the IL-2 moiety selected from the group consisting of cysteine, methionine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, glutamine, and arginine.

[22] L 1 - is bound to a lysine residue selected from the group consisting of K7, K8, K31, K34, K42, K47, K48, K53, K63, K75, and K96 based on SEQ ID NO: 2, the IL-2 conjugate according to any one of Embodiments 1 to 21 or a pharmaceutically acceptable salt thereof.

[23] -L 1 - is equation (IX-a) [8] JPEG0007857984000074.jpg51134 (In the formula, The dashed lines marked with an asterisk indicate the bond to nitrogen in -D, and the unmarked dashed lines indicate -L 2 Shows the bond to -Z, n is 0, 1, 2, 3, or 4, =Y 1 However, selected from the group consisting of =O and =S, -Y 2 - is selected from the group consisting of -O- and -S-, -Y 3 -、-Y 5 - is independently selected from the group consisting of -O- and -S-, -Y 4 -but, -O-, -NR 5 -, and -C(R 6 R 6a Selected from the group consisting of )-, -R 3 、-R 5 、-R 6 、-R 6a However, these are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl, -R 4 The selected compound is from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl. -W- is C 3-10 Cycloalkyl, 8-30 membered carbopolycyclyl, 3-10 membered heterocyclyl, -C(O)-, -C(O)N(R) 7 )-, -O-, -S-, and -N(R 7 C is optionally interrupted by one or more elements selected from the group consisting of )- 1-20 Selected from the group consisting of alkyl groups, -Nu is -N(R 7 R 7a ), -N(R 7 OH), -N(R 7 )-N(R 7a R 7b ), -S(R 7 ), -COOH, [9] JPEG0007857984000075.jpg96155 A nucleophile selected from the group consisting of, -Ar-,

[10] JPEG0007857984000076.jpg58143JPEG0007857984000077.jpg62143 (In the formula, The dashed line is -L 1 - indicates the bond to the remainder, -Z 1 -but -O-, -S-, and -N(R 7 Selected from the group consisting of )-, -Z 2 -but, -N(R 7 )- and, -R 7 、-R 7a 、-R 7b However, -H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 (Selected independently from a group consisting of alkinyls) (Selected from the group consisting of) It is, -L 1 -However, it can be further replaced by an optional choice. An IL-2 conjugate or a pharmaceutically acceptable salt thereof as described in any of Embodiments 1 to 22.

[24] -L 1 -But, equation (IX-c)

[11] JPEG0007857984000078.jpg47151 (In the formula, The dashed line marked with an asterisk indicates the bond of -D to nitrogen. The dashed line without a mark is -L 2 Shows the bond to -Z, (s1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.) An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of embodiments 1 to 23.

[25] -L 2 - is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-,-S(O) 2 N(R y1 )-,-S(O)N(R y1 )-,-S(O) 2 -, -S(O)-, -N(R y1 )S(O) 2 N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-,-N(R y1 )C(O)N(R y1a )-,-OC(O)N(R y1 )-、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Selected from the group consisting of alkynnyls, where -T-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl, one or more -R, which are the same or different y2 Replaced by optional means, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) y3 )-,-S(O) 2 N(R y3 )-,-S(O)N(R y3 )-,-S(O) 2 -, -S(O)-, -N(R y3 )S(O) 2 N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(R y3a )-,-N(R y3 )C(O)N(R y3a )-, and -OC(O)N(R y3 Interrupted by one or more elements selected from the group consisting of )-, -R y1 and -R y1a However, -H, -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 They are independently selected from a group consisting of alkynnyls, where -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl, one or more of the same or different -R y2 Replaced by optional means, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) y4 )-,-S(O) 2 N(R y4 )-,-S(O)N(R y4 )-,-S(O) 2 -, -S(O)-, -N(R y4 )S(O) 2 N(R y4a )-, -S-, -N(R y4 )-, -OC(OR y4 )(R y4a )-,-N(R y4 )C(O)N(R y4a )-, and -OC(O)N(R y4 Interrupted by one or more elements selected from the group consisting of )-, Each T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 A molecule is independently selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl, and each T is the same or different -R y2 It is independently and arbitrarily replaced by, Each -R y2 However, halogen, -CN, oxo (=O), -COOR y5 , -OR y5 , -C(O)R y5 ,-C(O)N(R y5 R y5a ), -S(O) 2 N(R y5 R y5a ), -S(O)N(R y5 R y5a ), -S(O) 2 R y5 ,-S(O)R y5 , -N(R y5 )S(O) 2 N(R y5a R y5b ), -SR y5 , -N(R y5 R y5a ), -NO 2 ,-OC(O)R y5 , -N(R y5 )C(O)R y5a , -N(R y5 )S(O) 2 R y5a , -N(R y5 )S(O)R y5a , -N(R y5 )C(O)OR y5a , -N(R y5 )C(O)N(R y5a R y5b ), -OC(O)N(R y5 R y5a ), and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 The alkyl group is optionally substituted with one or more halogens, either the same or different. Each -R y3 、-R y3a 、-R y4 、-R y4a 、-R y5 、-R y5a , and -Ry5b However, -H and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 The alkyl group is optionally substituted with one or more halogens, one or more of the same or different halogens. An IL-2 conjugate or a pharmaceutically acceptable salt thereof as described in any of Embodiments 1 to 24.

[26] -L 2 -but -O-, -T-, and -C(O)N(R y1 )- is optionally interrupted by one or more elements selected independently of C 1-20 It is an alkyl chain, C 1-20 The alkyl chain consists of -OH, -T, and -C(O)N(R y6 R y6a ) is optionally replaced by one or more elements independently selected from -R y1 、-R y6 、-R y6a However, H and C 1-4 Independently selected from the group consisting of alkyl groups, where T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 25, selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl.

[27] -L 2 -But, equation (IX-e)

[12] JPEG0007857984000079.jpg21153 (In the formula, The dashed line with the star symbol is -L 1 - indicates a bond to -, The unmarked dashed lines indicate connections to -Z. (s2 is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.) An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 26.

[28] A pharmaceutical composition comprising an IL-2 conjugate or a pharmaceutically acceptable salt thereof as described in any of Embodiments 1 to 27, and at least one excipient.

[29] An IL-2 conjugate according to any one of Embodiments 1 to 27 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to Embodiment 28, for use as a pharmaceutical.

[30] An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any of Embodiments 1 to 27, or a pharmaceutical composition according to Embodiment 28, for use in the treatment of a disease that can be treated with IL-2.

[31] Diseases that can be treated with IL-2 include sarcomas, chordomas, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminomas, fetal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, gastric cancer, non-small cell lung cancer, and small cell carcinoma. IL-2 conjugate or a pharmaceutically acceptable salt thereof or pharmaceutical composition for use as described in Embodiment 30, selected from the group consisting of alveolar lung cancer, bladder cancer, renal cell carcinoma, urothelial carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia, and leukemia.

[32] A method for treating, controlling, delaying or preventing in a mammalian patient, preferably a human patient, who is in need of treatment for one or more diseases that can be treated with IL-2, the method comprising administering to the patient in need a therapeutically effective amount of an IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any of Embodiments 1 to 27, or a pharmaceutical composition according to Embodiment 28.

[33] The method according to Embodiment 32, wherein the disease is selected from the group consisting of sarcoma, chordoma, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, swe...

Claims

1. Equation (Ia) 【Chemistry 1】 (In the formula, -D is the bias IL-2 portion, and the bias IL-2 portion includes the IL-2 portion, and with respect to the bias IL-2 portion, K of the bias IL-2 with respect to IL-2Rβ D K for the bias IL-2 with respect to IL-2Rαβ D The ratio of aldesleukin to IL-2Rβ is K D K for aldesleukin against IL-2Rαβ D Larger than the ratio, The aforementioned -D is the modifying portion M mod This includes a combination of at least one stable bond and at least one amino acid mutation, The at least one amino acid mutation is a mutation at position K34, R37, M38, T40, F41, K42, F43, Y44, E61, or L71 based on SEQ ID NO: 2, or at the corresponding position in its homolog or variant. The aforementioned at least one portion M mod It is linked to the aforementioned mutant amino acid, M mod This is a PEG-based polymer moiety having a molecular weight in the range of 1 kDa to 40 kDa. -L 1 - is a linker portion covalently and reversibly bonded to -D. -L 2 - is a chemical bond or a spacer moiety, -Z is a polymer moiety having a molecular weight in the range of 1 kDa to 1000 kDa. x is 1) IL-2 conjugate or a pharmaceutically acceptable salt thereof.

2. The IL-2 conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the at least one amino acid mutation replaces a native amino acid with an amino acid residue selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, lysine, serine, threonine, tryptophan, and tyrosine.

3. The IL-2 conjugate or a pharmaceutically acceptable salt thereof according to claim 2, wherein a natural amino acid is replaced by cysteine.

4. An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the IL-2 moiety comprises at least one amino acid mutation selected from the group consisting of K34C, R37C, M38C, T40C, F41C, K42C, F43C, Y44C, E61C, and L71C based on SEQ ID NO: 2, or the corresponding position in its homolog or variant.

5. Part M mod An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the IL-2 conjugate is bound to cysteine.

6. M mod However, equation (A-1) 【Chemistry 2】 (In the formula, -FG- is a bond, -SP- is the spacer part. -POL is a PEG-based polymer. It belongs to An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5.

7. M mod However, equation (A-1a) 【Transformation 3】 (In the formula, The dashed lines marked with asterisks indicate the binding of the side chain of the amino acid residue in the IL-2 region to the sulfur. b1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. b2 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. (b3 is an integer approximately 112.) An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6.

8. -Z has a molecular weight in the range of 5 kDa to 120 kDa, the IL-2 conjugate according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. -Z has a molecular weight in the range of 15 kDa to 80 kDa, the IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8.

10. -Z is given by equation (A). 【Chemistry 4】 (In the formula, -BP 1 <, -BP 2 <, -BP 3 <wherein, -N< and -C(R 8 ) are independently selected from the group consisting of, R 8 However, H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Selected from the group consisting of alkynnyls, -P 1 , -P 2 , -P 3 , -P 4 However, each is independently a PEG-based chain containing at least 40% PEG and having a molecular weight in the range of 3 to 40 kDa. -C 1 -, -C 2 -but, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Selected independently from the group consisting of alkynnyls, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl, one or more R, either the same or different 9 Replaced by choice by C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) 10 )-,-S(O) 2 N(R 10 )-,-S(O)N(R 10 )-,-S(O) 2 -, -S(O)-, -N(R 10 )S(O) 2 N(R 10a )-, -S-, -N(R 10 )-, -OC(OR 10 )(R 10a )-,-N(R 10 )C(O)N(R 10a )-, and -OC(O)N(R 10 Interrupted by one or more elements selected from the group consisting of )-, Each T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 A molecule is independently selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl, and each T is the same or different R 9 It is independently and arbitrarily replaced by, Each R 9 is independently selected from the group consisting of halogen, -CN, oxo (=O), -COOR 11 , -OR 11 , -C(O)R 11 , -C(O)N(R 11 R 11a ), -S(O) 2 N(R 11 R 11a ), -S(O)N(R 11 R 11a ), -S(O) 2 R 11 , -S(O)R 11 , -N(R 11 ), -N(R 2 R 11a ), -SR 11b , -N(R 11 R 11 ), -NO 11a ), -OC(O)R​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ each R 10 、R 10a 、R 11 、R 11a 、and R 11b is independently selected from the group consisting of -H and C 1-6 alkyl, where C 1-6 alkyl is optionally substituted by one or more of the same or different halogens) An IL-2 conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, including the portion thereof.

11. C in equation (A) 1 and C 2 However, equation (Aa) 【Transformation 5】 (In the formula, The dashed line with a star is BP 1 This shows a binding to, Each of the unmarked dashed lines is BP 2 or BP 3 This shows a binding to, q1 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8. q2 is selected from the group consisting of 1, 2, 3, 4, and 5. q3 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8. (q4 is selected from the group consisting of 1, 2, and 3.) It belongs to The IL-2 conjugate or a pharmaceutically acceptable salt thereof according to claim 10.

12. P in equation (A) 1 , P 2 , P 3 , and P 4 However, equation (Ab) is independent of each other. 【Transformation 6】 (In the formula, The dashed line indicates the connection to the remainder of -Z. m is 0 or 1, p is an integer in the range of 70 to 900. (q is selected from the group consisting of 1, 2, 3, 4, 5, and 6) The IL-2 conjugate or a pharmaceutically acceptable salt thereof according to claim 10 or 11.

13. BP of equation (A) 1 An IL-2 conjugate or pharmaceutically acceptable salt thereof according to any one of claims 10 to 12, wherein -N<

14. BP of equation (A) 2 and BP 3 The IL-2 conjugate or pharmaceutically acceptable salt thereof according to any one of claims 10 to 13, wherein all are -CH<.

15. -Z is the expression (Ac). 【Transformation 7】 (In the formula, p1, p2, p3, and p4 are independent integers in the range of 70 to 900. An IL-2 conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, including the portion thereof.

16. -L 1 - is bonded to an amino acid residue of the IL-2 moiety, the IL-2 conjugate according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof.

17. -L 1 - is bound to an amino acid residue of the IL-2 moiety selected from the group consisting of cysteine, methionine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, glutamine, and arginine, according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof.

18. L 1 - is bound to a lysine residue selected from the group consisting of K7, K8, K31, K34, K42, K47, K48, K53, K63, K75, and K96 based on SEQ ID NO: 2, the IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 17.

19. -L 1 - However, equation (IX-a) 【Transformation 8】 (In the formula, The dashed lines marked with an asterisk indicate the bond to nitrogen in -D, and the unmarked dashed lines indicate -L 2 Shows the bond to -Z, n is 0, 1, 2, 3, or 4, =Y 1 However, selected from the group consisting of =O and =S, -Y 2 - is selected from the group consisting of -O- and -S-, -Y 3 - is selected from the group consisting of -O- and -S-, -Y 4 -but, -O-, -NR 5 -, and -C(R 6 R 6a Selected from the group consisting of )-, =Y 5 is selected from the group consisting of =O and =S, -R 3 , -R 5 , -R 6 , -R 6a However, these are independently selected from the group consisting of -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl, -R 4 The selected compound is from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and 3,3-dimethylpropyl. -W- is C 3-10 Cycloalkyl, 8-30 membered carbopolycyclyl, 3-10 membered heterocyclyl, -C(O)-, -C(O)N(R) 7 )-, -O-, -S-, and -N(R 7 C is optionally interrupted by one or more elements selected from the group consisting of )- 1-20 Selected from the group consisting of alkyl groups, -Nu is -N(R 7 R 7a ), -N(R 7 OH), -N(R 7 )-N(R 7a R 7b ), -S(R 7 ), -COOH, 【Chemistry 9】 A nucleophile selected from the group consisting of, -Ar-, 【Chemistry 10】 (In the formula, The dashed line is -L 1 - indicates the bond to the remainder, -Z 1 -but -O-, -S-, and -N(R 7 Selected from the group consisting of )-, -Z 2 -but, -N(R 7 )- and, -R 7 , -R 7a , -R 7b However, -H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 (Selected independently from a group consisting of alkinyls) (Selected from the group consisting of) It is, -L 1 -However, it can be further replaced by an optional choice. An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 18.

20. -L 1 -But, equation (IX-c) 【Chemistry 11】 (In the formula, The dashed line marked with an asterisk indicates the bond of -D to nitrogen. The dashed line without a mark is -L 2 Shows the bond to -Z, (s1 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.) An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 19.

21. -L 2 - is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-,-S(O) 2 N(R y1 )-,-S(O)N(R y1 )-,-S(O) 2 -, -S(O)-, -N(R y1 )S(O) 2 N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-,-N(R y1 )C(O)N(R y1a )-,-OC(O)N(R y1 )-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Selected from the group consisting of alkynnyls, where -T-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl, one or more -R, which are the same or different y2 Replaced by choice by C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) y3 )-,-S(O) 2 N(R y3 )-,-S(O)N(R y3 )-,-S(O) 2 -, -S(O)-, -N(R y3 )S(O) 2 N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(R y3a )-,-N(R y3 )C(O)N(R y3a )-, and -OC(O)N(R y3 Interrupted by one or more elements selected from the group consisting of )-, -R y1 and -R y1a However, -H, -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 They are independently selected from a group consisting of alkynnyls, where -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl, one or more -R, which are the same or different y2 Replaced by choice by C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkinyl is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R) y4 )-,-S(O) 2 N(R y4 )-,-S(O)N(R y4 )-,-S(O) 2 -, -S(O)-, -N(R y4 )S(O) 2 N(R y4a )-, -S-, -N(R y4 )-, -OC(OR y4 )(R y4a )-,-N(R y4 )C(O)N(R y4a )-, and -OC(O)N(R y4 Interrupted by one or more elements selected from the group consisting of )-, Each T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 A molecule is independently selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl, and each T is the same or different -R y2 It is independently and arbitrarily replaced by, Each -R y2 However, halogen, -CN, oxo (=O), -COOR y5 , -OR y5 , -C(O)R y5 ,-C(O)N(R y5 R y5a ), -S(O) 2 N(R y5 R y5a ), -S(O)N(R y5 R y5a ), -S(O) 2 R y5 ,-S(O)R y5 , -N(R y5 )S(O) 2 N(R y5a R y5b ), -SR y5 , -N(R y5 R y5a ), -NO 2 -OC(O)R y5 , -N(R y5 )C(O)R y5a , -N(R y5 )S(O) 2 R y5a , -N(R y5 )S(O)R y5a , -N(R y5 )C(O)OR y5a , -N(R y5 )C(O)N(R y5a R y5b ), -OC(O)N(R y5 R y5a ), and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 The alkyl group is optionally substituted with one or more halogens, either the same or different. Each -R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a , and -R y5b However, -H and C 1-6 Independently selected from the group consisting of alkyls, where C 1-6 The alkyl group is optionally substituted with one or more halogens, one or more of the same or different halogens. An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20.

22. -L 2 -but -O-, -T-, and -C(O)N(R y1 )- is optionally interrupted by one or more elements selected independently of C 1-20 It is an alkyl chain, C 1-20 The alkyl chain consists of -OH, -T, and -C(O)N(R y6 R y6a ) is optionally replaced by one or more elements independently selected from -R y1 , -R y6 , -R y6a However, H and C 1-4 Independently selected from the group consisting of alkyl groups, where T is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 An IL-2 conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 21, selected from the group consisting of cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl.

23. -L 2 -But, equation (IX-e) 【Chemistry 12】 (In the formula, The dashed line with the star symbol is -L 1 - indicates a bond to -, The unmarked dashed lines indicate connections to -Z. (s2 is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.) An IL-2 conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 22.

24. A pharmaceutical composition comprising an IL-2 conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 23, and at least one excipient.

25. A pharmaceutical composition according to claim 24, for use as a pharmaceutical.

26. The pharmaceutical composition according to claim 24, for use in treating a disease that can be treated by IL-2.

27. Diseases that can be treated with IL-2 include sarcoma, chordoma, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminomas, fetal carcinoma, Wilms' tumor, cervical cancer, and sperm A pharmaceutical composition according to claim 26, selected from the group consisting of tumorigenesis, gastric cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, renal cell carcinoma, urothelial carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia, and leukemia.

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