Il-2 conjugates

Abstract

The present invention relates to IL-2 conjugates, pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising the IL-2 conjugates or pharmaceutically acceptable salts thereof, and uses thereof.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980022172.X, with an application date of March 27, 2019, a priority date of March 28, 2018, and an invention name of “IL-2 Conjugate”. Technical Field

[0002] The present invention relates to an IL-2 conjugate, a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the IL-2 conjugate or a pharmaceutically acceptable salt thereof, and uses thereof. Background Art

[0003] In healthy people, the immune system can often distinguish between healthy cells and cancerous cells. Once a designated cell is determined to be cancerous, the immune system usually eliminates it. However, when the immune system is damaged or defeated, such as by acute or chronic defects, cancer develops because the damaged immune system cannot distinguish and then eliminate cancer cells. In cancer patients, administering immunomodulatory proteins to the patient can help activate the patient's immune system, thereby enhancing the immune system's ability to eliminate cancer cells. In this way, further growth of the cancer is slowed, the potential spread of the cancer is inhibited, or even the cancer is eliminated.

[0004] One such immunomodulatory protein used to treat patients suffering from certain cancers is interleukin-2. Interleukin-2 (IL-2) is a naturally occurring cytokine with activity as a stimulator of natural killer cell (NK cell) and T cell proliferation and functional activity. IL-2 plays a key role in the generation, differentiation, survival and homeostasis of immune effector cells. IL-2 is synthesized by activated CD4+ helper T cells, and IL-2 can modulate immune responses for either immunity or tolerance through different receptor interactions.

[0005] IL-2 exerts its effects by binding to the IL-2 receptor (IL-2R). The association of the α-(CD25), β-(CD122), and common γ-(γc, CD132) subunits generates the trimeric, high-affinity IL-2R. The dimeric, intermediate-affinity IL-2Rβγ, composed of β- and γ-subunits, binds IL-2 with 50-fold lower affinity. CD25 is not required for IL-2 signaling but confers high affinity to the trimeric receptor, whereas the β- and γ-subunits mediate signal transduction. IL-2Rβγ is expressed on NK cells, monocytes, macrophages, and resting CD4+ and CD8+ T cells, whereas IL-2Rαβγ is transiently induced on activated T and NK cells and constitutively expressed on T regulatory cells. The ability of IL-2 to expand and activate both innate and adaptive effector cells underlies its antitumor activity.

[0006] In patients, IL-2 can stimulate an anti-tumor effect characterized by an increase in cytotoxic lymphocytes, including effector T and NK cells, when given at high doses (i.e., in humans, 600 000-720 000 IU / kg body weight three times a day for each cycle, up to 14 doses). It is speculated that during this therapy, IL-2 activates all T cells after administration of high doses; when the therapy cycle ends and IL-2 levels decrease to a certain point, IL-2 will become limited, and for the remaining wild-type IL-2, T regulatory (Treg) cells expressing IL-2Rαβγ will outcompete effector T cells expressing IL-2Rβγ.

[0007] Recombinant human IL-2 aldesleukin was the first cancer immunotherapy approved by the FDA in 1992. For metastatic melanoma and renal cancer, aldesleukin has shown complete regression of the cancer in about 10% of patients with the use of appropriate supportive care. Approximately 70% of patients with a complete response have been cured, remaining in complete regression for more than 25 years after initial treatment.

[0008] Based on its antitumor efficacy, high-dose IL-2 (aldesleukin) has been approved for use in patients with metastatic renal cell carcinoma and malignant melanoma. However, its antitumor immunity is dose-limited due to severe cardiovascular, pulmonary, hepatic, gastrointestinal, neurological, and hematological side effects, so it can only be given to patients in specialized centers. Similarly, once the level of administered IL-2 drops below the level required for IL-2Rβγ activity, it will contribute to the activation of Tregs expressing high-affinity IL-2Rαβγ, which can limit antitumor immunity.

[0009] Preclinical experiments have shown that IL-2-induced pulmonary edema, as a model of vascular leak syndrome, can be caused by the interaction of IL-2 with CD25 on lung endothelial cells and can be abolished by CD25 blocking antibodies, genetic disruption, or the use of IL-2-antibody complexes. Another proposed mechanism for IL-2-induced vascular leak syndrome involves the activation of eosinophils, as these cells express IL-2Rαβγ and IL-2 therapy in patients is accompanied by increased systemic eosinophil and IL-5 levels.

[0010] CD4+ regulatory T cells (Treg cells) responsible for suppressing the immune response leading to immune tolerance preferentially express the IL-2Rαβγ form of IL-2R. Therefore, administration of compounds that bind to IL-2Rαβγ and act as IL-2Rαβγ agonists may be expected to suppress the immune response, thereby also interfering with the anti-tumor response in cancer patients.

[0011] Effector CD4+T cells, CD8+T cells, and NK cells that significantly enhance anti-tumor immune responses preferentially express the IL-2Rβγ form of IL-2R. Therefore, administration of compounds that bind to IL-2Rβγ and act as IL-2Rβγ agonists may be expected to enhance anti-tumor immune responses (e.g., by increasing the proliferation and effector activity of CD4+T cells, CD8+T cells, and NK cells).

[0012] Therefore, administration of an IL-2Rβγ selective agonist (with reduced or no binding to IL-2Rα, or with enhanced binding to IL-2Rβγ) would be beneficial for patients with certain cancers, as this would be expected to reduce systemic vascular leakage side effects such as pulmonary edema, providing an improved therapeutic window. Moreover, an IL-2Rβγ selective agonist would have the benefit of avoiding selective activation of immunosuppressive regulatory T cells at low doses, being similarly potent against Tregs and CD4+ effector T cells, cytotoxic CD8+ effector T cells, and NK cells, thereby providing a greater potential to enhance the patient's immune system to eliminate cancer cells.

[0013] Optimally, such IL-2Rβγ selective agonists will also show relatively long exposure after administration, thereby further improving the patient's response to treatment. The enhancement of the effector arm of the immune system in cancer patients by administering IL-2βγ selective agonists can be further enhanced by administering antagonists of immunosuppressive pathways (e.g., CTLA-4 and PD-1 antagonists) or by administering immune agonists such as TLR ligands or substances that excite immune activation receptors such as 41BB (CD137), OX40, ICOS, CD40, CD28, NKG2D, NKp30, NKp44, NKp46, LFA1, CD16, CD64, CD32A and CD3 activating receptor agonists or antibodies that recruit antibody-directed cellular cytotoxicity (ADCC).

[0014] Adoptive transfer of tumor-reactive T cells has been developed as a clinically useful therapy that can induce anti-tumor immunity in patients. However, the widespread use of adoptive T cell transfer (ACT) therapy to treat cancer has some limitations, including the production of sufficient numbers of cells for infusion and the inability of transferred T cells to persist and remain functional in vivo. Clinically, co-administration of the T cell growth factor interleukin-2 (IL-2) can improve the survival, function, and anti-tumor activity of transplanted T cells. However, the use of IL-2 to enhance ACT is complicated by the pleiotropic properties of IL-2, which induces both immunostimulatory and inhibitory T cell responses and can cause severe toxicity.

[0015] Attempts have been made to solve the toxicity problem of IL-2. In one case, a formulation method has been attempted, see, for example, U.S. Patent No. 6,706,289 and International Patent Application Publication WO 02 / 00243 and WO 99 / 60128. In other methods, some IL-2 conjugates have been proposed, see, for example, U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502. In addition, some reversible IL-2 conjugates have been proposed, see, for example, WO12065086A1.

[0016] However, despite these approaches, there remains a need for IL-2 conjugates that provide safer treatment for cancer patients. Summary of the invention

[0017] It is therefore an object of the present invention to at least partially overcome the above mentioned disadvantages.

[0018] This object is achieved by an IL-2 conjugate of formula (Ia) or (Ib) or a pharmaceutically acceptable salt thereof,

[0019]

[0020] in

[0021] -D is a biased IL-2 portion, the biased IL-2 portion comprising an IL-2 portion, and for the biased IL-2 portion, the biased IL-2 has a K for IL-2Rαβ D The bias of IL-2 on IL-2Rβ D The ratio is higher than that of aldesleukin for IL-2Rαβ D Compared with aldesleukin, the K D proportion;

[0022] -L 1 - is a linker moiety that is covalently and reversibly attached to -D;

[0023] -L 2 - is a chemical bond or a spacer moiety;

[0024] -Z is a polymeric moiety or a substituted fatty acid moiety;

[0025] x is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; and

[0026] y is an integer selected from 2, 3, 4 and 5.

[0027] In the present invention, the terms are used with the following meanings:

[0028] As used herein, the term "interleukin-2" or "IL-2" refers to all IL-2 proteins, preferably from mammalian species, more preferably from primate species and most preferably from humans, as well as their variants, analogs, orthologs, homologs and derivatives and fragments thereof, characterized in that they play a key role in lymphocyte production, survival and homeostasis. The term "IL-2" also includes naturally occurring variants of IL-2, such as splice variants or allelic variants.

[0029] Human IL-2 has the sequence of SEQ ID NO: 1:

[0030] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE

[0031] ELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF

[0032] CQSIISTLT

[0033] Unprocessed human IL-2 additionally contains an N-terminal 20 amino acid signal peptide that is not present in the mature IL-2 molecule.

[0034] Preferably and unless otherwise indicated, the term "IL-2" preferably refers to aldesleukin, a variant of human IL-2, in which the amino acid (alanine) at position 1 present in human IL-2 of SEQ ID NO: 1 has been deleted (desA1) and the cysteine ​​at position 125 present in human IL-2 of SEQ ID NO: 1 has been replaced with serine (C125S). The sequence of aldesleukin is shown in SEQ ID NO: 2:

[0035] PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE

[0036] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFS

[0037] QSIISTLT

[0038] As used herein, the term "IL-2 protein variant" or "IL-2 variant" refers to a protein from the same species that is different from a reference IL-2 protein. Preferably, such a reference IL-2 protein sequence is the sequence of SEQ ID NO: 2. Generally, differences are limited so that the amino acid sequences of the reference and variant are closely similar overall 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 a query amino acid sequence is intended to mean that the amino acid sequence of the subject protein is identical to the query sequence, except that the subject protein sequence may include up to five amino acid changes for every 100 amino acids of the query amino acid sequence. These changes in the reference sequence may occur at the amino (N-terminal) or carboxyl terminal (C-terminal) position of the reference amino acid sequence or anywhere between those terminal positions, interspersed individually among residues in the reference sequence or in one or more consecutive groups within the reference sequence. The query sequence can be the entire amino acid sequence of the reference sequence or any specified fragment thereof, as described herein. Preferably, the query sequence is the sequence of SEQ ID NO:2.

[0039] Such IL-2 protein variants may be naturally occurring variants, such as naturally occurring allelic variants encoded by one of several alternative forms of IL-2 occupying a given locus on a chromosome or organism, or isoforms encoded by naturally occurring splice variants derived from a single primary transcript. Alternatively, the IL-2 protein variant may be a variant not yet known to occur naturally and that may be prepared by mutagenesis techniques known in the art.

[0040] It is known in the art that one or more amino acids can be deleted from the N-terminus or C-terminus of a biologically active peptide or protein, or from an internal position, i.e., between the N- and C-terminal amino acids, without substantial loss of biological function. The term IL-2 protein variant also includes such N-terminal, C-terminal and / or internal deletions.

[0041] Those skilled in the art also recognize that some amino acid sequences of IL-2 proteins can be changed without significantly affecting the structure or function of the peptide. These mutants include deletions, insertions, inversions, duplications and substitutions that have little effect on activity, selected according to general rules known in the art. For example, Bowie et al., 1990, Science 247:1306-1310 provides guidance on how to prepare phenotypically silent amino acid substitutions, which is incorporated herein by reference in its entirety, wherein the authors point out that there are two main methods for studying the tolerance of amino acid sequences to changes.

[0042] As used herein, the term "IL-2 analogues" refers to IL-2s from different and unrelated organisms that play the same function in each organism, but do not originate from an ancestral structure common to the ancestors of the organisms. Rather, similar IL-2s appeared separately and later evolved to play the same or similar functions. In other words, similar IL-2 proteins are proteins with very different amino acid sequences that play the same biological activity.

[0043] As used herein, the term "IL-2 ortholog" refers to IL-2 in two different species whose sequences are related to each other through a common homologous IL-2 in an ancestral species, but have evolved to diverge from each other.

[0044] As used herein, the term "IL-2 homolog" (homolog) refers to IL-2 of different organisms, which play the same function in each organism and originate from an ancestral structure shared by the ancestors of the organisms. In other words, homologous IL-2 proteins are proteins with very similar amino acid sequences that play the same biological activity. Preferably, an IL-2 protein homolog can be defined as a protein that exhibits at least 40%, 50%, 60%, 70%, 80%, 90% or 95% identity with a reference IL-2 protein, preferably an IL-2 protein of SEQ ID NO: 2.

[0045] As used herein, the term "IL-2 protein fragment" refers to any peptide that comprises a continuous stretch of a portion of the amino acid sequence of an IL-2 protein, preferably the protein of SEQ ID NO: 2. More particularly, the IL-2 protein fragment comprises at least 50, such as at least 60, at least 70 or at least 80 consecutive amino acids of an IL-2 protein, more preferably the protein of SEQ ID NO: 2.

[0046] The term "IL-2" also includes poly (amino acid) conjugates having the above sequence, but having a main chain including both amide and non-amide linkages (e.g., ester linkages), such as depsipeptides. Depsipeptides are amino acid residue chains in which the main chain includes both amide (peptide) and ester bonds. Therefore, the term "side chain" as used herein refers to a portion of the α-carbon of the amino acid moiety (if the amino acid moiety is linked by an amine bond, such as in a peptide or protein) or refers to any carbon-containing portion of the main chain of the poly (amino acid) conjugate (e.g., in the case of depsipeptides). Preferably, the term "IL-2" refers to a protein having a main chain formed by an amide (peptide) bond.

[0047] Since the term IL-2 includes the above-mentioned variants, analogs, orthologues, homologues, derivatives and fragments of IL-2, all references to specific positions within the reference sequence also include equivalent positions in these variants, analogs, orthologues, homologues, derivatives and fragments of the IL-2 portion, even if not specifically mentioned.

[0048] As used herein, the term "biased IL-2" refers to a protein wherein the biased IL-2 has a K for IL-2Rαβ. D The bias of IL-2 on IL-2Rβ D The ratio is greater than the K of aldesleukin of SEQ ID NO: 2 to IL-2Rαβ D Compared with aldesleukin, the K D This is represented by the following formula:

[0049]

[0050] in

[0051]

[0052] in

[0053] “K D "Biased IL-2 towards IL-2Rαβ" is the K biased IL-2 towards IL-2Rαβ D ,

[0054] “K D The bias of IL-2 towards IL-2Rαβ is the K bias of IL-2 towards IL-2Rβ D ,

[0055] “K D "Aldesleukin to IL-2Rαβ" is the K of aldesleukin to IL-2Rαβ D ,and

[0056] “K D "Aldesleukin for IL-2Rβ" is the K of aldesleukin for IL-2Rβ D .

[0057] Determination of biased IL-2 K for IL-2Rαβ D , biased IL-2 K for IL-2Rβ D 、K of aldesleukin on IL-2Rαβ D and aldesleukin for IL-2Rβ DThe desired binding affinity / kinetics can be assessed using surface plasmon resonance (SPR) measured as follows on a Biacore instrument (GE Healthcare): A human Fc capture surface on a CM5 (or C1 or CM4) chip is prepared by covalently coating an anti-human Fc antibody, or a protein A chip is used. Next, IL-2Rβ-Fc or a suitable mixture of IL2-Rα-Fc and IL2-Rβ-Fc, such as a 1:1 mixture, is immobilized on the chip. To measure affinity / kinetic constants, a serial dilution of the analyte is prepared, for example, starting at 1.5nM to 2μM or between 100nM and 1μM for IL-2 compounds (ending at, for example, 0.03nM to 100nM or 0.1nM to 1nM). The analytes are each exposed to the receptor-modified chip for an appropriate amount of time, such as 1-30 minutes, which may be, for example, 2 minutes or may be 3 minutes, and then washed for an appropriate amount of time, such as 2-60 minutes, such as 10 minutes. Binding curves obtained from the dilution series were fit to a 1:1 kinetic model to relate the observed response units (R) to the association and dissociation rate constants ka and kd:

[0058]

[0059] in

[0060] t is time;

[0061] C is the concentration of the analyte; and

[0062] R max is the maximum surface binding capacity.

[0063] If determined via the kinetic 1:1 model, the ratio of the dissociation and association rates provides the equilibrium dissociation constant, K D .

[0064] Alternatively, the binding curves obtained from the dilution series were fit to a 1:1 steady-state interaction model, which was determined by the steady-state binding level (R eq ) for the curve of the assay concentration (C) and calculated K for a 1:1 interaction D :

[0065]

[0066] in

[0067] R eq is the steady-state binding level;

[0068] C is the concentration of the analyte; and

[0069] R max is the maximum surface binding capacity.

[0070] It will be appreciated that not every calculation method is possible for every biased IL-2 molecule. If, for example, the reaction is too fast, then using a 1:1 kinetic model may not be possible and a 1:1 steady-state interaction model may be used. If, for example, equilibrium is not achieved, then using a 1:1 interaction model may not be possible and a 1:1 kinetic model may be used.

[0071] It is understood that the determination is biased towards the K of IL-2 for IL2Rαβ and IL2Rβ rather than the IL-2 conjugates of the present invention. D Therefore, K D Determined in the absence of -L 1 -、-L 2 - and -Z moieties, preferably prior to synthesis of the IL-2 conjugate of the invention, except when the IL-2 conjugate is a translational fusion protein, in which case K D The assay is performed after release of biased IL-2, for example after protease or chemical cleavage.

[0072] The biased IL-2 comprises a protein portion, which is an IL-2 portion, preferably an IL-2 portion having an amino acid sequence of SEQ ID NO: 2, which comprises at least one amino acid mutation or at least one deletion or at least one modified portion M mod , for example 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 modified parts M mod , or any combination thereof. mod The moiety may be a protein moiety or a non-protein moiety. mod In some embodiments, the IL-2 portion has the sequence of SEQ ID NO:1.

[0073] In the IL-2 conjugate of the present invention, the biased IL-2 is present in the form of the corresponding biased IL-2 part. The IL-2 conjugate of the present invention releases all -Z parts, that is, when the biased IL-2 part, -D and -L are cleaved 1 -Reversible link between IL-2 and IL-2 release bias.

[0074] As used herein, the term "affinity" refers to the strength of the sum of 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, the term "affinity" as used herein refers to intrinsic binding affinity, which reflects a 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 can often be expressed in terms of the equilibrium dissociation constant (K D) are the dissociation and association rate constants (k d and k a ). Thus, equivalent affinities may include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be determined by well-established methods known in the art, including those described herein.

[0075] As used herein, the terms "α-subunit of the IL-2 receptor" and "IL-2Rα" refer to human CD25.

[0076] As used herein, the terms "β-subunit of IL-2 receptor" and "IL-2Rβ" refer to human CD122.

[0077] 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, such as a 1:1 mixture, of IL-2Rα and IL-2Rβ.

[0078] The IL-2 conjugate of the present invention releases biased IL-2, that is, the IL-2 conjugate of the present invention is a prodrug biased towards IL-2. The term "prodrug" as used herein refers to a biologically active portion that is reversibly and covalently linked to a special protective group through a linker portion (which can also be referred to as a "reversible prodrug linker"), such as a biased IL-2 portion, wherein the linker portion includes a reversible link with the biologically active portion, and wherein the special protective group changes or eliminates the undesirable properties in the parent molecule of the biologically active portion, that is, in the corresponding drug. This also includes enhancing the desired properties in the drug and inhibiting undesirable properties. Special non-toxic protective groups are called "carriers". Prodrugs release reversibly and covalently bound biologically active portions in the form of corresponding drugs. In other words, prodrugs are conjugates comprising biologically active portions, and the covalent and reversible conjugation of the biologically active portion via a reversible prodrug linker portion is direct or carried out through a spacer portion. Another term for "biologically active portion" is "drug portion".

[0079] As used herein, the term "reversible" or "degradable" in relation to the connection of the first part to the second part means that the link connecting the first part and the second part is cleavable under physiological conditions (an aqueous buffer at pH 7.4 and 37° C.) with a half-life of 1 hour to 3 months, preferably 1 hour to 2 months, and even more preferably 1 hour to 1 month. The cleavage may be enzymatic or non-enzymatic, preferably non-enzymatic. Therefore, the term "stable" or "permanent" in relation to the connection of the first part to the second part means that the half-life of the cleavage of the link connecting the first part and the second part under physiological conditions is longer than 3 months.

[0080] The term "modifying moiety" as used herein preferably refers to a substituent or a polymeric moiety.

[0081] As used herein, the term "disulfide bridging" refers to the insertion of a moiety between two sulfur atoms of a disulfide bridge. This is achieved by reacting each thiol-reactive functional group with one of the sulfur atoms of the disulfide bridge using a reagent having the moiety between two thiol-reactive functional groups so that the moiety is inserted between the sulfur atoms after the disulfide bond has been reduced. If more than one disulfide bridge is present in a peptide or protein, the disulfide bridge may be inserted between the sulfur atoms of one disulfide bridge, or may be inserted between the sulfur atoms of different disulfide bridges. Such disulfide bridges may be naturally present in the peptide or protein, or may have been artificially introduced, for example by replacing an existing amino acid moiety with a cysteine ​​moiety or by adding a cysteine ​​moiety to a peptide or protein.

[0082] As used herein, the term "reagent" refers to a chemical compound that contains at least one functional group for reacting with a functional group of another chemical compound or drug. It is understood that drugs containing functional groups (such as primary or secondary amines or hydroxyl functional groups) are also reagents.

[0083] As used herein, the term "moiety" refers to a portion of a molecule that lacks one or more atoms compared to the corresponding reagent. If, for example, a reagent of the formula "HXH" reacts with another reagent and becomes part of the reaction product, the corresponding moiety of the reaction product has the structure "HX-" or "-X-", and each "-" refers to the connection to the other moiety. Thus, the biologically active moiety is released from the reversible linkage as a drug.

[0084] It is understood that if a sequence or atom group chemical structure is provided in a manner in which the atom group connects to two moieties or separates the moieties, the sequence or chemical structure can be connected to the two moieties in either direction unless otherwise expressly stated. For example, "-C(O)N(R 1 )-" can be used as "-C(O)N(R 1 )-" or as "-N(R 1 )C(O)-" connects two parts or separates the parts. Similarly, Part can be used as or as Connects two parts or separates parts.

[0085] As used herein, the term "substituted" refers to the replacement of one or more -H atoms of a molecule or moiety with a different atom or group of atoms, the latter termed a "substituent."

[0086] The term "substituent" as used herein preferably refers to a moiety selected from the group consisting of halogen, -CN, -COOR x1 、-OR x1 、-C(O)Rx1 、-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 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more identical or different -R x2 Substitute, and where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted with one or more selected from -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(Rx3 )C(O)N(R x3a )-and-OC(O)N(R x3 )-groups;

[0087] -R x1 , -R x1a , -R x1b are independently selected from -H, -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more identical or different -R x2 Substitute, and where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted with one or more selected from -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 )-groups;

[0088] T 0 Each is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein T 0 Each independently optionally replaced by one or more identical or different -R x2 replace;

[0089] -R x2 are independently selected from halogen, -CN, oxo (=O), -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 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different;

[0090] -R x3 , -R x3a , -R x4 , -R x4a , -R x4b are each independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

[0091] Preferably, up to 6 -H atoms of the optionally substituted molecule are independently substituted with substituents, for example, 5 -H atoms are independently substituted with substituents, 4 -H atoms are independently substituted with substituents, 3 -H atoms are independently substituted with substituents, 2 -H atoms are independently substituted with substituents, or 1 -H atom is independently substituted with substituents.

[0092] As used herein, the term "fatty acid" refers to a saturated or unsaturated monocarboxylic acid with an aliphatic tail, which may include 4-28 carbon atoms. Fatty acid may be saturated or unsaturated, straight or branched. The term "fatty acid variant" refers to a modified fatty acid in which some of the carbon atoms may be replaced by other atoms or atomic groups, which may be substituted.

[0093] The term "peptide" as used herein refers to a chain of at least 2 and up to 50, including 50 amino acid monomeric parts connected by peptide (amide) linkages. The term "peptide" also includes peptidomimetics such as D-peptides, peptoids or β-peptides, covering such peptidomimetics chains having up to 50, including 50 monomeric parts.

[0094] The term "protein" as used herein refers to a chain having more than 50 amino acid monomer portions (which may also be referred to as "amino acid residues") connected by peptide linkages, wherein preferably no more than 12,000 amino acid monomer portions are connected by peptide linkages, for example no more than 10,000 amino acid monomer portions, no more than 8,000 amino acid monomer portions, no more than 5,000 amino acid monomer portions, or no more than 2,000 amino acid monomer portions.

[0095] As used herein, the term "about" when used in conjunction with a numerical value is used to refer to a range of the numerical value plus and minus no more than 25%, more preferably no more than 20%, more preferably no more than 10%, of the numerical value, inclusive. For example, the phrase "about 200" is used to refer to a range of 200 + / - 25%, i.e., a range of 150 to 250, inclusive; preferably 200 + / - 20%, i.e., a range of 160 to 240, inclusive; even more preferably 200 + / - 10%, i.e., a range of 180 to 220, inclusive. It is understood that a percentage given as "about 50%" does not refer to a range of "50% + / - 25%, i.e., 25 to 75%, inclusive, but rather "about 50%" refers to a range of 37.5 to 62.5%, inclusive, i.e., plus and minus 25% of the numerical value 50.

[0096] As used herein, the term "polymer" refers to a molecule comprising a repeating structural unit, i.e. a monomer, connected by a chemical bond in a linear, cyclic, branched, cross-linked or dendritic manner or a combination thereof, which can be synthetic or biologically derived or a combination of the two. It is understood that the polymer can also include one or more other chemical groups and / or one or more parts, such as one or more functional groups. Similarly, it is also understood that a peptide or protein is a polymer, even if the side chains of each amino acid residue may be different. Preferably, a soluble polymer has a molecular weight of at least 0.5 kDa, such as a molecular weight of at least 1 kDa, such as a molecular weight of at least 2 kDa, such as a molecular weight of at least 3 kDa or a molecular weight of at least 5 kDa. If the polymer is soluble, it preferably has a molecular weight of at most 1000 kDa, such as at most 750 kDa, such as at most 500 kDa, such as at most 300 kDa, such as at most 200 kDa, such as at most 100 kDa. It is understood that for insoluble polymers such as hydrogels, a meaningful molecular weight range cannot be provided.

[0097] As used herein, the term "polymeric" refers to an agent or moiety that comprises one or more polymers or one or more polymer moieties. A polymeric agent or moiety may also optionally comprise one or more other moieties, which are preferably selected from:

[0098] ·C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl, and tetrahydronaphthyl; and

[0099] ·Selected from the following links

[0100]

[0101] in

[0102] Dashed lines indicate attachment to the remainder of a moiety or reagent, and

[0103] -R and -R a are independently selected from -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0104] It is understood by those skilled in the art that the polymer products obtained from the polymerization reaction do not all have the same molecular weight, but rather exhibit a molecular weight distribution. Therefore, as used herein, the molecular weight range, molecular weight, number range of monomers in a polymer, and number of monomers in a polymer refer to the number average molecular weight and the number average of monomers, i.e., the arithmetic mean of the molecular weight of a polymer or a polymeric portion and the arithmetic mean of the number of monomers in a polymer or a polymeric portion.

[0105] Accordingly, in a polymeric moiety comprising "x" monomer units, any integer assigned to "x" thus corresponds to the arithmetic mean number of monomers. Any integer range assigned to "x" provides an integer range within which the arithmetic mean number of monomers lies. An integer assigned to "x" as "about x" means that the arithmetic mean number of monomers lies within an integer range of x + / - 25%, preferably x + / - 20%, and more preferably x + / - 10%.

[0106] As used herein, the term "number average molecular weight" refers to the common arithmetic mean of the molecular weights of the individual polymers.

[0107] The term "PEG-based" as used herein in connection with a part or agent means that the part or agent comprises PEG. Preferably, the part or agent based on PEG comprises at least 10% (w / w) PEG, such as at least 20% (w / w) PEG, such as at least 30% (w / w) PEG, such as at least 40% (w / w) PEG, such as at least 50% (w / w), such as at least 60 (w / w) PEG, such as at least 70% (w / w) PEG, such as at least 80% (w / w) PEG, such as at least 90% (w / w) PEG, such as at least 95%. The remaining weight percentage of the part or agent based on PEG is other parts, preferably selected from the following parts and links:

[0108] ·C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl, and tetrahydronaphthyl; and

[0109] ·Selected from the following links

[0110]

[0111] in

[0112] Dashed lines indicate attachment to the remainder of a moiety or reagent, and

[0113] -R and -R a are independently selected from -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0114] The term "hyaluronic acid-based" is used accordingly.

[0115] The term "PEG-based comprising at least X% PEG" as used herein in relation to a moiety or agent means that the moiety or agent comprises at least X% (w / w) ethylene glycol units (-CH2CH2O-), wherein the ethylene glycol units may be block by block, arranged in alternation or may be randomly distributed within the moiety or agent, preferably all ethylene glycol units of the moiety or agent are present in one block; the remaining weight percentage of the PEG-based moiety or agent is other moieties, preferably selected from the following moieties and links:

[0116] ·C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl, and tetrahydronaphthyl; and

[0117] ·Selected from the following links

[0118]

[0119] in

[0120] Dashed lines indicate attachment to the remainder of a moiety or reagent, and

[0121] -R and -R a are independently selected from -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0122] The term "hyaluronic acid-based comprising at least X% hyaluronic acid" is used accordingly.

[0123] As used herein, the term "hydrogel" refers to a hydrophilic or amphiphilic polymeric 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 network structure and physical integrity.

[0124] The term "interrupting" means that the moiety is inserted between two carbon atoms or - if the insertion is at one of the moiety's termini - between a carbon or heteroatom and a hydrogen atom, preferably between a carbon and a hydrogen atom.

[0125] As used herein, the term "C 1-4 "Alkyl" alone or in combination means a straight or branched chain alkyl moiety having 1 to 4 carbon atoms. If present at the end of a molecule, a straight or branched chain C 1-4 Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. 1-4 When an alkyl group is connected, the C 1-4Examples of alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2-. 1-4 Each hydrogen of the alkyl carbon may optionally be replaced by a substituent as defined above. 1-4 The alkyl group may be interrupted by one or more moieties as defined below.

[0126] As used herein, the term "C 1-6 "Alkyl" alone or in combination means a straight chain or branched chain alkyl moiety having 1 to 6 carbon atoms. If present at the end of a molecule, straight chain and branched C 1-6 Examples of alkyl groups are 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. 1-6 When an alkyl group is connected, the C 1-6 Examples of alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, and -C(CH3)2-. 1-6 Each hydrogen atom of a carbon may be optionally replaced by a substituent as defined above. 1-6 The alkyl group may be interrupted by one or more moieties as defined below.

[0127] 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, wherein C 1-10 , C 1-20 or C 1-50 Each hydrogen atom of a 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 moieties as defined below.

[0128] As used herein, the term "C 2-6 "Alkenyl" alone or in combination refers to a straight or branched hydrocarbon moiety having 2 to 6 carbon atoms and containing at least one carbon-carbon double bond. If present at the end of a molecule, examples are -CH=CH2, -CH=CH-CH3, -CH2-CH=CH2, -CH=CHCH2-CH3 and -CH=CH-CH=CH2. When the two parts of the molecule are connected by C 2-6 When the alkenyl group is connected, the C 2-6An example of an alkenyl group is -CH=CH-. 2-6 Each hydrogen atom of the alkenyl moiety may be optionally replaced by a substituent as defined above. 2-6 The alkenyl group may be interrupted by one or more moieties as defined below.

[0129] Therefore, the term “C 2-10 Alkenyl", "C 2-20 "Alkenyl" or "C 2-50 "Alkenyl" alone or in combination refers to a straight or branched chain hydrocarbon moiety having 2-10, 2-20 or 2-50 carbon atoms and containing at least one carbon-carbon double bond. 2-10 Alkenyl, C 2-20 Alkenyl or C 2-50 Each hydrogen atom of the alkenyl group may optionally be replaced by a substituent as defined above. 2-10 Alkenyl, C 2-20 Alkenyl or C 2-50 The alkenyl group may be interrupted by one or more moieties as defined below.

[0130] As used herein, the term "C 2-6 "Alkynyl" alone or in combination refers to a straight or branched hydrocarbon moiety having 2 to 6 carbon atoms and containing at least one carbon-carbon triple bond. If present at the end of a molecule, examples are -C≡CH, -CH2-C≡CH, CH2-CH2-C≡CH and CH2-C≡C-CH3. When two parts of a molecule are connected by an alkynyl group, an example is -C≡C-. C 2-6 Each hydrogen atom of the alkynyl group may optionally be replaced by a substituent as defined above. Optionally, one or more double bonds may occur. Optionally, C 2-6 The alkynyl group may be interrupted by one or more moieties as defined below.

[0131] Therefore, as used herein, the term "C 2-10 Alkynyl", "C 2-20 Alkynyl" and "C 2-50 "Alkynyl" alone or in combination refers to a straight or branched chain hydrocarbon moiety having 2-10, 2-20 or 2-50 carbon atoms, respectively, containing at least one carbon-carbon triple bond. 2-10 Alkynyl, C 2-20 Alkynyl or C 2-50 Each hydrogen atom of the alkynyl group may optionally be replaced by a substituent as defined above. Optionally, one or more double bonds may occur. Optionally, C 2-10 Alkynyl, C 2-20 Alkynyl or C 2-50 The alkynyl group may be interrupted by one or more moieties as defined below.

[0132] As mentioned above, C 1-4 Alkyl, C1-6 Alkyl, C 1-10 Alkyl, C 1-20 Alkyl, C 1-50 Alkyl, C 2-6 Alkenyl, C 2-10 Alkenyl, C 2-20 Alkenyl, C 2-50 Alkenyl, C 2-6 Alkynyl, C 2-10 Alkynyl, C 2-20 Alkenyl or C 2-50 The alkynyl group may optionally be interrupted by one or more moieties, preferably selected from:

[0133]

[0134] in

[0135] A dashed line indicates attachment to the remainder of a moiety or reagent; and

[0136] -R and -R a are independently selected from -H and methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0137] As used herein, the term "C 3-10 "Cycloalkyl" refers to a cyclic alkyl chain having 3 to 10 carbon atoms which may be saturated or unsaturated, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. 3-10 Each hydrogen atom of a cycloalkyl carbon may be replaced by a substituent as defined above. 3-10 "Cycloalkyl" also includes bridged bicyclic rings such as norbornane or norbornene.

[0138] The term "8- to 30-membered carbon polycyclic group" or "8- to 30-membered carbon polycyclic ring" refers to a cyclic moiety of two or more rings having 8-30 ring atoms, wherein two adjacent rings share at least one ring atom, and may contain up to the maximum number of double bonds (aromatic or non-aromatic rings, which are fully saturated, partially saturated or unsaturated). Preferably, the 8- to 30-membered carbon polycyclic group refers to a cyclic moiety of two, three, four or five rings, more preferably a cyclic moiety of two, three or four rings.

[0139] As used herein, the term "3- to 10-membered heterocyclyl" or "3- to 10-membered heterocycle" refers to a ring having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms, which may contain up to the maximum number of double bonds (aromatic or non-aromatic rings, which are fully saturated, partially saturated or unsaturated), wherein at least one ring atom up to 4 ring atoms are replaced by heteroatoms selected from sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-) and wherein the ring is attached to the remainder of the molecule via a carbon or nitrogen atom. Examples of 3- to 10-membered heterocycles include, but are not limited to, aziridine, oxirane, thiirane, aziridine, oxirane, thiirane, azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrrolidine, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazolin, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane, azepine Each hydrogen atom of the 3- to 10-membered heterocyclic group or the 3- to 10-membered heterocyclic group may be replaced by a substituent as defined below.

[0140] The term "8- to 11-membered heterobicyclyl" or "8- to 11-membered heterobicycle" as used herein refers to a heterocyclic moiety having two rings of 8-11 ring atoms, wherein at least one ring atom is shared by both rings, which may contain up to the maximum number of double bonds (aromatic or non-aromatic rings, which are fully saturated, partially saturated or unsaturated), wherein at least one ring atom up to 6 ring atoms are replaced by heteroatoms selected from sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-) and wherein the ring is attached to the remainder of the molecule via a carbon or nitrogen atom. Examples of 8- to 11-membered heterobicyclic rings are indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazolinone, 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 spirocyclic structures of two rings such as 1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocyclic rings such as 8-azabicyclo[3.2.1]octane. Each hydrogen atom of the 8- to 11-membered heterobicyclic ring or the 8- to 11-membered heterobicyclic ring carbon may be replaced by a substituent as defined below.

[0141] Similarly, the term "8- to 30-membered heteropolycyclyl" or "8- to 30-membered heteropolycycle" refers to a heterocyclic moiety having more than two rings, preferably three, four or five rings, with 8 to 30 ring atoms, wherein two adjacent rings share at least one ring atom, may contain up to the maximum number of double bonds (aromatic or non-aromatic rings, which are fully saturated, partially saturated or unsaturated), wherein at least one ring atom up to 10 ring atoms are replaced by heteroatoms selected from sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-) and wherein the ring is attached to the remainder of the molecule via a carbon or nitrogen atom.

[0142] It will be understood that the phrase "R x / R y The pairs together with the atoms to which they are attached form C 3-10 Cycloalkyl or 3- to 10-membered heterocyclic group"

[0143]

[0144] Refers to R x and R y The following structure is formed:

[0145]

[0146] Where R is C 3-10 a cycloalkyl group or a 3- to 10-membered heterocyclic group.

[0147] It is also understood that the phrase "R x / R y The pairs together with the atoms to which they are attached form ring A"

[0148]

[0149] Refers to R x and R y The following structure is formed:

[0150]

[0151] As used herein, "halogen" refers to fluorine, chlorine, bromine or iodine. It is generally preferred that halogen is fluorine or chlorine.

[0152] As used herein, the term "functional group" refers to an atomic group that can react with other atomic groups. Exemplary functional groups include, for example, carboxylic acid (-(C=O)OH), primary or secondary amine (-NH2, -NH-), maleimide, thiol (-SH), sulfonic acid (-(O=S=O)OH), formates or carbonates, carbamates (-O(C=O)N<), hydroxyl (-OH), aldehydes (-(C=O)H), ketones (-(C=O)-), hydrazines (>NN<), isocyanates, isothiocyanates, phosphoric acid (-O(P=O)OHOH), phosphonic acid (-O(P=O)OHH), haloacetyl, alkyl halides, acryloyl, aryl fluorides, hydroxylamine, disulfide, sulfonamide, sulfuric acid, vinyl sulfone, vinyl ketone, diazoalkane, ethylene oxide, and aziridine.

[0153] When the IL-2 conjugates of the present invention contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically acceptable salts. Therefore, the IL-2 conjugates of the present invention containing acidic groups can be used according to the present invention, for example, as alkali metal salts, alkaline earth metal salts or ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts formed with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. There may be IL-2 conjugates of the present invention containing one or more basic groups (i.e., groups that can be protonated), and they can be used according to the present invention in the form of addition salts with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic 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, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and other acids known to those skilled in the art. Other methods are known to those skilled in the art for converting basic groups into cations, such as alkylation of amine groups to produce positively charged ammonium groups and appropriate counterions for salts. If the IL-2 conjugates of the invention contain both acidic and basic groups, the invention also includes inner salts or betaines (zwitterions) in addition to the salt forms mentioned. The respective salts 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 with other salts or cation exchange. The present invention also includes all salts of the IL-2 conjugates of the present invention, which are not suitable for direct use in medicines due to low physiological compatibility, but can be used, for example, as intermediates in chemical reactions or for the preparation of pharmaceutically acceptable salts.

[0154] The term "pharmaceutically acceptable" refers to substances that do not cause harm when administered to a patient, preferably substances approved for use in animals, preferably humans, by regulatory agencies such as EMA (Europe) and / or FDA (US) and / or any other national regulatory agencies.

[0155] As used herein, the term "excipient" refers to a diluent, adjuvant or carrier used together with a therapeutic substance such as a drug or prodrug. Such pharmaceutical excipients can be sterile liquids, such as water and oil, including petroleum, animal, plant or synthetic sources, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered orally, water is a preferred excipient. When the pharmaceutical composition is administered intravenously, saline and dextrose aqueous solutions are preferred excipients. Saline solutions and dextrose aqueous solutions and glycerol solutions are preferably used as liquid excipients for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum powder, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the pharmaceutical composition may also contain a small amount of a wetting agent or emulsifier, a pH buffer such as acetate, succinate, tris, carbonate, phosphate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) or may contain a detergent such as Tween, poloxamer, poloxamines, CHAPS, Igepal or amino acids, such as glycine, lysine or histidine. These pharmaceutical compositions may take the form of solutions, suspensions, emulsions, tablets, pellets, capsules, powders, sustained release formulations, and the like. The pharmaceutical composition may be formulated as a suppository with conventional binders and excipients such as triglycerides. Oral formulations may include standard excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Such compositions will contain a therapeutically effective amount of the drug or biologically active portion and an appropriate amount of excipients to provide a form suitable for use with the patient. The formulation should suit the mode of administration.

[0156] Typically, the term "comprising" also covers "consisting of

[0157] Proportion 偏倚IL-2 Ratio 阿地白介素 The ratio is greater than 1, preferably greater than 2, preferably greater than 3, preferably greater than 4 and even more preferably greater than 5. In some embodiments, the ratio 偏倚IL-2 Ratio 阿地白介素 The ratio is greater than 10, greater than 20, greater than 50, greater than 70, greater than 100 or greater than 150.

[0158] Biased IL-2 moiety-D is an IL-2 moiety, preferably aldesleukin, comprising

[0159] (a) at least one modified portion M mod , which may be the same or different; or

[0160] (b) at least one amino acid mutation; or

[0161] (c) at least one deletion; or any combination of (a), (b), and (c).

[0162] At least one of the modifications listed in (a), (b) and (c) is present in -D and ensures that the ratio is in the biased IL-2 portion 偏倚IL-2 Greater than proportion 阿地白介素 However, it is understood that the IL-2 moiety may also contain one or more of the modifications listed in (a), (b) and (c), which when viewed individually do not ensure proportionality. 偏倚IL-2 Greater than proportion 阿地白介素 Such additional modifications may, for example, provide improved -D production or storage characteristics.

[0163] In one embodiment, the IL-2-biasing moiety comprises at least one modified moiety M. mod In another embodiment, the biased IL-2 portion comprises at least one amino acid mutation in the IL-2 portion. In another embodiment, the biased IL-2 portion comprises at least one deletion in the IL-2 portion. In another embodiment, the biased IL-2 portion comprises at least one modified portion M mod In another embodiment, the IL-2 portion comprises at least one modified portion M. mod In another embodiment, the biased IL-2 portion comprises at least one amino acid mutation and at least one deletion in the IL-2 portion. In another embodiment, the biased IL-2 portion comprises at least one modified portion M mod A stable linkage to the IL-2 portion and at least one amino acid mutation and at least one deletion in the IL-2 portion.

[0164] The IL-2-biased moiety may comprise at least one modified moiety M mod Stable link to the IL-2 moiety. mod The attachment of M can be at the N-terminus, C-terminus, amino acid side chain or internal position of the IL-2 moiety. mod The moiety may preferably be a substituent or a polymeric moiety. mod The connection of M is at the N-terminus of the IL-2 portion.mod The connection is at the C-terminus of the IL-2 portion. In some embodiments, M mod The connection is at the amino acid side chain of the IL-2 portion. In some embodiments, M mod The connection is at the inner position of the IL-2 part. If more than one M mod The moiety is linked to the IL-2 moiety, then the linkage may occur at any combination of linkage positions selected from the group consisting of the N-terminus, the C-terminus, a side chain of an amino acid residue, and an internal position.

[0165] In one embodiment, M mod Preferably, the substituent has a molecular weight of 15 Da to 1 kDa.

[0166] In one embodiment, the M mod The moiety may be introduced in the form of a disulfide bridge. Preferably, the disulfide bridge is formed between the thiol groups of two cysteine ​​residues. The disulfide bridge is an example of a connection of a modified moiety at an internal position. In one embodiment, the 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 or inserted into the IL-2 moiety, preferably the IL-2 moiety of SEQ ID NO: 2, or replace a naturally occurring amino acid residue of the IL-2 moiety, preferably the IL-2 moiety of SEQ ID NO: 2.

[0167] Preferred ways of obtaining such disulfide bridges are disclosed in Jones et al. (J. Am. Chem. Soc., 2012, 134(3), pp. 1847-1852), WO2011 / 018611, WO2011 / 018612 and WO2011 / 018613.

[0168] Preferably the disulfide bridge occurs at a position involved in binding to IL-2Rα.Thus, preferably the disulfide bridge results in a biased IL-2 moiety with reduced affinity for IL-2Rαβ compared to aldesleukin.

[0169] In one embodiment, the disulfide bridge is formed between C57 and C104 if the IL-2 portion has the sequence of SEQ ID NO:2.

[0170] In another embodiment, M modIt is a polymeric part. The polymeric part can include straight chain, branched or multi-arm polymers. In one embodiment, the polymer is a straight chain polymer. In another embodiment, the polymer is a branched polymer. The branched polymer preferably has one, two, three, four or five branch points. Preferably two, three or four polymer arms extend from each branch point. In another embodiment, the polymer is a multi-arm polymer. The multi-arm polymer preferably has 3, 4, 5, 6, 7 or 8 polymer arms.

[0171] If M modIn one embodiment, the polymer has a molecular weight of about 0.5 kDa. In one embodiment, the polymer has a molecular weight of about 1 kDa. In one embodiment, the polymer has a molecular weight of about 2 kDa. In one embodiment, the polymer has a molecular weight of about 3 kDa. In one embodiment, the polymer has a molecular weight of about 4 kDa. In one embodiment, the polymer has a molecular weight of about 5 kDa. In one embodiment, the polymer has a molecular weight of about 7.5 kDa. In another embodiment, the polymer has a molecular weight of about 10 kDa. In another embodiment, the polymer has a molecular weight of about 15 kDa. In another embodiment, the polymer has a molecular weight of about 20 kDa. In another embodiment, the polymer has a molecular weight of about 30 kDa. In another embodiment, the polymeric portion has a molecular weight of about 40kDa. In another embodiment, the polymeric portion has a molecular weight of about 50kDa. In another embodiment, the polymeric portion has a molecular weight of about 70kDa. In another embodiment, the polymeric portion has a molecular weight of about 80kDa. In another embodiment, the polymeric portion has a molecular weight of about 90kDa. In another embodiment, the polymeric portion has a molecular weight of about 100kDa. In one embodiment, the polymer has a molecular weight of 0.5kDa. In one embodiment, the polymer has a molecular weight of 1kDa. In one embodiment, the polymer has a molecular weight of 2kDa. In one embodiment, the polymer has a molecular weight of 3kDa. In one embodiment, the polymer has a molecular weight of 4kDa. In one embodiment, the polymer has a molecular weight of 5kDa. In one embodiment, the polymer has a molecular weight of 7.5kDa. In another embodiment, the polymeric portion has a molecular weight of 10kDa. In another embodiment, the polymeric portion has a molecular weight of 15kDa. In another embodiment, the polymeric portion has a molecular weight of 20kDa. In another embodiment, the polymeric portion has a molecular weight of 30kDa. In another embodiment, the polymeric portion has a molecular weight of 40 kDa. In another embodiment, the polymeric portion has a molecular weight of 50 kDa. In another embodiment, the polymeric portion has a molecular weight of 70 kDa. In another embodiment, the polymeric portion has a molecular weight of 80 kDa. In another embodiment, the polymeric portion has a molecular weight of 90 kDa.In another embodiment, the polymeric moiety has a molecular weight of 100 kDa.

[0172] If M mod is a polymeric moiety, then the polymeric moiety preferably comprises a polymer selected from the group consisting of 2-methacryloyl-oxyethylphosphorylcholins, poly(acrylic acid), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides), poly(asparagine), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamide), poly(esters), 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 methacrylate), poly(hydroxypropyl oxazoline), poly(imino carbonate), poly(lactic acid), poly(lactic-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyl oxazoline), poly(organophosphazene), poly(orthoesters), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinyl pyrrolidone), silicone, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, alginates, mannans, pectins, rhamnogalacturonic acid, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate-based polymers, xylan and copolymers thereof.

[0173] In one embodiment, M mod It is a PEG-based polymer.

[0174] In another embodiment, M mod It is a polymer based on hyaluronic acid.

[0175] In another embodiment, M mod In one embodiment, the peptide or protein portion M mod It may be a translational fusion with the IL-2 portion that is biased towards IL-2. In another embodiment, the peptide or protein portion M mod It can be chemically conjugated to the IL-2 moiety that is biased towards IL-2. Preferably, the peptide or protein moiety M mod Not a fragment or IL-2-part of IL-2.

[0176] M in the form of a peptide or protein moietymod It can be a synthetic or natural protein part or a part or variant thereof. Exemplary non-IL-2 parts include albumin; antibody domains, such as Fc domains or antigen binding domains of immunoglobulins; CTP, and CD25; each in its naturally occurring form or a variant or fragment thereof.

[0177] Peptide or protein portion M fused to IL-2 portion mod It may be linked at the N-terminus or C-terminus, or may be inserted at an internal position of the IL-2 moiety. It is understood that more than one peptide or protein moiety M mod The more than one peptide or protein moiety M may be translationally fused or chemically conjugated to and / or inserted into the IL-2 moiety. mod The IL-2 moiety may have the same or different sequences. For example, the IL-2 moiety may have a first peptide or protein moiety M translationally fused or chemically conjugated to the N-terminus of the IL-2 moiety. mod and a second peptide or protein moiety M translationally fused or chemically conjugated to the C-terminus of the IL-2 moiety mod In another example, the biasing IL-2 portion can comprise a first peptide or protein portion M translationally fused or chemically conjugated to the N-terminus of the IL-2 portion. mod and a second peptide or protein moiety M that is translationally inserted or chemically conjugated to an internal position of the IL-2 moiety mod In another example, the biasing IL-2 portion can comprise a first peptide or protein portion M translationally fused or chemically conjugated to the C-terminus of the IL-2 portion. mod and a second peptide or protein moiety M that is translationally inserted or chemically conjugated to an internal position of the IL-2 moiety mod In a further example, the biasing IL-2 portion can comprise a first peptide or protein portion M translationally fused or chemically conjugated to the N-terminus of the IL-2 portion. mod A second peptide or protein moiety M that is translationally fused or chemically conjugated to the C-terminus of the IL-2 moiety mod and a third peptide or protein moiety M that is translationally inserted or chemically conjugated to an internal position of the IL-2 moiety mod .

[0178] M mod The attachment of M can be at a proteinogenic or non-proteinogenic amino acid residue of the IL-2 moiety. modThe attachment of M occurs at a proteinogenic amino acid. Such proteinogenic amino acid residues are preferably selected from cysteine, methionine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, glutamine and arginine. In some embodiments, M mod The attachment occurs at a non-protein amino acid. If the modified moiety is attached to a non-protein amino acid residue, it is understood that the non-protein amino acid residue was artificially introduced into the IL-2 moiety. The non-protein amino acid residue may be a residue that has a molecule available for conjugation to the M mod Any non-proteinogenic amino acid residue that is a functional group of the IL-2 moiety. In some embodiments, the non-proteinogenic amino acid comprises a functional group selected from the group consisting of a carbonyl group, a carbonyl derivative, such as a carbonyl-like group, a labeled carbonyl group, and a protected carbonyl group, an azide, an oxime, and a hydroxylamine in their side chain.

[0179] In some embodiments, the non-proteinogenic amino acid is a non-proteinogenic amino acid as described in WO 2006 / 069246 A2, which is incorporated herein by reference. In some embodiments, the non-proteinogenic amino acid has the structure as described in WO2006 / 069246A2 as follows: Formula (I)

[00265] to

[00283] , Formula (XXX)

[00284] , Formula (XXX-A)

[00285] , Formula (XXX-B)

[00286] , Formula (XXXI)

[00287] , Formula (XXXI-A)

[00288] , Formula (XXXI-B)

[00289] , Formula (XXXII)

[00290] , Formula (XXXII-A)

[00291] , Formula (XXXII-B)

[00292] , Formula (XXXX)

[00293] , Formula (XXXXI)

[00294] , Formula (XXXIX)

[00295] , Formula (XXXIX)

[00296] , Formula (XXXIX)

[00297] , Formula (XXXIX)

[00298] , Formula (XXXIX)

[00299] , Formula (XXXIX)

[00300] , Formula (XXXIX)

[00301] , Formula (XXXIX)

[00302] , Formula (XXXIX)

[00303] , Formula (XXXIX)

[00304] , Formula (XXXIX)

[00305] , Formula (XXXIX)

[00306] , Formula (XXXIX)

[00307] , Formula (XXXIX)

[00308] , Formula (XXXIX)

[00309] , Formula (XXXIX)

[00310] , Formula (XXXIX)

[00311] , Formula (XXXIX)

[00312] , Formula (XXXIX)

[00313] , Formula (XXXIX)

[00314] , Formula (XXXIX)

[00315] , Formula (XXXIX)

[00316] , Formula (XXXIX)

[00317] , Formula (XXXIX)

[00340] to

[00341] , Formula (XIII) of

[00342] to

[00343] , Formula (XV) of

[00344] and

[00345] , Formula (XV) of

[00345] to

[00346] , Formula (XV) of

[00347] to

[00348] , Formula (XV) of

[00349] and

[00350] , or Formula (XXIX) of

[00530] , the non-proteinogenic amino acids are incorporated herein by reference.

[0180] In one embodiment, M modThe connection occurs at a lysine residue of the IL-2 portion, for example, a lysine residue selected from K7, K8, K31, K34, K42, K47, K48, K53, K63, K75 and K96 based on SEQ ID NO: 2 or a corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional lysine residues compared to SEQ ID NO: 2, the connection may also occur at the additional lysine residue. It is understood that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the lysine residues of SEQ ID NO: 2 may be used to connect to M mod If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more lysine residues than the IL-2 portion of SEQ ID NO: 2, more than 11 lysine residues may be used to link to the M mod , i.e., up to the maximum number of lysine residues present in the homologue or variant of SEQ ID NO: 2 that can be used to link M mod In one embodiment, an M mod The M moiety is linked to a lysine residue in SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M moieties may be the same or different. mod The moiety is linked to two lysine residues of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the three M residues, which may be the same or different, are linked to two lysine residues of SEQ ID NO: 2 or a homologue or variant thereof. mod The amino acid residues of the present invention are linked to three lysine residues of SEQ ID NO:2 or its homologues or variants. In another embodiment, the four amino acid residues, which may be the same or different, are linked to three lysine residues of SEQ ID NO:2 or its homologues or variants. mod The lysine residues of SEQ ID NO: 2 or its homologues or variants are connected to the lysine residues of SEQ ID NO: 2 or its homologues or variants. In another embodiment, the five lysine residues that may be the same or different mod The amino acid residues of the present invention are linked to the five lysine residues of SEQ ID NO: 2 or its homologues or variants. In another embodiment, the six lysine residues, which may be the same or different, are linked to the five lysine residues of SEQ ID NO: 2 or its homologues or variants. mod The moiety is linked to six lysine residues of SEQ ID NO: 2 or a homologue or variant thereof.

[0181] In another embodiment, M modThe attachment occurs at a threonine residue of the IL-2 portion, for example, a threonine residue selected from T2, T6, T9, T36, T40, T50, T100, T101, T110, T112, T122, T130 and T132 based on SEQ ID NO: 2 or a corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional threonine residues compared to SEQ ID NO: 2, the attachment may also occur at the additional threonine residue. It is understood that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the threonine residues of SEQ ID NO: 2 may be used for attachment to M mod If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more threonine residues than the IL-2 portion of SEQ ID NO: 2, then more than 13 threonine residues may be used to link to the M mod , i.e., up to the maximum number of threonine residues present in the homologue or variant available for attachment of M mod In one embodiment, an M mod The M portion is linked to a threonine residue of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M portions may be the same or different. mod The threonine residues of SEQ ID NO: 2 or a homologue or variant thereof are linked to the threonine residues of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the three threonine residues, which may be the same or different, are linked to the threonine residues of SEQ ID NO: 2 or a homologue or variant thereof. mod The threonine residues of SEQ ID NO: 2 or its homologues or variants are linked to the threonine residues of SEQ ID NO: 2 or its homologues or variants. In another embodiment, the four threonine residues, which may be the same or different, are linked to the threonine residues of SEQ ID NO: 2 or its homologues or variants. mod The threonine residues of SEQ ID NO: 2 or its homologues or variants are linked to the threonine residues of SEQ ID NO: 2 or its homologues or variants. In another embodiment, the five threonine residues, which may be the same or different, are linked to the threonine residues of SEQ ID NO: 2 or its homologues or variants. mod The threonine residues of SEQ ID NO: 1 or its homologues or variants are linked to the threonine residues of SEQ ID NO: 1 or its homologues or variants. In another embodiment, the six threonine residues, which may be the same or different, are linked to the threonine residues of SEQ ID NO: 1 or its homologues or variants. mod The moiety is linked to six threonine residues of SEQ ID NO: 2 or a homologue or variant thereof.

[0182] In another embodiment, M modThe connection occurs at a serine residue of the IL-2 portion, for example, a serine residue selected from S3, S4, S5, S74, S86, S98, S124, S126 and S129 based on SEQ ID NO: 2 or a corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional serine residues compared to SEQ ID NO: 2, the connection may also occur at the additional serine residue. It is understood that 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the serine residues of SEQ ID NO: 2 may be used for connection to M mod If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more serine residues than the IL-2 portion of SEQ ID NO: 2, more than 9 serine residues may be used to link to the M mod , i.e., up to the maximum number of serine residues present in the homologue or variant available for attachment of M mod In one embodiment, an M mod The M portion is linked to a serine residue in SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M portions may be the same or different. mod The moiety is linked to two serine residues of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the three M residues, which may be the same or different, are linked to the two serine residues of SEQ ID NO: 2 or a homologue or variant thereof. mod The moiety is linked to three serine residues of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the four M residues, which may be the same or different, mod The four serine residues of SEQ ID NO: 2 or its homologues or variants are linked to the moiety. In another embodiment, the five M residues, which may be the same or different, are linked to the four serine residues of SEQ ID NO: 2 or its homologues or variants. mod The moiety is linked to five serine residues of SEQ ID NO: 1 or a homologue or variant thereof. In another embodiment, the six M residues, which may be the same or different, mod The moiety is linked to the six serine residues of SEQ ID NO: 2 or a homologue or variant thereof.

[0183] In another embodiment, M mod The connection occurs at a tyrosine residue of the IL-2 portion, for example, selected from the tyrosine residues Y30, Y44 and Y106 based on SEQ ID NO: 2 or at the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional tyrosine residues compared to SEQ ID NO: 2, the connection may also occur at the additional tyrosine residue. It is understood that 1, 2 or 3 of the tyrosine residues of SEQ ID NO: 2 may be used for connection to M modIf the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more tyrosine residues than the IL-2 portion of SEQ ID NO: 2, more than 3 tyrosine residues may be used to link to the M mod , i.e., up to the maximum number of tyrosine residues present in the homologue or variant available for attachment of M mod In one embodiment, an M mod The M portion is linked to a tyrosine residue in SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M portions may be the same or different. mod The moiety is linked to two tyrosine residues of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the three M residues, which may be the same or different, are linked to two tyrosine residues of SEQ ID NO: 2 or a homologue or variant thereof. mod The moiety is linked to three tyrosine residues of SEQ ID NO: 2 or a homologue or variant thereof.

[0184] In another embodiment, M mod The attachment occurs at a histidine residue of the IL-2 portion, for example, selected from the histidine residues H15, H54 and H78 based on SEQ ID NO: 2 or at the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional histidine residues compared to SEQ ID NO: 2, the attachment may also occur at the additional histidine residue. It is understood that 1, 2 or 3 of the histidine residues of SEQ ID NO: 2 may be used for attachment to M mod If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more histidine residues than the IL-2 portion of SEQ ID NO: 2, more than 3 histidine residues may be used to link to the M mod , i.e., up to the maximum number of histidine residues present in the homologue or variant available for attachment of M mod In one embodiment, an M mod The moiety is linked to a histidine residue of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M residues, which may be the same or different, are linked to a histidine residue of SEQ ID NO: 2 or a homologue or variant thereof. mod The moiety is linked to two histidine residues of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the three M residues, which may be the same or different, are linked to two histidine residues of SEQ ID NO: 2 or a homologue or variant thereof. mod The moiety is linked to three histidine residues of SEQ ID NO: 2 or a homologue or variant thereof.

[0185] In another embodiment, M modThe linkage occurs at a tryptophan residue of the IL-2 portion, for example, a tryptophan residue at position W120 based on SEQ ID NO: 2 or at the corresponding position of a homologue or variant of SEQ ID NO: 2. When a homologue or variant of IL-2 comprises one or more additional tryptophan residues compared to SEQ ID NO: 2, the linkage may also occur at the additional tryptophan residues. If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more tryptophan residues than the IL-2 portion of SEQ ID NO: 2, more than one tryptophan residue may be used for linkage to M. mod , i.e., up to the maximum number of tryptophan residues present in the homologue or variant available for attachment of M mod In one embodiment, an M mod The moiety is linked to a tryptophan residue of SEQ ID NO: 2 or a homologue or variant thereof.

[0186] In another embodiment, M mod The connection occurs at an aspartate residue of the IL-2 portion, for example, selected from the aspartate residues D19, D83 and D108 based on SEQ ID NO: 2 or at the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional aspartate residues compared to SEQ ID NO: 2, the connection may also occur at the additional aspartate residue. It is understood that 1, 2 or 3 of the aspartate residues of SEQ ID NO: 2 may be used to connect to M mod If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more aspartate residues than the IL-2 portion of SEQ ID NO: 2, more than 3 aspartate residues may be used to link to the M mod , i.e., up to the maximum number of aspartic acid residues present in the homologue or variant available for attachment of M mod In one embodiment, an M mod The moiety is linked to an aspartic acid residue of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M residues, which may be the same or different, are mod The moiety is linked to two aspartic acid residues of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the three M residues, which may be the same or different, are linked to the two aspartic acid residues of SEQ ID NO: 2 or a homologue or variant thereof. mod The moiety is linked to three aspartic acid residues of SEQ ID NO: 2 or a homologue or variant thereof.

[0187] In one embodiment, M modThe attachment occurs at a glutamic acid residue of the IL-2 portion, for example, a glutamic acid residue selected from E14, E51, E59, E60, E61, E66, E67, E94, E99, E105, E109 and E115 based on SEQ ID NO: 2 or at the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional glutamic acid residues compared to SEQ ID NO: 2, the attachment may also occur at the additional glutamic acid residue. It is understood that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the glutamic acid residues of SEQ ID NO: 2 may be used for attachment to M mod If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more glutamic acid residues than the IL-2 portion of SEQ ID NO: 2, then more than 12 glutamic acid residues may be used to link to the M mod , i.e., up to the maximum number of glutamic acid residues present in the homologue or variant can be used to attach one or more modifying moieties. mod The moiety is linked to a glutamic acid residue of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M residues, which may be the same or different, are linked to a glutamic acid residue of SEQ ID NO: 2 or a homologue or variant thereof. mod The moiety is linked to two glutamic acid residues of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the three M residues, which may be the same or different, are linked to the two glutamic acid residues of SEQ ID NO: 2 or a homologue or variant thereof. mod The glutamic acid residues of SEQ ID NO:2 or its homologues or variants are linked to the glutamic acid residues of SEQ ID NO:2 or its homologues or variants. In another embodiment, the four M residues, which may be the same or different, are linked to the glutamic acid residues of SEQ ID NO:2 or its homologues or variants. mod The four glutamic acid residues of SEQ ID NO: 2 or its homologues or variants are linked to the moiety. In another embodiment, the five M residues, which may be the same or different, are linked to the moiety of SEQ ID NO: 2 or its homologues or variants. mod The five glutamic acid residues of SEQ ID NO: 2 or its homologues or variants are linked to the moiety. In another embodiment, the six M residues, which may be the same or different, are linked to the moiety of SEQ ID NO: 2 or its homologues or variants. mod The moiety is linked to the six glutamic acid residues of SEQ ID NO: 2 or a homologue or variant thereof.

[0188] In another embodiment, M modThe connection occurs at an arginine residue of the IL-2 portion, for example, selected from the group consisting of R37, R80, R82 and R119 based on SEQ ID NO: 2 or at the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional arginine residues compared to SEQ ID NO: 2, the connection may also occur at the additional arginine residue. It is understood that 1, 2, 3 or 4 of the arginine residues of SEQ ID NO: 2 may be used to connect to M mod If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more arginine residues than the IL-2 portion of SEQ ID NO: 2, more than 4 arginine residues may be used to link to the M mod , i.e., up to the maximum number of arginine residues present in the homologue or variant available for attachment of M mod In one embodiment, an M mod The M portion is linked to an arginine residue in SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M portions may be the same or different. mod The moiety is linked to two arginine residues of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the three M residues, which may be the same or different, are linked to two arginine residues of SEQ ID NO: 2 or a homologue or variant thereof. mod The arginine residues of SEQ ID NO:2 or its homologues or variants are linked to the arginine residues of SEQ ID NO:2 or its homologues or variants. In another embodiment, the four arginine residues, which may be the same or different, are linked to the arginine residues of SEQ ID NO:2 or its homologues or variants. mod The moiety is linked to four arginine residues of SEQ ID NO: 2 or a homologue or variant thereof.

[0189] In another embodiment, M mod The attachment occurs at a cysteine ​​residue of the IL-2 portion, for example, selected from the cysteine ​​residues C57 and C104 based on SEQ ID NO: 2 or at the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional cysteine ​​residues compared to SEQ ID NO: 2, the attachment may also occur at the additional cysteine ​​residue. It is understood that one or two of the cysteine ​​residues of SEQ ID NO: 2 may be used to attach M mod If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more cysteine ​​residues than the IL-2 portion of SEQ ID NO: 2, then more than 2 cysteine ​​residues may be used to link to the M mod , i.e., up to the maximum number of cysteine ​​residues present in the homologue or variant available for attachment of M mod In one embodiment, an M modThe moiety is linked to a cysteine ​​residue of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M residues, which may be the same or different, are linked to a cysteine ​​residue of SEQ ID NO: 2 or a homologue or variant thereof. mod The moiety is linked to two cysteine ​​residues of SEQ ID NO: 2 or a homologue or variant thereof.

[0190] In another embodiment, M mod The attachment occurs at a methionine residue of the IL-2 portion, for example, a methionine residue selected from M22, M38, M45 and M103 based on SEQ ID NO:2 or at a corresponding position of a homologue or variant of SEQ ID NO:2. When a homologue or variant of IL-2 comprises one or more additional methionine residues compared to SEQ ID NO:2, attachment may also occur at the additional methionine residues. It is understood that 1, 2, 3 or 4 of the methionine residues of SEQ ID NO:2 may be used to attach one or more modified portions. If the IL-2 portion is a homologue or variant of SEQ ID NO:2 and comprises more methionine residues than the IL-2 portion of SEQ ID NO:2, more than 4 methionine residues may be used to attach M mod , i.e., up to the maximum number of methionine residues present in the homologue or variant available for attachment of M mod In one embodiment, an M mod The moiety is linked to a methionine residue of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M residues, which may be the same or different, are mod The moiety is linked to two methionine residues of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the three methionine residues, which may be the same or different, are linked to the methionine residues of SEQ ID NO: 2 or a homologue or variant thereof. mod The methionine residues of SEQ ID NO: 2 or its homologues or variants are linked to the methionine residues of SEQ ID NO: 2 or its homologues or variants. In another embodiment, the four methionine residues, which may be the same or different, are linked to the methionine residues of SEQ ID NO: 2 or its homologues or variants. mod The moiety is linked to four methionine residues of SEQ ID NO: 2 or a homologue or variant thereof.

[0191] In another embodiment, M mod The connection occurs at a glutamine residue of the IL-2 portion, for example, selected from the glutamine residues Q10, Q12, Q21, Q56, Q73 and Q125 based on SEQ ID NO: 2 or at the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional glutamine residues compared to SEQ ID NO: 2, the connection may also occur at the additional glutamine residue. It is understood that 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the glutamine residues of SEQ ID NO: 2 may be used for connection to Mmod If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and comprises more glutamine residues than the IL-2 portion of SEQ ID NO: 2, more than 6 glutamine residues may be used to link to the M mod , i.e., up to the maximum number of glutamine residues present in the homologue or variant available for attachment of M mod In one embodiment, an M mod The moiety is linked to a glutamine residue of SEQ ID NO: 2 or a homologue or variant thereof. In another embodiment, the two M residues may be the same or different. mod In another embodiment, the three M residues may be the same or different. mod In another embodiment, the four M residues may be the same or different. mod In another embodiment, the five M residues may be the same or different. mod In another embodiment, the six residues M mod The six glutamine residues which may be identical or different are linked to SEQ ID NO: 2 or a homologue or variant thereof.

[0192] It will be appreciated that in some embodiments, a biased IL-2 moiety may have amino acids linked to more than one type of amino acid residue, such as cysteine ​​and lysine. mod part.

[0193] M mod The connection to the IL-2 portion is via a stable covalent linkage. In some embodiments, the IL-2 portion and M mod The linkage between the IL-2 moiety and the M is via an amide. mod The links between the parts are Partial.

[0194] Preferably, at least one M mod The attachment of the moiety occurs at an amino acid position of the IL-2 moiety known to be involved in the attachment to IL-2Rα. Thus, preferably, at least one M mod The linking of the moieties results in a significantly reduced affinity of the IL-2 moiety, preferably a variant of the IL-2 moiety of SEQ ID NO: 2, for IL-2Rαβ compared to aldesleukin, ie a biased IL-2 moiety is generated.

[0195] Preferably, M mod The connection occurs at an amino acid position selected from K34, R37, M38, T40, F41, K42, F43, Y44, E61 and L71 based on SEQ ID NO: 2 or a corresponding position of a homologue or variant thereof. Even more preferably, M mod The connection occurs at an amino acid position selected from F41, Y44, E61 and L71 based on SEQ ID NO: 2 or the corresponding position of a homologue or variant thereof. It is understood that not all of these amino acid positions include amino acids that would allow direct conjugation to M mod functional groups, connecting M at these amino acid positions mod Some steps may be required before, such as replacing the naturally occurring amino acid with a different amino acid or performing some chemical modification. mod The attachment of M can occur at these positions at a naturally occurring amino acid or at an amino acid that replaces a naturally occurring amino acid at that particular position, and the attachment site can be a proteinaceous or non-proteinaceous amino acid, both of which are implemented as described above. mod The connection occurs at the corresponding position of amino acid position K34 or a homologue or variant thereof based on SEQ ID NO: 2. In some embodiments, M mod The connection occurs at the corresponding position of amino acid position R37 or a homologue or variant thereof based on SEQ ID NO: 2. In some embodiments, M mod The connection occurs at the corresponding position of amino acid position M38 or a homologue or variant thereof based on SEQ ID NO: 2. In some embodiments, M mod The connection occurs at the corresponding position of amino acid position T40 based on SEQ ID NO: 2 or a homologue or variant thereof. In some embodiments, M mod The connection occurs at the corresponding position of amino acid position F41 or a homologue or variant thereof based on SEQ ID NO: 2. In some embodiments, M mod The connection occurs at the corresponding position of amino acid position K42 based on SEQ ID NO: 2 or a homologue or variant thereof. In some embodiments, M mod The connection occurs at the corresponding position of amino acid position F43 or a homologue or variant thereof based on SEQ ID NO: 2. In some embodiments, M mod The connection occurs at the corresponding position of amino acid position Y44 or a homologue or variant thereof based on SEQ ID NO: 2. In some embodiments, M mod The connection occurs at the corresponding position of amino acid position E61 based on SEQ ID NO: 2 or a homologue or variant thereof. In some embodiments, Mmod The connection occurs at the corresponding position based on amino acid position L71 of SEQ ID NO: 2 or a homologue or variant thereof.

[0196] In some embodiments, M mod Having formula (A-1)

[0197]

[0198] in

[0199] -FG- is the link;

[0200] -SP- is a spacer moiety; and

[0201] -POL is a polymer.

[0202] In some embodiments, -FG- of formula (A-1) is The dashed lines marked with an asterisk indicate the sulfur linkage to the IL-2 moiety, and the unmarked dashed lines indicate the linkage to -SP-. The sulfur may be the sulfur provided by the side chain of cysteine.

[0203] In some embodiments, -FG- of formula (A-1) is Wherein the dotted line marked with an asterisk refers to the connection with the nitrogen of the IL-2 part, and the unmarked dotted line refers to the connection with -SP-. The nitrogen can be the nitrogen of the N-terminal amine from the IL-2 part or the nitrogen of the side chain of lysine. In some embodiments, the nitrogen is the nitrogen of the N-terminal amine of the IL-2 part. In some embodiments, the nitrogen is the nitrogen of the side chain of lysine of the IL-2 part.

[0204] In some embodiments, -FG- of formula (A-1) is Wherein the dashed line marked with an asterisk refers to the sulfur connection to the nitrogen of the IL-2 part, the unmarked dashed line refers to the connection to -SP-, and a1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. The sulfur can be the sulfur provided by the side chain of cysteine, and the nitrogen can be the nitrogen from the N-terminal amine of the IL-2 part or the nitrogen of the side chain of lysine. In some embodiments, the dashed line marked with an asterisk refers to the connection to the sulfur, and the sulfur is provided by the side chain of cysteine. In some embodiments, a1 is an integer from 1 to 8. In some embodiments, a1 is an integer from 1 to 6. In some embodiments, a1 is an integer from 1 to 4. In some embodiments, a1 is 1. In some embodiments, a1 is 2. In some embodiments, a1 is 3. In some embodiments, a1 is 4. In some embodiments, a1 is 5. In some embodiments, a1 is 6.

[0205] In some embodiments, -FG- of formula (A-1) is wherein dashed lines marked with an asterisk refer to sulfur linkage to a nitrogen of the IL-2 moiety and unmarked dashed lines refer to linkage to -SP-; and a2 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In some embodiments, a2 is an integer from 1 to 8. In some embodiments, a2 is an integer from 1 to 6. In some embodiments, a2 is an integer from 1 to 4. In some embodiments, a2 is 1. In some embodiments, a2 is 2. In some embodiments, a2 is 3. In some embodiments, a2 is 4. In some embodiments, a2 is 5. In some embodiments, a2 is 6. The sulfur may be the sulfur provided by the side chain of cysteine ​​and the nitrogen may be the nitrogen from the N-terminal amine of the IL-2 moiety or the nitrogen of the side chain of lysine. In some embodiments, the dashed line marked with an asterisk refers to the linkage to the sulfur, which is provided by the side chain of cysteine.

[0206] In some embodiments, -SP- of formula (A-1) is selected from C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more identical or different R 9 Substitute, and where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more selected from -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 )-groups;

[0207] T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbocyclic group and 8- to 30-membered heteropolycyclic group; wherein each T is independently optionally replaced by one or more identical or different R 9 replace;

[0208] -R 9 are independently selected from halogen, -CN, oxo (=O), -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 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0209] -R 10 , -R 10a , -R 11 , -R 11a and -R 11b are each independently selected from -H and C 1-6 Alkyl, where C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

[0210] In some embodiments, -SP- of formula (A-1) is C 1-20 Alkyl, the C 1-20 The alkyl group is optionally substituted with one or more -R 9 Substitution and the C 1-20 The alkyl group is optionally substituted with one or more selected from -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 )-; wherein -R 9 Each selected from C 1-6 Alkyl; and -R 10 and -R 10a are each independently selected from -H and C 1-6 alkyl.

[0211] In some embodiments, -SP- of formula (A-1) is C 1-10 Alkyl, the C 1-10 The alkyl group is optionally substituted with one or more -R 9 Substitution and the C 1-10 The alkyl group is optionally substituted with one or more selected from -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 )-; wherein -R 9 Each selected from C 1-6 Alkyl; and -R 10 and -R 10a are each independently selected from -H and C 1-6 alkyl.

[0212] In some embodiments, the -POL of formula (A-1) is a PEG-based polymer. In some embodiments, the -POL has formula (A-1i)

[0213]

[0214] in

[0215] Dashed lines indicate connections with -SP-;

[0216] m is 0 or 1;

[0217] p is an integer between 12 and 22700; and

[0218] q is selected from 1, 2, 3, 4, 5 and 6.

[0219] In some embodiments, m of Formula (A-1i) is 0. In some embodiments, m of Formula (A-1i) is 1.

[0220] In some embodiments, p of formula (A-1i) is an integer from 23 to 227000, such as 45 to 11300, or 69 to 4540, or 114 to 2700. In some embodiments, p of formula (A-1i) is about 12. In some embodiments, p of formula (A-1i) is about 23. In some embodiments, p of formula (A-1i) is about 46. In some embodiments, p of formula (A-1i) is about 68. In some embodiments, p of formula (A-1i) is about 90. In some embodiments, p of formula (A-1i) is about 112. In some embodiments, p of formula (A-1i) is about 170. In some embodiments, p of formula (A-1i) is about 227. In some embodiments, p of formula (A-1i) is about 340. In some embodiments, p of formula (A-1i) is about 450. In some embodiments, p of Formula (A-1i) is about 680. In some embodiments, p of Formula (A-1i) is about 900. In some embodiments, p of Formula (A-1i) is about 1130. In some embodiments, p of Formula (A-1i) is about 1350. In some embodiments, p of Formula (A-1i) is about 1590. In some embodiments, p of Formula (A-1i) is about 1800. In some embodiments, p of Formula (A-1i) is about 2045. In some embodiments, p of Formula (A-1i) is about 2275.

[0221] In some embodiments, q of formula (A-1i) is 1. In some embodiments, q of formula (A-1i) is 2. In some embodiments, q of formula (A-1i) is 3. In some embodiments, q of formula (A-1i) is 4. In some embodiments, q of formula (A-1i) is 5. In some embodiments, q of formula (A-1i) is 6.

[0222] In some embodiments, the -POL of formula (A-1) has the formula (A-1ii)

[0223]

[0224] in

[0225] Dashed lines indicate connections with -SP-;

[0226] FG is a functional group;

[0227] m is 0 or 1;

[0228] p is an integer between 12 and 22700; and

[0229] q is selected from 1, 2, 3, 4, 5 and 6.

[0230] If M in formula (A-1) mod The moiety will be conjugated to other moieties, such as one or more -L 1 -L 2 It will be appreciated that if -POL has the formula (A-1ii), then the compound is a reagent and upon addition of one or more such moieties, e.g. one or more -L 1 -L 2 After conjugation of the -Z moiety to the functional group of the reagent, FG is no longer present, but a link has been formed with a suitable functional group present in the reagent form of the one or more other moieties.

[0231] It is also understood that the conjugation of the moiety to M mod , for example -L 1 -L 2 Other attachment sites for the -Z moiety are also possible.

[0232] In some embodiments, m of Formula (A-1ii) is 0. In some embodiments, m of Formula (A-1ii) is 1.

[0233] In some embodiments, p of formula (A-1ii) is an integer from 23 to 227000, such as 45 to 11300, or 69 to 4540, or 114 to 2700. In some embodiments, p of formula (A-1ii) is about 12. In some embodiments, p of formula (A-1ii) is about 23. In some embodiments, p of formula (A-1ii) is about 46. In some embodiments, p of formula (A-1ii) is about 68. In some embodiments, p of formula (A-1ii) is about 90. In some embodiments, p of formula (A-1ii) is about 112. In some embodiments, p of formula (A-1ii) is about 170. In some embodiments, p of formula (A-1ii) is about 227. In some embodiments, p of formula (A-1ii) is about 340. In some embodiments, p of formula (A-1ii) is about 450. In some embodiments, p of formula (A-1ii) is about 680. In some embodiments, p of formula (A-1ii) is about 900. In some embodiments, p of formula (A-1ii) is about 1130. In some embodiments, p of formula (A-1ii) is about 1350. In some embodiments, p of formula (A-1ii) is about 1590. In some embodiments, p of formula (A-1ii) is about 1800. In some embodiments, p of formula (A-1ii) is about 2045. In some embodiments, p of formula (A-1ii) is about 2275.

[0234] In some embodiments, q of formula (A-1ii) is 1. In some embodiments, q of formula (A-1ii) is 2. In some embodiments, q of formula (A-1ii) is 3. In some embodiments, q of formula (A-1ii) is 4. In some embodiments, q of formula (A-1ii) is 5. In some embodiments, q of formula (A-1ii) is 6.

[0235] If other parts, such as -L 1 -L 2 The -Z moiety is conjugated to M via the -POL moiety of formula (A-1) mod , then the -POL portion may have formula (A-1iii), (A-1iv), (A-1v) or (A-1vi):

[0236]

[0237] in

[0238] The dashed line marked with an asterisk indicates the difference between the two parts, such as -L 1 -L 2 -Z part connection;

[0239] The unlabeled dashed line indicates the connection to -SP-; and

[0240] m, p and q are used as defined in formula (A-1i).

[0241] In some embodiments, other moieties, such as -L 1 -L 2 The -Z moiety is conjugated to M via the -POL moiety of formula (A-1) mod , producing the -POL portion of formula (A-1iii). In some embodiments, other portions, such as -L 1 -L 2 The -Z moiety is conjugated to M via the -POL moiety of formula (A-1) mod , producing the -POL portion of formula (A-1iv). In some embodiments, other portions, such as -L 1 -L 2 The -Z moiety is conjugated to M via the -POL moiety of formula (A-1) mod , producing the -POL portion of formula (A-iv). In some embodiments, other portions, such as -L 1 -L 2 The -Z moiety is conjugated to M via the -POL moiety of formula (A-1) mod , producing the -POL portion of formula (A-1vi).

[0242] In some embodiments, the -POL of formula (A-1) is a hyaluronic acid-based polymer.

[0243] In some embodiments, M mod Having formula (A-1a)

[0244]

[0245] in

[0246] Dashed lines marked with asterisks indicate sulfur linkages to side chains of amino acid residues of the IL-2 moiety;

[0247] b1 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20;

[0248] b2 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; and

[0249] b3 is an integer between 12 and 22700.

[0250] In some embodiments, b1 of formula (A-1a) is an integer from 1 to 8. In some embodiments, b1 of formula (A-1a) is an integer from 1 to 6. In some embodiments, b1 of formula (A-1a) is an integer from 1 to 4. In some embodiments, b1 of formula (A-1a) is 1. In some embodiments, b1 of formula (A-1a) is 2. In some embodiments, b1 of formula (A-1a) is 3. In some embodiments, b1 of formula (A-1a) is 4. In some embodiments, b1 of formula (A-1a) is 5. In some embodiments, b1 of formula (A-1a) is 6.

[0251] In some embodiments, b2 of formula (A-1a) is an integer from 1 to 8. In some embodiments, b2 of formula (A-1a) is an integer from 1 to 6. In some embodiments, b2 of formula (A-1a) is an integer from 1 to 4. In some embodiments, b2 of formula (A-1a) is 1. In some embodiments, b2 of formula (A-1a) is 2. In some embodiments, b2 of formula (A-1a) is 3. In some embodiments, b2 of formula (A-1a) is 4. In some embodiments, b2 of formula (A-1a) is 5. In some embodiments, b2 of formula (A-1a) is 6.

[0252] In some embodiments, b3 of formula (A-1a) is an integer from 23 to 227000, such as 45 to 11300, or 69 to 4540, or 114 to 2700. In some embodiments, b3 of formula (A-1a) is about 12. In some embodiments, b3 of formula (A-1a) is about 23. In some embodiments, b3 of formula (A-1a) is about 46. In some embodiments, b3 of formula (A-1a) is about 68. In some embodiments, b3 of formula (A-1a) is about 90. In some embodiments, b3 of formula (A-1a) is about 112. In some embodiments, b3 of formula (A-1a) is about 170. In some embodiments, b3 of formula (A-1a) is about 227. In some embodiments, b3 of formula (A-1a) is about 340. In some embodiments, b3 of formula (A-1a) is about 450. In some embodiments, b3 of formula (A-1a) is about 680. In some embodiments, b3 of formula (A-1a) is about 900. In some embodiments, b3 of formula (A-1a) is about 1130. In some embodiments, b3 of formula (A-1a) is about 1350. In some embodiments, b3 of formula (A-1a) is about 1590. In some embodiments, b3 of formula (A-1a) is about 1800. In some embodiments, b3 of formula (A-1a) is about 2045. In some embodiments, b3 of formula (A-1a) is about 2275.

[0253] In some embodiments, b1 of formula (A-1a) is 2, b2 of formula (A-1a) is 3, and b3 of formula (A-1a) is about 12. In some embodiments, b1 of formula (A-1a) is 2, b2 of formula (A-1a) is 3, and b3 of formula (A-1a) is about 23. In some embodiments, b1 of formula (A-1a) is 2, b2 of formula (A-1a) is 3, and b3 of formula (A-1a) is about 46. In some embodiments, b1 of formula (A-1a) is 2, b2 of formula (A-1a) is 3, and b3 of formula (A-1a) is about 68. In some embodiments, b1 of formula (A-1a) is 2, b2 of formula (A-1a) is 3, and b3 of formula (A-1a) is about 90. In some embodiments, b1 of formula (A-1a) is 2, b2 of formula (A-1a) is 3, and b3 of formula (A-1a) is about 112. In some embodiments, b1 of formula (A-1a) is 2, b2 of formula (A-1a) is 3, and b3 of formula (A-1a) is about 170. In some embodiments, b1 of formula (A-1a) is 2, b2 of formula (A-1a) is 3, and b3 of formula (A-1a) is about 227. In some embodiments, b1 of formula (A-1a) is 2, b2 of formula (A-1a) is 3, and b3 of formula (A-1a) is about 340. In some embodiments, b1 of formula (A-1a) is 2, b2 of formula (A-1a) is 3, and b3 of formula (A-1a) is about 450.

[0254] In some embodiments, M mod Having formula (A-1b)

[0255]

[0256] in

[0257] Dashed lines marked with asterisks indicate sulfur linkages to side chains of amino acid residues of the IL-2 moiety;

[0258] c1 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20;

[0259] c2 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; and

[0260] c3 is an integer between 12 and 22700.

[0261] In some embodiments, c1 of formula (A-1b) is an integer from 1 to 8. In some embodiments, c1 of formula (A-1b) is an integer from 1 to 6. In some embodiments, c1 of formula (A-1b) is an integer from 1 to 4. In some embodiments, c1 of formula (A-1b) is 1. In some embodiments, c1 of formula (A-1b) is 2. In some embodiments, c1 of formula (A-1b) is 3. In some embodiments, c1 of formula (A-1b) is 4. In some embodiments, c1 of formula (A-1b) is 5. In some embodiments, c1 of formula (A-1b) is 6.

[0262] In some embodiments, c2 of formula (A-1b) is an integer from 1 to 8. In some embodiments, c2 of formula (A-1b) is an integer from 1 to 6. In some embodiments, c2 of formula (A-1b) is an integer from 1 to 4. In some embodiments, c2 of formula (A-1b) is 1. In some embodiments, c2 of formula (A-1b) is 2. In some embodiments, c2 of formula (A-1b) is 3. In some embodiments, c2 of formula (A-1b) is 4. In some embodiments, c2 of formula (A-1b) is 5. In some embodiments, c2 of formula (A-1b) is 6.

[0263] In some embodiments, c3 of formula (A-1b) is an integer from 23 to 227000, such as 45 to 11300, or 69 to 4540, or 114 to 2700. In some embodiments, c3 of formula (A-1b) is about 12. In some embodiments, c3 of formula (A-1b) is about 23. In some embodiments, c3 of formula (A-1b) is about 46. In some embodiments, c3 of formula (A-1b) is about 68. In some embodiments, c3 of formula (A-1b) is about 90. In some embodiments, c3 of formula (A-1b) is about 112. In some embodiments, c3 of formula (A-1b) is about 170. In some embodiments, c3 of formula (A-1b) is about 227. In some embodiments, c3 of formula (A-1b) is about 340. In some embodiments, c3 of formula (A-1b) is about 450. In some embodiments, c3 of formula (A-1b) is about 680. In some embodiments, c3 of formula (A-1b) is about 900. In some embodiments, c3 of formula (A-1b) is about 1130. In some embodiments, c3 of formula (A-1b) is about 1350. In some embodiments, c3 of formula (A-1b) is about 1590. In some embodiments, c3 of formula (A-1b) is about 1800. In some embodiments, c3 of formula (A-1b) is about 2045. In some embodiments, c3 of formula (A-1b) is about 2275.

[0264] In some embodiments, c1 of formula (A-1b) is 2, c2 of formula (A-1b) is 3, and c3 of formula (A-1b) is about 12. In some embodiments, c1 of formula (A-1b) is 2, c2 of formula (A-1b) is 3, and c3 of formula (A-1b) is about 23. In some embodiments, c1 of formula (A-1b) is 2, c2 of formula (A-1b) is 3, and c3 of formula (A-1b) is about 46. In some embodiments, c1 of formula (A-1b) is 2, c2 of formula (A-1b) is 3, and c3 of formula (A-1b) is about 68. In some embodiments, c1 of formula (A-1b) is 2, c2 of formula (A-1b) is 3, and c3 of formula (A-1b) is about 90. In some embodiments, c1 of formula (A-1b) is 2, c2 of formula (A-1b) is 3, and c3 of formula (A-1b) is about 112. In some embodiments, c1 of formula (A-1b) is 2, c2 of formula (A-1b) is 3, and c3 of formula (A-1b) is about 170. In some embodiments, c1 of formula (A-1b) is 2, c2 of formula (A-1b) is 3, and c3 of formula (A-1b) is about 227. In some embodiments, c1 of formula (A-1b) is 2, c2 of formula (A-1b) is 3, and c3 of formula (A-1b) is about 340. In some embodiments, c1 of formula (A-1b) is 2, c2 of formula (A-1b) is 3, and c3 of formula (A-1b) is about 450.

[0265] In some embodiments, M mod Having formula (A-1c)

[0266]

[0267] in

[0268] Dashed lines marked with asterisks indicate sulfur linkages to side chains of amino acid residues of the IL-2 moiety;

[0269] d1 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20;

[0270] d2 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; and

[0271] d3 is an integer between 12 and 22700.

[0272] In some embodiments, d1 of formula (A-1c) is an integer from 1 to 8. In some embodiments, d1 of formula (A-1c) is an integer from 1 to 6. In some embodiments, d1 of formula (A-1c) is an integer from 1 to 4. In some embodiments, d1 of formula (A-1c) is 1. In some embodiments, d1 of formula (A-1c) is 2. In some embodiments, d1 of formula (A-1c) is 3. In some embodiments, d1 of formula (A-1c) is 4. In some embodiments, d1 of formula (A-1c) is 5. In some embodiments, d1 of formula (A-1c) is 6.

[0273] In some embodiments, d2 of formula (A-1c) is an integer from 1 to 8. In some embodiments, d2 of formula (A-1c) is an integer from 1 to 6. In some embodiments, d2 of formula (A-1c) is an integer from 1 to 4. In some embodiments, d2 of formula (A-1c) is 1. In some embodiments, d2 of formula (A-1c) is 2. In some embodiments, d2 of formula (A-1c) is 3. In some embodiments, d2 of formula (A-1c) is 4. In some embodiments, d2 of formula (A-1c) is 5. In some embodiments, d2 of formula (A-1c) is 6.

[0274] In some embodiments, d3 of formula (A-1c) is an integer from 23 to 227000, such as 45 to 11300, or 69 to 4540, 114 to 2700, or 160 to 900. In some embodiments, d3 of formula (A-1c) is about 12. In some embodiments, d3 of formula (A-1c) is about 23. In some embodiments, d3 of formula (A-1c) is about 46. In some embodiments, d3 of formula (A-1c) is about 68. In some embodiments, d3 of formula (A-1c) is about 90. In some embodiments, d3 of formula (A-1c) is about 112. In some embodiments, d3 of formula (A-1c) is about 170. In some embodiments, d3 of formula (A-1c) is about 227. In some embodiments, d3 of formula (A-1c) is about 340. In some embodiments, the d3 of formula (A-1c) is about 450. In some embodiments, the d3 of formula (A-1c) is about 680. In some embodiments, the d3 of formula (A-1c) is about 900. In some embodiments, the d3 of formula (A-1c) is about 1130. In some embodiments, the d3 of formula (A-1c) is about 1350. In some embodiments, the d3 of formula (A-1c) is about 1590. In some embodiments, the d3 of formula (A-1c) is about 1800. In some embodiments, the d3 of formula (A-1c) is about 2045. In some embodiments, the d3 of formula (A-1c) is about 2275.

[0275] In some embodiments, d1 of formula (A-1c) is 2, d2 of formula (A-1c) is 3, and d3 of formula (A-1cd) is about 12. In some embodiments, d1 of formula (A-1c) is 2, d2 of formula (A-1c) is 3, and d3 of formula (A-1c) is about 23. In some embodiments, d1 of formula (A-1c) is 2, d2 of formula (A-1c) is 3, and d3 of formula (A-1c) is about 46. In some embodiments, d1 of formula (A-1c) is 2, d2 of formula (A-1c) is 3, and d3 of formula (A-1c) is about 68. In some embodiments, d1 of formula (A-1c) is 2, d2 of formula (A-1c) is 3, and d3 of formula (A-1c) is about 90. In some embodiments, d1 of formula (A-1c) is 2, d2 of formula (A-1c) is 3, and d3 of formula (A-1c) is about 112. In some embodiments, d1 of formula (A-1c) is 2, d2 of formula (A-1c) is 3, and d3 of formula (A-1c) is about 170. In some embodiments, d1 of formula (A-1c) is 2, d2 of formula (A-1c) is 3, and d3 of formula (A-1c) is about 227. In some embodiments, d1 of formula (A-1c) is 2, d2 of formula (A-1c) is 3, and d3 of formula (A-1c) is about 340. In some embodiments, d1 of formula (A-1c) is 2, d2 of formula (A-1c) is 3, and d3 of formula (A-1c) is about 450.

[0276] The biased IL-2 portion may comprise an IL-2 portion comprising at least one amino acid mutation, such as one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, seven amino acid mutations, eight amino acid mutations, nine amino acid mutations or ten amino acid mutations. The amino acid mutation preferably occurs at a position involved in binding to IL-2Rα. Thus, preferably, the at least amino acid mutation results in a reduced affinity of the biased IL-2 portion for IL-2Rαβ compared to aldesleukin.

[0277] Preferably, the 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 the corresponding position of a homologue or variant thereof. Even more preferably, the 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 the corresponding position of a homologue or variant thereof. In some embodiments, the at least one amino acid mutation occurs at the corresponding position of amino acid position K34 based on SEQ ID NO: 2, or a homologue or variant thereof. In some embodiments, the at least one amino acid mutation occurs at the corresponding position of amino acid position R37 based on SEQ ID NO: 2, or a homologue or variant thereof. In some embodiments, the at least one amino acid mutation occurs at the corresponding position of amino acid position M38 based on SEQ ID NO: 2, or a homologue or variant thereof. In some embodiments, the at least one amino acid mutation occurs at the corresponding position of amino acid position T40 based on SEQ ID NO: 2, or a homologue or variant thereof. In some embodiments, the at least one amino acid mutation occurs at the corresponding position of amino acid position F41 based on SEQ ID NO: 2 or a homologue or variant thereof. In some embodiments, the at least one amino acid mutation occurs at the corresponding position of amino acid position K42 based on SEQ ID NO: 2 or a homologue or variant thereof. In some embodiments, the at least one amino acid mutation occurs at the corresponding position of amino acid position F43 based on SEQ ID NO: 2 or a homologue or variant thereof. In some embodiments, the at least one amino acid mutation occurs at the corresponding position of amino acid position Y44 based on SEQ ID NO: 2 or a homologue or variant thereof. In some embodiments, the at least one amino acid mutation occurs at the corresponding position of amino acid position E61 based on SEQ ID NO: 2 or a homologue or variant thereof. In some embodiments, the at least one amino acid mutation occurs at the corresponding position of amino acid position L71 based on SEQ ID NO: 2 or a homologue or variant thereof.

[0278] In some embodiments, the mutation is the replacement of a naturally occurring amino acid with an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, lysine, serine, threonine, tryptophan and tyrosine. In some embodiments, a naturally occurring amino acid is replaced by alanine. In some embodiments, a naturally occurring amino acid is replaced by arginine. In some embodiments, a naturally occurring amino acid is replaced by asparagine. In some embodiments, a naturally occurring amino acid is replaced by aspartic acid. In some embodiments, a naturally occurring amino acid is replaced by cysteine. In some embodiments, a naturally occurring amino acid is replaced by glutamine. In some embodiments, a naturally occurring amino acid is replaced by glutamic acid. In some embodiments, a naturally occurring amino acid is replaced by glycine. In some embodiments, a naturally occurring amino acid is replaced by histidine. In some embodiments, a naturally occurring amino acid is replaced by lysine. In some embodiments, a naturally occurring amino acid is replaced by serine. In some embodiments, a naturally occurring amino acid is replaced by threonine. In some embodiments, a naturally occurring amino acid is replaced by tryptophan. In some embodiments, a naturally occurring amino acid is replaced by tyrosine. In some embodiments, the mutation is to replace a naturally occurring amino acid with an amino acid residue selected from arginine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, serine, threonine, tryptophan and tyrosine. In some embodiments, the mutation is to replace a naturally occurring amino acid with an amino acid residue selected from cysteine, glutamic acid, lysine, serine, threonine and tyrosine. In some embodiments, the naturally occurring amino acid is replaced by a non-protein amino acid. The embodiment of the non-protein amino acid is as described above.

[0279] In one embodiment, the IL-2 biased IL-2 portion comprises an amino acid mutation selected from K34A, K34C, K34G, K34S, K34T, K34Q, K34E, K34N, K34D, K34H, K34W, K34Y and K34R based on SEQ ID NO: 2 or the corresponding positions of its homologues or variants. In some embodiments, the IL-2 portion comprises a K34A mutation. In some embodiments, the IL-2 portion comprises a K34C mutation. In some embodiments, the IL-2 portion comprises a K34G mutation. In some embodiments, the IL-2 portion comprises a K34S mutation. In some embodiments, the IL-2 portion comprises a K34T mutation. In some embodiments, the IL-2 portion comprises a K34Q mutation. In some embodiments, the IL-2 portion comprises a K34E mutation. In some embodiments, the IL-2 portion comprises a K34D mutation. In some embodiments, the IL-2 portion comprises a K34H mutation. In some embodiments, the IL-2 portion comprises a K34W mutation. In some embodiments, the IL-2 portion comprises a K34Y mutation. In some embodiments, the IL-2 portion comprises a K34R mutation.

[0280] In one embodiment, the IL-2 portion biased IL-2 comprises an amino acid mutation selected from R37A, R37C, R37G, R37S, R37T, R37Q, R37E, R37N, R37D, R37H, R37W, R37Y and R37K based on SEQ ID NO: 2 or the corresponding positions of its homologues or variants. In some embodiments, the IL-2 portion comprises an R37A mutation. In some embodiments, the IL-2 portion comprises an R37C mutation. In some embodiments, the IL-2 portion comprises an R37G mutation. In some embodiments, the IL-2 portion comprises an R37S mutation. In some embodiments, the IL-2 portion comprises an R37T mutation. In some embodiments, the IL-2 portion comprises an R37Q mutation. In some embodiments, the IL-2 portion comprises an R37E mutation. In some embodiments, the IL-2 portion comprises an R37N mutation. In some embodiments, the IL-2 portion comprises an R37D mutation. In some embodiments, the IL-2 portion comprises an R37H mutation. In some embodiments, the IL-2 portion comprises an R37K mutation. In some embodiments, the IL-2 portion comprises an R37W mutation. In some embodiments, the IL-2 portion comprises an R37Y mutation. In some embodiments, the IL-2 portion comprises an R37K mutation.

[0281] In one embodiment, the at least one amino acid mutation is selected from 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 based on SEQ ID NO:2, or the corresponding positions of their homologs or variants.

[0282] In some embodiments, the IL-2 portion that is biased against IL-2 comprises an amino acid mutation selected from the corresponding positions of F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R, F41H, F41W, F41Y and F41K based on SEQ ID NO: 2 or its homologues or variants. In some embodiments, the IL-2 portion comprises an amino acid mutation selected from the corresponding positions of F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R and F41K based on SEQ ID NO: 2 or its homologues or variants. In one embodiment, the IL-2 portion comprises an F41A mutation. In another embodiment, the IL-2 portion comprises an F41C mutation. In another embodiment, the IL-2 portion comprises an F41G mutation. In another embodiment, the IL-2 portion comprises a F41S mutation. In another embodiment, the IL-2 portion comprises a F41T mutation. In another embodiment, the IL-2 portion comprises a F41Q mutation. In another embodiment, the IL-2 portion comprises a F41E mutation. In another embodiment, the IL-2 portion comprises a F41N mutation. In another embodiment, the IL-2 portion comprises a F41D mutation. In another embodiment, the IL-2 portion comprises a F41R mutation. In another embodiment, the IL-2 portion comprises a F41H mutation. In another embodiment, the IL-2 portion comprises a F41W mutation. In another embodiment, the IL-2 portion comprises a F41Y mutation. In another embodiment, the IL-2 portion comprises a F41K mutation.

[0283] In some embodiments, the IL-2 portion comprises an amino acid mutation selected from Y44A, Y44C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R, Y44H, Y44W and Y44K based on SEQ ID NO: 2 or a homologue or variant thereof. In some embodiments, the IL-2 portion comprises an amino acid mutation selected from Y44A, Y44C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R and Y44K based on SEQ ID NO: 2 or a homologue or variant thereof. In one embodiment, the IL-2 portion comprises a Y44A mutation. In another embodiment, the IL-2 portion comprises a Y44C mutation. In another embodiment, the IL-2 portion comprises a Y44G mutation. In another embodiment, the IL-2 portion comprises a Y44S mutation. In another embodiment, the IL-2 portion comprises a Y44T mutation. In another embodiment, the IL-2 portion comprises a Y44Q mutation. In another embodiment, the IL-2 portion comprises a Y44E mutation. In another embodiment, the IL-2 portion comprises a Y44N mutation. In another embodiment, the IL-2 portion comprises a Y44D mutation. In another embodiment, the IL-2 portion comprises a Y44R mutation. In another embodiment, the IL-2 portion comprises a Y44H mutation. In another embodiment, the IL-2 portion comprises a Y44W mutation. In another embodiment, the IL-2 portion comprises a Y44K mutation.

[0284] In some embodiments, the IL-2 portion comprises an amino acid mutation selected from L71G, L71C, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R, L71H, L71W, L71Y and L71K based on SEQ ID NO: 2 or a homologue or variant thereof. In some embodiments, the IL-2 portion comprises an amino acid mutation selected from L71G, L71C, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R and L71K based on SEQ ID NO: 2 or a homologue or variant thereof. In one embodiment, the IL-2 portion comprises an L72G mutation. In another embodiment, the IL-2 portion comprises an L72C mutation. In another embodiment, the IL-2 portion comprises an L72A mutation. In another embodiment, the IL-2 portion comprises an L72S mutation. In another embodiment, the IL-2 portion comprises an L72T mutation. In another embodiment, the IL-2 portion comprises an L72Q mutation. In another embodiment, the IL-2 portion comprises an L72E mutation. In another embodiment, the IL-2 portion comprises an L72N mutation. In another embodiment, the IL-2 portion comprises an L72D mutation. In another embodiment, the IL-2 portion comprises an L72R mutation. In another embodiment, the IL-2 portion comprises an L72H mutation. In another embodiment, the IL-2 portion comprises an L72W mutation. In another embodiment, the IL-2 portion comprises an L72Y mutation. In another embodiment, the IL-2 portion comprises an L72K mutation.

[0285] In another embodiment, the IL-2 portion comprises an amino acid mutation selected from F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R and F41K and another amino acid mutation selected from Y44A, Y44C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R and Y44K, based on SEQ ID NO: 2, or the corresponding positions of its homologues or variants. In one embodiment, the IL-2 portion comprises F41A and Y44A mutations. In another embodiment, the IL-2 portion comprises F41C mutations and Y44A mutations. In another embodiment, the IL-2 portion comprises F41A and Y44C mutations.

[0286] In another embodiment, the IL-2 portion comprises an amino acid mutation selected from F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R and F41K and another amino acid mutation selected from L71G, L71C, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R and L71K, based on SEQ ID NO: 2, or the corresponding positions of its homologues or variants. In one embodiment, the IL-2 portion comprises F42A and L72G mutations. In another embodiment, the IL-2 portion comprises F42C and L72G mutations. In another embodiment, the IL-2 portion comprises F42A and L72C mutations.

[0287] In another embodiment, the IL-2 portion comprises an amino acid mutation selected from Y44A, Y44C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R and Y44K and another amino acid mutation selected from L71G, L71C, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R and L71K, based on SEQ ID NO: 2, or the corresponding positions of a homologue or variant thereof. In one embodiment, the IL-2 portion comprises Y45A and L72G mutations. In another embodiment, the IL-2 portion comprises Y45C and L72G mutations. In another embodiment, the IL-2 portion comprises Y45A and L72C mutations.

[0288] In another embodiment, the IL-2 portion comprises an amino acid mutation selected from F41A, F41C, F41G, F41S, F41T, F41Q, F41E, F41N, F41D, F41R, and F41K; another amino acid mutation selected from Y44A, Y41C, Y44G, Y44S, Y44T, Y44Q, Y44E, Y44N, Y44D, Y44R, and Y44K; and another amino acid mutation selected from L71G, L71C, L71A, L71S, L71T, L71Q, L71E, L71N, L71D, L71R, and L71K, based on SEQ ID NO: 2, or the corresponding positions of a homologue or variant thereof. In one embodiment, the IL-2 portion comprises F41A, Y44A, and L71G mutations. In another embodiment, the IL-2 portion comprises F41C, Y44A, and L71G mutations. In another embodiment, the IL-2 portion comprises F41A, Y44C, and L71G mutations. In another embodiment, the IL-2 portion comprises F41A, Y44C, and L71C mutations.

[0289] The IL-2 portion may additionally or alternatively comprise an amino acid mutation that eliminates an O-glycosylation site. Preferably, the amino acid mutation is at a position corresponding to residue 2 of aldesleukin, even more preferably the amino acid mutation is selected from T2A, T2G, T2Q, T2E, T2N, T2D, T2R, T2K and T2P, most preferably T2A, based on SEQ ID NO: 2, or a corresponding position of a homologue or variant thereof.

[0290] The IL-2 portion may additionally or alternatively comprise one or more additional amino acid mutations which 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 US 5,206,344.

[0291] In some embodiments, the IL-2-biased moiety is a moiety comprising at least one amino acid mutation and at least one modified moiety M. mod The IL-2 portion of the stably linked IL-2, such as an amino acid mutation and an M mod part, an amino acid mutation and two M mod part, two amino acid mutations and one M mod part, three amino acid mutations and one M mod Partial, or one amino acid mutation and three M mod It is understood that amino acid mutations and M mod The number of moieties can be selected independently of one another and can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In some embodiments, the amino acid mutation and M mod The number of moieties is independently selected from 1, 2, 3, 4, 5, 6, 7 and 8. In some embodiments, the number of amino acid mutations is 1. In some embodiments, the number of amino acid mutations is 2. In some embodiments, the number of amino acid mutations is 3. In some embodiments, the number of amino acid mutations is 4. In some embodiments, the number of amino acid mutations is 5. In some embodiments, the number of amino acid mutations is 6. In some embodiments, M mod The number of parts is 1. In some embodiments, M mod The number of parts is 2. In some embodiments, M mod The number of parts is 3. In some embodiments, M mod The number of parts is 4. In some embodiments, M mod The number of parts is 5. In some embodiments, M mod The number of parts is 6. mod Embodiments of moieties and mutation sites / mutations are as described above.

[0292] In some embodiments, the biased IL-2 portion is a portion comprising at least one amino acid mutation and at least one M-terminal residue linked to the mutated amino acid. mod Part of the IL-2 part. It can be understood that amino acid mutations and M mod The number of parts may vary; other M mod The moiety can be conjugated to the IL-2 moiety at the non-mutated amino acid residue; not all mutated amino acid residues can be conjugated to the M moiety. mod part, as long as there is at least one M mod The moiety is conjugated to a mutant amino acid residue. mod The number of moieties can be selected independently of one another and can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In some embodiments, the amino acid mutation and M mod The number of moieties is independently selected from 1, 2, 3, 4, 5, 6, 7 and 8. In some embodiments, the number of amino acid mutations is 1. In some embodiments, the number of amino acid mutations is 2. In some embodiments, the number of amino acid mutations is 3. In some embodiments, the number of amino acid mutations is 4. In some embodiments, the number of amino acid mutations is 5. In some embodiments, the number of amino acid mutations is 6. In some embodiments, M mod The number of parts is 1. In some embodiments, M mod The number of parts is 2. In some embodiments, M mod The number of parts is 3. In some embodiments, M mod The number of parts is 4. In some embodiments, M mod The number of parts is 5. In some embodiments, M mod The number of parts is 6. mod Embodiments of moieties and mutation sites / mutations are as described above.

[0293] In some embodiments, the biased IL-2 portion is an IL-2 portion comprising at least one amino acid mutation at a position selected from K34, R37, M38, T40, F41, K42, F43, Y44, E61, and L71, wherein the naturally occurring amino acid is replaced with cysteine, resulting in the mutations K34C, R37C, M38C, T40C, F41C, K42C, F43C, Y44C, E61C, and L71C, and wherein at least one M mod In some embodiments, the biased IL-2 moiety comprises a K34C mutation and additionally comprises an M conjugated to the sulfur of the cysteine ​​replacing the lysine at position 34. modIn some embodiments, the biased IL-2 portion comprises an R37C mutation and additionally comprises an M conjugated to the sulfur of the cysteine ​​replacing the arginine at position 37. mod In some embodiments, the biased IL-2 portion comprises an M38C mutation and additionally comprises an M38C conjugated to the sulfur of the cysteine ​​replacing the methionine at position 38. mod In some embodiments, the IL-2-biasing moiety comprises a T40C mutation and additionally comprises an M conjugated to the sulfur of a cysteine ​​replacing a threonine at position 40. mod In some embodiments, the biased IL-2 portion comprises an F41C mutation and additionally comprises an M conjugated to the sulfur of the cysteine ​​replacing the phenylalanine at position 41. mod In some embodiments, the biased IL-2 portion comprises a K42C mutation and additionally comprises an M conjugated to the sulfur of the cysteine ​​replacing the lysine at position 42. mod In some embodiments, the biased IL-2 portion comprises an F43C mutation and additionally comprises an M conjugated to the sulfur of the cysteine ​​replacing the phenylalanine at position 43. mod In some embodiments, the biased IL-2 portion comprises a Y44C mutation and additionally comprises an M conjugated to the sulfur of the cysteine ​​replacing the tyrosine at position 44. mod In some embodiments, the biased IL-2 portion comprises an E61C mutation and additionally comprises an M conjugated to the sulfur of the cysteine ​​replacing the glutamic acid at position 61. mod In some embodiments, the biased IL-2 portion comprises an L71C mutation and additionally comprises an M conjugated to the sulfur of the cysteine ​​replacing the lysine at position 71. mod In some embodiments, the biased IL-2 portion comprises a mutation selected from K34C, R37C, M38C, T40C, F41C, K42C, F43C, Y44C, E61C, and L71C, wherein one of the M mod The moiety is conjugated to the sulfur of the cysteine ​​replacing the naturally occurring amino acid, ie, arginine at position 37, for example in the case of the R37C mutation.

[0294] The IL-2 portion may comprise at least one deletion (see point (c)). It will be appreciated that the deletion preferably relates to the sequence of SEQ ID NO: 2. The deletion may be a naturally occurring deletion, such as in the form of a splice variant, or may be an artificially introduced deletion. One such naturally occurring splice variant is the IL-2δ2 mutant (see Tsytsikov et al., JBC 1996, 271 (38): 23055-23060), in which exon 2 is excluded and which results in a deletion of amino acid residues N30 to K49 based on the sequence of SEQ ID NO: 1, which corresponds to a deletion of amino acid residues N29 to K48 based on the sequence of SEQ ID NO: 2.

[0295] Preferably, the deletion occurs at a position involved in binding to IL-2Rα.Thus, preferably, the at least one deletion results in a reduced affinity of the biased IL-2 moiety for IL-2Rαβ compared to aldesleukin.

[0296] In one embodiment, -D comprises an IL-2 moiety, preferably an IL-2 moiety having the sequence SEQ ID NO: 2, comprising at least one mutation introducing a cysteine ​​residue and further comprising a modifying moiety M linked to said cysteine ​​mutation. mod . Preferably, the cysteine ​​mutation is selected from K34C, R37C, M38C, T40C, F41C, K42C, F43C, Y44C, E61C and L71C. In some embodiments, the cysteine ​​mutation is a K34C mutation. In some embodiments, the cysteine ​​mutation is an R37C mutation. In some embodiments, the cysteine ​​mutation is an M38C mutation. In some embodiments, the cysteine ​​mutation is a T40C mutation. In some embodiments, the cysteine ​​mutation is an F41C mutation. In some embodiments, the cysteine ​​mutation is a K42C mutation. In some embodiments, the cysteine ​​mutation is an F43C mutation. In some embodiments, the cysteine ​​mutation is a Y44C mutation. In some embodiments, the cysteine ​​mutation is an E61C mutation. In some embodiments, the cysteine ​​mutation is an L71C mutation.

[0297] In one embodiment, the IL-2 portion is a superkine as described by Levin et al. (Nature, 2012, 484: 529-535). Preferably, in addition to or as an alternative to the above modifications, the IL-2 portion comprises one or more mutations of amino acid residues Q73, L79, R80, L84, I85 and I91, in particular one or more mutations selected from Q73H, L79F, R80D, L84V, I85V and I91F. In one embodiment, in addition to or as an alternative to the above modifications, the IL-2 portion comprises Q73H, L79F, R80D, L84V, I85V and I91F mutations. In another embodiment, in addition to or as an alternative to the above modifications, the IL-2 portion comprises L79F, R80D, L84V, I85V and I91F mutations. If IL-2 is in part superkine-based, then the corresponding bias would be that IL-2 has a higher affinity for IL-2Rβ than aldesleukin.

[0298] The IL-2 conjugates of the present invention comprise at least one covalently and reversibly linked polymeric moiety and / or a substituted fatty acid moiety -Z.

[0299] Surprisingly, it was found that the addition of such at least one covalently and reversibly linked polymeric moiety and / or substituted fatty acid moiety was able to further extend the circulation half-life of the IL-2-biased moiety, while its reversible linkage ensures sufficient affinity for IL-2Rβ after cleavage of the at least one covalently and reversibly linked polymeric moiety or substituted fatty acid moiety.

[0300] In one embodiment, the IL-2 conjugate of the invention comprises a -Z moiety which is a substituted fatty acid or a polymeric moiety. In one embodiment, -Z is a substituted fatty acid. In another embodiment, -Z is a polymeric moiety.

[0301] In another embodiment, the IL-2 conjugate of the invention comprises two -Z moieties, which may be the same or different. In one embodiment, the two -Z moieties are substituted fatty acids, which may be the same or different. In another embodiment, the two -Z moieties are polymeric moieties, which may be the same or different. In another embodiment, one -Z moiety is a substituted fatty acid and the other -Z moiety is a polymeric moiety.

[0302] In another embodiment, the IL-2 conjugate of the invention comprises three -Z moieties, which may be the same or different. In one embodiment, all three -Z moieties are substituted fatty acids, which may be the same or different. In another embodiment, all three -Z moieties are polymeric moieties, which may be the same or different. In another embodiment, one or two -Z moieties are substituted fatty acids and the remaining -Z moieties are polymeric moieties.

[0303] In another embodiment, the IL-2 conjugate of the invention comprises four -Z moieties, which may be the same or different. In one embodiment, all four -Z moieties are substituted fatty acids, which may be the same or different. In another embodiment, all four -Z moieties are polymeric moieties, which may be the same or different. In another embodiment, one, two or three -Z moieties are substituted fatty acids and the remaining -Z moieties are polymeric moieties.

[0304] If -Z is a substituted fatty acid moiety, it is preferably a substituted fatty acid moiety disclosed in WO 2005 / 027978 A2 and WO 2014 / 060512 A1, which are incorporated herein by reference.

[0305] If -Z is a polymeric moiety, the polymeric moiety preferably has a molecular weight 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 polymeric moiety with a molecular weight of about 2 kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of about 5 kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of about 10 kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of about 15 kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of about 20 kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of about 30 kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of about 40 kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of about 50 kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of about 60 kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of about 70kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of about 80kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of about 90kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of about 100kDa. In one embodiment, -Z is a polymeric portion with a molecular weight of 2kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of 5kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of 10kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of 15kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of 20kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of 30kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of 40kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of 50kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of 60kDa. In another embodiment, -Z is a polymeric portion with a molecular weight of 70kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of 80 kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of 90 kDa. In another embodiment, -Z is a polymeric moiety with a molecular weight of 100 kDa.

[0306] If -Z is a polymeric moiety, the polymeric moiety preferably comprises a polymer selected from the group consisting of 2-methacryloyl-oxyethylphosphorylcholins, poly(acrylic acid), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides), poly(asparagine), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamide), poly(esters), 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 methacrylate), poly(hydroxypropyl oxazoline), poly(imino carbonate), poly(lactic acid), poly(lactic-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyl oxazoline), poly(organophosphazene), poly(orthoesters), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinyl pyrrolidone), silicone, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, alginate, mannan, pectin, rhamnogalacturonic acid, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate-based polymers, xylan and copolymers thereof.

[0307] In one embodiment, -Z is a peptide or protein moiety. Preferably, the peptide or protein moiety is not an IL-2 moiety or a fragment thereof. The peptide or protein moiety -Z may be ligated to the IL-2 moiety via -L 1 -L 2 - chemically conjugated to -D or via a reversible linker moiety -L 1 -Translation fused to -D, where -L 1 - is a peptide or protein moiety and -L 2 - is preferably a chemical bond. In one embodiment, the peptide or protein moiety -Z is linked to -L 1 -L 2 - chemically conjugated to -D. In another embodiment, the peptide or protein moiety -Z is linked to -D via a reversible linker moiety -L. 1 -Translation fused to -D, where L 1 - is a peptide or protein moiety and -L 2 - is preferably a chemical bond. It is understood that the peptide or protein reversible linker portion -L 1 - can be enzymatically or non-enzymatically degradable. To promote enzymatic degradation, -L1 - may contain a protease recognition site.

[0308] If -Z is a peptide or protein moiety, it is preferably selected from a moiety comprising the carboxy-terminal peptide of chorionic gonadotropin, as described in US 2012 / 0035101 A1, which is incorporated herein by reference; an albumin moiety; a random coil protein moiety and an Fc fusion protein moiety.

[0309] In one embodiment, -Z comprises a random coil peptide or protein moiety.

[0310] Preferably, the random coil peptide or protein portion comprises at least 25 amino acid residues and at most 2000 amino acids. Even more preferably, the random coil peptide or protein portion comprises at least 30 amino acid residues and at most 1500 amino acid residues. Even more preferably, the random coil peptide or protein portion comprises at least 50 amino acid residues and at most 500 amino acid residues.

[0311] In a preferred embodiment, -Z comprises a random coil protein portion, wherein at least 80%, preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% of the total number of amino acids forming the random coil protein portion are selected from alanine and proline. Even more preferably, at least 10%, but less than 75%, preferably less than 65% of the total number of amino acid residues of the random coil protein portion are proline residues. Preferably, the random coil protein portion is as described in WO 2011 / 144756 A1, which is incorporated herein by reference in its entirety. Even more preferably, -Z comprises at least one portion selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 51 and SEQ ID NO: 61 as described in WO2011 / 144756, which is incorporated herein by reference. The portion comprising this random coil protein containing alanine and proline will be referred to as "PA" or "PA portion".

[0312] Thus, in one embodiment, -Z comprises a PA moiety.

[0313] In another embodiment, -Z comprises a random coil protein portion, wherein at least 80%, preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% of the total number of amino acids forming the random coil protein portion are selected from alanine, serine and proline. Even more preferably, at least 4%, but less than 40% of the total number of amino acid residues of the random coil protein portion are proline residues. Preferably, the random coil protein portion is as described in WO 2008 / 155134 A1, which is incorporated herein by reference in its entirety. Even more preferably, -Z comprises at least one portion selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:56, as disclosed in WO2008 / 155134 A1, which is incorporated herein by reference. The portion comprising this random coil protein portion containing alanine, serine and proline will be referred to as "PAS" or a "PAS portion".

[0314] Thus, in one embodiment, -Z comprises a PAS moiety.

[0315] In another embodiment, -Z comprises a random coil protein portion, wherein at least 80%, preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% of the total number of amino acids forming the random coil protein portion are selected from alanine, glycine, serine, threonine, glutamic acid and proline. Preferably, the random coil protein portion is as described in WO 2010 / 091122 A1, which is incorporated herein by reference. Even more preferably,-Z contains at least one moiety selected from the following: SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184; SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:759, SEQ ID NO:760, SEQ ID NO:761, SEQ ID NO:762, SEQ ID NO:763, SEQ ID NO:764, SEQ ID NO:765, SEQ ID NO:766, SEQ ID NO:767, SEQ ID NO:768, SEQ ID NO:769, SEQ ID NO:770, SEQ ID NO:771, SEQ ID NO:772, SEQ ID NO:773, SEQ ID NO:774, SEQ ID NO:775, SEQ ID NO:776, SEQ ID NO:777, SEQ ID NO:778, SEQ ID NO:779, SEQ ID NO:1715, SEQ ID NO:1716, SEQ ID NO:1718, SEQ ID NO:1719, SEQ ID NO:1720, SEQ ID NO:1721 and SEQ ID NO:1722, as described in WO2010 / 091122A1,This document is incorporated herein by reference. According to the designation in WO 2010 / 091122 A1, the portion comprising the random coil protein portion containing alanine, glycine, serine, threonine, glutamic acid, and proline will be referred to as "XTEN" or "XTEN portion".

[0316] Thus, in one embodiment, -Z comprises an XTEN moiety.

[0317] In another embodiment, -Z is a hyaluronic acid based polymer.

[0318] In another embodiment, -Z is a PEG-based portion, such as a linear, branched or multi-armed PEG-based portion. In one embodiment, -Z is a PEG-based portion of a branched chain. Preferably, the PEG-based portion -Z of the branched chain is a PEG-based portion of a branched chain with one, two, three, four, five or six branching points. Preferably, -Z is a PEG-based portion of a branched chain with one, two or three branching points. In one embodiment, -Z is a PEG-based portion of a branched chain with one branching point. In another embodiment, -Z is a PEG-based portion of a branched chain with two branching points. In another embodiment, -Z is a PEG-based portion of a branched chain with three branching points.

[0319] Each branch point is preferably independently selected from -N<, -CH< and >C<.

[0320] In some embodiments, -Z comprises a moiety of formula (A):

[0321]

[0322] in

[0323] -BP 1 <,-BP 2 <,-BP 3 < are independently selected from -N< and -C(R 8 )<

[0324] R 8 Selected from H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl;

[0325] -P 1 , -P 2 , -P 3 , -P 4 are independently of one another a PEG-based chain comprising at least 40% PEG and having a molecular weight of 3 to 40 kDa;

[0326] -C1 -、-C 2 - independently selected from C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more identical or different R 9 Substitute, and where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more selected from -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 )-groups;

[0327] T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbocyclic group and 8- to 30-membered heteropolycyclic group; wherein each T is independently optionally replaced by one or more identical or different R 9 replace;

[0328] R 9 are independently selected from halogen, -CN, oxo (=O), -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 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0329] R 10 , R 10a , R 11 , R 11a and R 11b are each independently selected from -H and C 1-6 Alkyl, where C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

[0330] In some embodiments, -P 1 , -P 2 , -P 3 , -P 4 are independently a PEG-based chain comprising at least 50% PEG and having a molecular weight of 3 to 40 kDa. 1 , -P 2 , -P 3 , -P 4 are independently PEG-based chains comprising at least 60% PEG and having a molecular weight of 3 to 40 kDa. 1 , -P 2 , -P 3 , -P 4 are independently PEG-based chains comprising at least 70% PEG and having a molecular weight of 3 to 40 kDa. 1 , -P 2 , -P 3 , -P 4Independently of each other are PEG-based chains comprising at least 80% PEG and having a molecular weight of 3 to 40 kDa.

[0331] In some embodiments, P of formula (A) 1 , P 2 , P 3 and P 4 The molecular weight of each moiety is independently 5 to 30 kDa, such as 5 to 25 kDa, or 8 to 20 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be about 5 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be about 7 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be about 10 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be about 12 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be about 15 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be about 20 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be about 25 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be about 30 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be 7 kDa. 1 , P 2 , P 3 or P 4The molecular weight of the moiety may be 10 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be 12 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be 15 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be 20 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be 25 kDa. 1 , P 2 , P 3 or P 4 The molecular weight of the moiety may be 30 kDa.

[0332] In some embodiments, P of formula (A) 1 , P 2 , P 3 and P 4 Have the same structure.

[0333] In some embodiments, BP of formula (A) 1 Yes –N<.

[0334] In some embodiments, BP of formula (A) 2 and BP 2 In some embodiments, BP of formula (A) 2 and BP 2 All are –CH<.

[0335] In some embodiments, C of formula (A) 1 and C 2 In some embodiments, C of formula (A) 1 and C 2 is one or more selected from -O-, -C(O)N(R 10 )- and 3- to 10-membered heterocyclic groups separated by C 1-50 Alkyl; wherein the 3- to 10-membered heterocyclic group is substituted by at least one oxo group (=O).

[0336] In some embodiments, C of formula (A) 1 and C2 With formula (Aa)

[0337]

[0338] in

[0339] The dashed line marked with an asterisk refers to BP 1 connect;

[0340] Unlabeled dashed lines indicate BP 2 or BP 3 connect;

[0341] q1 is selected from 1, 2, 3, 4, 5, 6, 7 and 8;

[0342] q2 is selected from 1, 2, 3, 4 and 5;

[0343] q3 is selected from 1, 2, 3, 4, 5, 6, 7 and 8; and

[0344] q4 is selected from 1, 2 and 3.

[0345] In some embodiments, q1 of formula (Aa) is selected from 4, 5, 6, 7 and 8. In some embodiments, q1 of formula (Aa) is selected from 5, 6 and 7. In some embodiments, q1 of formula (Aa) is 1. In some embodiments, q1 of formula (Aa) is 2. In some embodiments, q1 of formula (Aa) is 3. In some embodiments, q1 of formula (Aa) is 4. In some embodiments, q1 of formula (Aa) is 5. In some embodiments, q1 of formula (Aa) is 6. In some embodiments, q1 of formula (Aa) is 7. In some embodiments, q1 of formula (Aa) is 8.

[0346] In some embodiments, q2 of Formula (Aa) is selected from 1, 2, and 3. In some embodiments, q2 of Formula (Aa) is 1. In some embodiments, q2 of Formula (Aa) is 2. In some embodiments, q2 of Formula (Aa) is 3. In some embodiments, q2 of Formula (Aa) is 4. In some embodiments, q2 of Formula (Aa) is 5.

[0347] In some embodiments, q3 of formula (Aa) is selected from 2, 3, 4 and 5. In some embodiments, q3 of formula (Aa) is selected from 2, 3 and 4. In some embodiments, q3 of formula (Aa) is 1. In some embodiments, q3 of formula (Aa) is 2. In some embodiments, q3 of formula (Aa) is 3. In some embodiments, q3 of formula (Aa) is 4. In some embodiments, q3 of formula (Aa) is 5. In some embodiments, q3 of formula (Aa) is 6. In some embodiments, q3 of formula (Aa) is 7. In some embodiments, q3 of formula (Aa) is 8.

[0348] In some embodiments, q4 of formula (Aa) is 1. In some embodiments, q4 of formula (Aa) is 2. In some embodiments, q4 of formula (Aa) is 3.

[0349] In some embodiments, P of formula (A) 1 , P 2 , P 3 and P 4 Independently of each other, they are of formula (Ab)

[0350]

[0351] in

[0352] The dashed line indicates the connection to the rest of -Z;

[0353] m is 0 or 1;

[0354] p is an integer from 70 to 900; and

[0355] q is selected from 1, 2, 3, 4, 5 and 6.

[0356] In some embodiments, m of Formula (Ab) is 0. In some embodiments, m of Formula (Ab) is 1.

[0357] In some embodiments, p of Formula (Ab) is an integer from 115 to 680. In some embodiments, p of Formula (Ab) is an integer from 115 to 560. In some embodiments, p of Formula (Ab) is an integer from 185 to 450. In some embodiments, p of Formula (Ab) is about 115. In some embodiments, p of Formula (Ab) is about 160. In some embodiments, p of Formula (Ab) is about 225. In some embodiments, p of Formula (Ab) is about 270. In some embodiments, p of Formula (Ab) is about 340. In some embodiments, p of Formula (Ab) is about 450. In some embodiments, p of Formula (Ab) is about 560.

[0358] In some embodiments, q of formula (Ab) is 1. In some embodiments, q of formula (Ab) is 2. In some embodiments, q of formula (Ab) is 3. In some embodiments, q of formula (Ab) is 4. In some embodiments, q of formula (Ab) is 5. In some embodiments, q of formula (Ab) is 6.

[0359] In some embodiments, -Z comprises a moiety of formula (Ac):

[0360]

[0361] wherein p1, p2, p3, and p4 are independently integers ranging from 70 to 900.

[0362] In some embodiments, the p1 of formula (Ac) is an integer from 115 to 680. In some embodiments, the p1 of formula (Ac) is an integer from 115 to 560. In some embodiments, the p1 of formula (Ac) is an integer from 185 to 450. In some embodiments, the p1 of formula (Ac) is an integer from 220 to 240. In some embodiments, the p1 of formula (Ac) is about 115. In some embodiments, the p1 of formula (Ac) is about 160. In some embodiments, the p1 of formula (Ac) is about 225. In some embodiments, the p1 of formula (Ac) is about 270. In some embodiments, the p1 of formula (Ac) is about 340. In some embodiments, the p1 of formula (Ac) is about 450. In some embodiments, the p1 of formula (Ac) is about 560.

[0363] In some embodiments, p2 of formula (Ac) is an integer from 115 to 680. In some embodiments, p2 of formula (Ac) is an integer from 115 to 560. In some embodiments, p2 of formula (Ac) is an integer from 185 to 450. In some embodiments, p2 of formula (Ac) is an integer from 220 to 240. In some embodiments, p2 of formula (Ac) is about 115. In some embodiments, p2 of formula (Ac) is about 160. In some embodiments, p2 of formula (Ac) is about 225. In some embodiments, p2 of formula (Ac) is about 270. In some embodiments, p2 of formula (Ac) is about 340. In some embodiments, p2 of formula (Ac) is about 450. In some embodiments, p2 of formula (Ac) is about 560.

[0364] In some embodiments, p3 of formula (Ac) is an integer from 115 to 680. In some embodiments, p3 of formula (Ac) is an integer from 115 to 560. In some embodiments, p3 of formula (Ac) is an integer from 185 to 450. In some embodiments, p3 of formula (Ac) is an integer from 220 to 240. In some embodiments, p3 of formula (Ac) is about 115. In some embodiments, p3 of formula (Ac) is about 160. In some embodiments, p3 of formula (Ac) is about 225. In some embodiments, p3 of formula (Ac) is about 270. In some embodiments, p3 of formula (Ac) is about 340. In some embodiments, p3 of formula (Ac) is about 450. In some embodiments, p3 of formula (Ac) is about 560.

[0365] In some embodiments, p4 of formula (Ac) is an integer from 115 to 680. In some embodiments, p4 of formula (Ac) is an integer from 115 to 560. In some embodiments, p4 of formula (Ac) is an integer from 185 to 450. In some embodiments, p4 of formula (Ac) is an integer from 220 to 240. In some embodiments, p4 of formula (Ac) is about 115. In some embodiments, p4 of formula (Ac) is about 160. In some embodiments, p4 of formula (Ac) is about 225. In some embodiments, p4 of formula (Ac) is about 270. In some embodiments, p4 of formula (Ac) is about 340. In some embodiments, p4 of formula (Ac) is about 450. In some embodiments, p4 of formula (Ac) is about 560.

[0366] In some embodiments, p1, p2, p3 and p4 of formula (Ac) are equal. In some embodiments, p1, p2, p3 and p4 range from 220 to 240.

[0367] In one embodiment, -Z is a moiety as disclosed in WO 2012 / 02047A1, which is incorporated herein by reference.

[0368] In another embodiment, -Z is a moiety as disclosed in WO 2013 / 024048 A1, which is incorporated herein by reference.

[0369] In some embodiments, -Z is water insoluble. In some embodiments, -Z is a hydrogel.

[0370] In some embodiments, the hydrogel comprises a polymer selected from the group consisting of 2-methacryloyl-oxyethylphosphorylcholins, poly(acrylic acid), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides), poly(asparagine), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamide), poly(esters), poly(ethylene), poly(ethylene glycols) such as 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) acrylate.

[0013] The invention also includes but is not limited to the following: poly(methyleneimine), ...

[0371] In some embodiments, -Z is a poly(alkylene glycol)-based or hyaluronic acid-based hydrogel.

[0372] In some embodiments, -Z is a poly(propylene glycol)-based hydrogel.

[0373] In some embodiments, -Z is a PEG-based hydrogel.

[0374] In some embodiments, -Z is a PEG-based hydrogel as described in WO2011 / 012715A1 or WO2014 / 056926A1, which are incorporated herein by reference.

[0375] In some embodiments, -Z is a hyaluronic acid based hydrogel.

[0376] In some embodiments, -Z is a hyaluronic acid-based hydrogel as described in WO2018 / 175788A1, which is incorporated herein by reference.

[0377] -L 1-moiety can be via the IL-2 moiety, in particular via an amino acid residue of the IL-2 moiety or via a modified moiety M present in -D mod In one embodiment, -L 1 - is linked to -D via the IL-2 moiety, in particular via an amino acid residue of the IL-2 moiety. In another embodiment, -L 1 - via the modified moiety M present in -D mod Connect to -D. It is understood that one or more -L 1 -Parts can be connected to M mod part.

[0378] In one embodiment, all -L present in the IL-2 conjugate of the invention 1 The -moiety is linked to the amino acid residue of -D.

[0379] If -L 1 -an amino acid residue linked to the IL-2 moiety, which may be a proteinaceous or non-proteinaceous amino acid residue of -D. In one embodiment, -L 1 - is linked to a non-protein amino acid residue, preferably a non-protein amino acid as described above. In another embodiment, -L 1 - is connected to a proteinaceous amino acid residue. If the connection occurs at a proteinaceous amino acid residue, the proteinaceous amino acid residue is selected from cysteine, methionine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, glutamine and arginine in some embodiments. In some embodiments, this type of proteinaceous amino acid residue is selected from cysteine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid and arginine.

[0380] In one embodiment, -L 1- a cysteine ​​residue connected to -D, for example, selected from the cysteine ​​residues of C57 and C104 based on SEQ ID NO: 2 or the corresponding position of a homologue or variant of SEQ ID NO: 2. When a homologue or variant of IL-2 comprises one or more additional cysteine ​​residues compared to SEQ ID NO: 2, the connection can also occur at this cysteine ​​residue, which can be naturally occurring or can be the result of an addition, insertion or mutation. It is understood that one or two of the cysteine ​​residues of SEQ ID NO: 2 can be used to connect one or two -Z moieties to -D, respectively. If the IL-2 moiety is a homologue or variant of SEQ ID NO: 2 and has more cysteine ​​residues than the IL-2 moiety of SEQ ID NO: 2, more than two cysteine ​​residues can be used to connect -Z, that is, up to the maximum number of cysteine ​​residues present in the homologue or variant can be used to connect -Z. In one embodiment, one -Z moiety is connected to one cysteine ​​residue. In another embodiment, a total of two -Z moieties are connected to two cysteine ​​residues.

[0381] In another embodiment, -L 1 - a histidine residue linked to -D, for example, selected from the histidine residues H15, H54 and H78 based on SEQ ID NO: 2 or the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional histidine residues compared to SEQ ID NO: 2, the linking may also occur at this histidine residue, which may be naturally occurring or may be the result of an addition, insertion or mutation. It is understood that one, two or three of the histidine residues of SEQ ID NO: 2 may be used to link one, two or three -L, respectively. 1 If the IL-2 moiety is a homologue or variant of SEQ ID NO: 2 and has more histidine residues than the IL-2 moiety of SEQ ID NO: 2, more than three histidine residues may be used to link to the -L 1 - to -D, i.e., up to the maximum number of histidine residues present in the homologue or variant available for attachment to -L 1 -. In one embodiment, a -L 1 - moiety is attached to a histidine residue. In another embodiment, a total of two -L 1 - moieties are attached to two histidine residues. In another embodiment, a total of three -L 1 The -part is linked to three histidine residues.

[0382] In another embodiment, -L 1- to a lysine residue, for example a lysine residue selected from K7, K8, K31, K34, K42, K47, K48, K53, K63, K75 or K96 based on SEQ ID NO: 2 or the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional lysine residues compared to SEQ ID NO: 2, the linkage may also occur at this lysine residue, which may be naturally occurring or may be the result of an addition, insertion or mutation. It is understood that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the lysine residues of SEQ ID NO: 2 may be used to link 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 -L, respectively. 1 If the IL-2 moiety is a homologue or variant of SEQ ID NO: 2 and has more lysine residues than the IL-2 moiety of SEQ ID NO: 2, more than 11 lysine residues may be used to link to the -L 1 -, i.e., up to the maximum number of lysine residues present in the homologue or variant available for attachment of -L 1 -. In one embodiment, a -L 1 - moiety is attached to a lysine residue. In another embodiment, a total of two -L 1 - moieties are attached to two lysine residues. In another embodiment, a total of three -L 1 - moieties are attached to three lysine residues. In another embodiment, a total of four -L 1 - moieties are attached to four lysine residues. In another embodiment, a total of five -L 1 - moieties are attached to five lysine residues. In another embodiment, a total of six -L 1 The -part is linked to six lysine residues.

[0383] In another embodiment, -L 1 - to a tryptophan residue, for example the tryptophan residue at position W120 based on SEQ ID NO: 2 or the corresponding position of a homologue or variant of SEQ ID NO: 2. When a homologue or variant of IL-2 comprises one or more additional tryptophan residues compared to SEQ ID NO: 2, the linkage may also occur at this tryptophan residue, which may be naturally occurring or may be the result of an addition, insertion or mutation. If the IL-2 moiety is a homologue or variant of SEQ ID NO: 2 and has more tryptophan residues than the IL-2 moiety of SEQ ID NO: 2, more than one tryptophan residue may be used for linkage to -L 1-, i.e., up to the maximum number of tryptophan residues present in the homologue or variant available for attachment of -L 1 -. In one embodiment, a -L 1 The -part is linked to a tryptophan residue.

[0384] In another embodiment, -L 1 - to a serine residue, for example, selected from the group consisting of S3, S4, S5, S74, S86, S98, S124, S126 and S129 based on SEQ ID NO: 2 or the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional serine residues compared to SEQ ID NO: 2, the linkage may also occur at the serine residue, which may be naturally occurring or may be the result of an addition, insertion or mutation. It is understood that 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the serine residues of SEQ ID NO: 2 may be used for linkage to -L 1 If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and has more serine residues than the IL-2 portion of SEQ ID NO: 2, more than 9 serine residues may be used to link to -L 1 - to -D, i.e., up to the maximum number of serine residues present in the homologue or variant available for attachment to -L 1 -. In one embodiment, a -L 1 - moiety is attached to a serine residue. In another embodiment, a total of two -L 1 - moieties are attached to two serine residues. In another embodiment, a total of three -L 1 - moiety is attached to three serine residues. In another embodiment, a total of four -L 1 - moieties are attached to four serine residues. In another embodiment, a total of five -L 1 - moieties are attached to five serine residues. In another embodiment, a total of six -L 1 The -part is linked to six serine residues.

[0385] In another embodiment, -L 1- to a threonine residue, for example a threonine residue selected from T2, T6, T9, T36, T40, T50, T100, T102, T110, T112, T122, T130 and T132 based on SEQ ID NO: 2, or a corresponding position of a homologue or variant of SEQ ID NO: 2. When a homologue or variant of IL-2 comprises one or more additional threonine residues compared to SEQ ID NO: 2, the linkage may also occur at this threonine residue, which may be naturally occurring or may be the result of an addition, insertion or mutation. It is understood that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the threonine residues of SEQ ID NO: 2 may be used to link 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 -L, respectively. 1 If the IL-2 moiety is a homologue or variant of SEQ ID NO: 2 and has more threonine residues than the IL-2 moiety of SEQ ID NO: 2, then more than 13 threonine residues may be used to link to the -L 1 - to -D, i.e., up to the maximum number of threonine residues present in the homologue or variant available for attachment to -L 1 -. In one embodiment, a -L 1 - moiety is attached to a threonine residue. In another embodiment, a total of two -L 1 - moieties are attached to two threonine residues. In another embodiment, a total of three -L 1 - portion is connected to three threonine residues. In another embodiment, a total of four -L 1 - moieties are attached to four threonine residues. In another embodiment, a total of five -L 1 - moieties are attached to five threonine residues. In another embodiment, a total of six -L 1 The - portion is linked to six threonine residues.

[0386] In another embodiment, -L 1 - to a tyrosine residue, for example, selected from the group consisting of Y30, Y44 and Y106 based on SEQ ID NO: 2 or the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional tyrosine residues compared to SEQ ID NO: 2, the linkage may also occur at this tyrosine residue, which may be naturally occurring or may be the result of an addition, insertion or mutation. It is understood that 1, 2 or 3 of the tyrosine residues of SEQ ID NO: 2 may be used to link 1, 2 or 3 -L 1If the IL-2 moiety is a homologue or variant of SEQ ID NO: 2 and has more tyrosine residues than the IL-2 moiety of SEQ ID NO: 2, more than 3 tyrosine residues may be used to link to -L 1 -, i.e., up to the maximum number of tyrosine residues present in the homologue or variant available for attachment of -L 1 - to -D. In one embodiment, one -L 1 - moiety is attached to a tyrosine residue. In another embodiment, a total of two -L 1 - moieties are attached to two tyrosine residues. In another embodiment, a total of three -L 1 The -part is linked to three tyrosine residues.

[0387] In another embodiment, -L 1 -linked to an aspartic acid residue, for example selected from the aspartic acid residues D19, D83 and D108 based on SEQ ID NO: 2 or the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional aspartic acid residues compared to SEQ ID NO: 2, the linking may also occur at this aspartic acid residue, which may be naturally occurring or may be the result of an addition, insertion or mutation. It is understood that 1, 2 or 3 of the aspartic acid residues of SEQ ID NO: 2 may be used to link 1, 2 or 3 -L, respectively. 1 If the IL-2 moiety is a homologue or variant of SEQ ID NO: 2 and has more aspartate residues than the IL-2 moiety of SEQ ID NO: 2, more than 3 aspartate residues may be used to link to the -L 1 -, i.e., up to the maximum number of aspartic acid residues present in the homologue or variant available for attachment of -L 1 - to -D. In one embodiment, one -L 1 - moiety is attached to an aspartic acid residue. In another embodiment, a total of two -L 1 - moiety is attached to two aspartic acid residues. In another embodiment, a total of three -L 1 The -part is linked to three aspartic acid residues.

[0388] In another embodiment, -L 1- to a glutamic acid residue, for example, a glutamic acid residue selected from E14, E51, E59, E60, E61, E66, E67, E94, E99, E105, E109 and E115 based on SEQ ID NO: 2 or the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional glutamic acid residues compared to SEQ ID NO: 2, the linkage may also occur at the glutamic acid residue, which may be naturally occurring or may be the result of an addition, insertion or mutation. It is understood that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the glutamic acid residues of SEQ ID NO: 2 may be used to link 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 -L 1 If the IL-2 portion is a homologue or variant of SEQ ID NO: 2 and has more glutamic acid residues than the IL-2 portion of SEQ ID NO: 2, more than 12 glutamic acid residues may be used to link to the -L portion. 1 -, i.e., up to the maximum number of glutamic acid residues present in the homologue or variant available for attachment of -L 1 -. In one embodiment, a -L 1 - moiety is attached to a glutamic acid residue. In another embodiment, a total of two -L 1 - moiety is attached to two glutamic acid residues. In another embodiment, a total of three -L 1 - moiety is attached to three glutamic acid residues. In another embodiment, a total of four -L 1 - moiety is attached to four glutamic acid residues. In another embodiment, a total of five -L 1 - moiety is attached to five glutamic acid residues. In another embodiment, a total of six -L 1 The -part is linked to six glutamic acid residues.

[0389] In another embodiment, -L 1 - to an arginine residue, for example, selected from the group consisting of R37, R80, R82 and R119 based on SEQ ID NO: 2 or the corresponding position of a homologue or variant of SEQ ID NO: 2. When the homologue or variant of IL-2 comprises one or more additional arginine residues compared to SEQ ID NO: 2, the linkage may also occur at this arginine residue, which may be naturally occurring or may be the result of an addition, insertion or mutation. It is understood that 1, 2, 3 or 4 of the arginine residues of SEQ ID NO: 2 may be used to link 1, 2, 3 or 4 -L 1If the IL-2 moiety is a homologue or variant of SEQ ID NO: 2 and has more arginine residues than the IL-2 moiety of SEQ ID NO: 2, more than 4 arginine residues may be used to link to the -L 1 -, i.e., up to the maximum number of arginine residues present in the homologue or variant available for attachment of -L 1 -. In one embodiment, a -L 1 - moiety is attached to an arginine residue. In another embodiment, a total of two -L 1 - moiety is attached to two arginine residues. In another embodiment, a total of three -L 1 - portion is attached to four arginine residues. In another embodiment, a total of four -L 1 The -part is linked to four tyrosine residues.

[0390] In another embodiment, at least one -L 1 -part is linked to the amino acid residue of -D and the remaining one or more -L 1 The -moiety is linked to the modifying moiety present in -D.

[0391] In another embodiment, all -L present in the IL-2 conjugate of the invention 1 - moiety is linked to the modified moiety present in -D. In one embodiment, one -L 1 - moiety is attached to a modified moiety of -D. In another embodiment, a total of two -L 1 - moieties are linked to one modifying moiety, ie to the same modifying moiety. In another embodiment, a total of three -L 1 - moiety is linked to a modifying moiety. In another embodiment, a total of four -L 1 - moiety is linked to a modifying moiety. In another embodiment, a total of five -L 1 - moiety is linked to a modifying moiety. In another embodiment, a total of six -L 1 - moiety is linked to a modifying moiety. In another embodiment, a total of two -L 1 The - moiety is linked to two modifying moieties, i.e., one -L per modifying moiety 1 In another embodiment, a total of three -L 1 The - moiety is linked to three modifying moieties, i.e., one -L per modifying moiety 1 In another embodiment, a total of four -L 1 - moiety is connected to four modifying moieties, i.e., each modifying moiety is connected to one -L 1 In another embodiment, a total of five -L1 - moiety is linked to five modifying moieties, i.e., one moiety per modifying moiety. In another embodiment, a total of six -L 1 - part is connected to six modifying parts, that is, each modifying part is connected to one -L 1 -.

[0392] In one embodiment, -L 1 - has a structure as disclosed in WO 2009 / 095479 A2. Therefore, in one embodiment, -L 1 - the moiety has the formula (II):

[0393]

[0394] The dashed line indicates the connection to the nitrogen of -D through the formation of an amide bond;

[0395] -X-Yes-C(R 4 R 4a )-;-N(R 4 )-;-O-;-C(R 4 R 4a )-C(R 5 R 5a )-;-C(R 5 R 5a )-C(R 4 R 4a )-;-C(R 4 R 4a )-N(R 6 )-;-N(R 6 )-C(R 4 R 4a )-;-C(R 4 R 4a )-O-;-OC(R 4 R 4a )-; or -C(R 7 R 7a )-;

[0396] X 1 is C; or S(O);

[0397] -X 2 -Yes-C(R 8 R 8a )-; or -C(R 8 R 8a )-C(R 9 R 9a )-;

[0398] =X 3 is = O; = S; or = N-CN;

[0399] -R 1 , -R 1a , -R 2 , -R 2a , -R 4 , -R 4a , -R 5 , -R 5a , -R 6 , -R 8 , -R 8a , -R 9 , -R 9a are independently selected from -H; and C 1-6 alkyl;

[0400] -R 3 , -R 3a are independently selected from -H; and C 1-6 Alkyl, provided that -R 3 , -R 3a In the case where one or both are not -H, they are passed to SP 3 - Hybridized carbon atoms are linked to the N to which they are attached;

[0401] -R 7 Yes-N(R 10 R 10a ); or -NR 10 -(C=O)-R 11 ;

[0402] -R 7a , -R 10 , -R 10a , -R 11 independently -H; or C 1-6 alkyl;

[0403] Optionally, -R 1a / -R 4a , -R 1a / -R 5a , -R 1a / -R 7a , -R 4a / -R 5a , -R 8a / -R 9a One or more of the pairs form chemical bonds;

[0404] Optionally, -R 1 / -R 1a , -R 2 / -R 2a , -R 4 / -R 4a , -R 5 / -R 5a, -R 8 / -R 8a , -R 9 / -R 9a One or more of the pairs together with the atoms to which they are attached form C 3-10 Cycloalkyl; or 3- to 10-membered heterocyclic group;

[0405] Optionally, -R 1 / -R 4 , -R 1 / -R 5 , -R 1 / -R 6 , -R 1 / -R 7a , -R 4 / -R 5 , -R 4 / -R 6 , -R 8 / -R 9 , -R 2 / -R 3 One or more of the pairs together with the atoms to which they are attached form ring A;

[0406] Optionally, R 3 / R 3a Together with the nitrogen atom to which they are attached, they form a 3- to 10-membered heterocyclic ring;

[0407] A is selected from phenyl; naphthyl; indenyl; indanyl; tetrahydronaphthyl; C 3-10 cycloalkyl; 3- to 10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl; and

[0408] Where -L 1 - by at least one -L 2 -Z replaces, and where -L 1 - is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (II) is not replaced by -L 2 -Z or a substituent.

[0409] Preferably, -L in formula (II) 1 -Being a-L 2 -Z partial replacement.

[0410] In one embodiment, -L in formula (II) 1 - has not been further replaced.

[0411] It is understood that if -R 3 / -R 3a Together with the nitrogen atom to which they are attached, they form a 3- to 10-membered heterocyclic ring, then only those atoms directly attached to the nitrogen atom are SP3 In other words, through -R 3 / -R 3a Together with the nitrogen atom to which they are attached, such 3- to 10-membered heterocyclic rings have the following structure:

[0412]

[0413] in

[0414] The dotted line refers to -L 1 -The remaining part is connected;

[0415] The ring contains 3 to 10 atoms, including at least one nitrogen; and

[0416] R # and R ## Indicates SP 3 -Hybridized carbon atoms.

[0417] It is also understood that the 3- to 10-membered heterocyclic ring may be further substituted.

[0418] By -R of formula (II) 3 / -R 3a Exemplary embodiments of suitable 3- to 10-membered heterocyclic rings formed together with the nitrogen atom to which they are attached are as follows:

[0419]

[0420] in

[0421] Dashed lines indicate attachment to the remainder of the molecule; and

[0422] -R is selected from -H and C 1-6 alkyl.

[0423] -L of formula (II) 1 - may optionally be further substituted. Generally, any substituent may be used as long as the cleavage principle is not affected, i.e., the hydrogens marked with an asterisk in formula (II) are not replaced, and the nitrogens of the following moieties of formula (II) remain part of a primary, secondary or tertiary amine,

[0424]

[0425] That is, -R 3 and -R 3a Independently of each other, either -H or via SP 3 The -hybridized carbon atom is attached to -N<.

[0426] In one embodiment, -R of formula (II) 1 or -R 1a By-L2 -Z or -L 2 In another embodiment, -R 2 or -R 2a By-L 2 -Z or -L 2 In another embodiment, -R 3 or -R 3a By-L 2 -Z or -L 2 In another embodiment, -R 4 By-L 2 -Z or -L 2 In another embodiment, -R 5 or -R 5a By-L 2 -Z or -L 2 In another embodiment, -R 6 By-L 2 -Z or -L 2 In another embodiment, -R 7 or -R 7a By-L 2 -Z or -L 2 In another embodiment, -R 8 or -R 8a By-L 2 -Z or -L 2 In another embodiment, -R 9 or -R 9a By-L 2 -Z or -L 2 -Z' instead.

[0427] In another embodiment, -L 1 - has a structure as disclosed in WO2016 / 020373A1. Therefore, in one embodiment, -L 1 - the moiety has the formula (III):

[0428]

[0429] in

[0430] Dashed lines indicate linkage to a primary or secondary amine or hydroxyl group of -D via formation of an amide or ester linkage, respectively;

[0431] -R 1 , -R 1a , -R2 , -R 2a , -R 3 and -R 3a are independently selected from -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;

[0432] -R 4 , -R 5 and -R 5a are independently selected from -H, -C(R 9 R 9a R 9b ) and -T;

[0433] a1 and a2 are independently 0 or 1;

[0434] -R 6 , -R 6a , -R 7 , -R 7a , -R 8 , -R 8a , -R 8b , -R 9 , -R 9a , -R 9b Each is independently selected from -H, halogen, -CN, -COOR 10 、-OR 10 、-C(O)R 10 、-C(O)N(R 10 R 10a )、-S(O)2N(R 10 R 10a )、-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 Alkynyl; wherein -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 The alkynyl group is optionally substituted with one or more identical or different -R 11 Substitute, and where C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 The alkynyl group is optionally substituted with one or more selected from -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 )-groups;

[0435] -R 10 , -R 10a , -R 10b Each independently selected from -H, -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkynyl; wherein -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 The alkynyl group is optionally substituted with one or more identical or different -R 11 Substitute, and where C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20The alkynyl group is optionally substituted with one or more selected from -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 )-groups;

[0436] T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally replaced by one or more identical or different -R 11 replace;

[0437] -R 11 are independently selected from halogen, -CN, oxo (=O), -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(R13 )C(O)N(R 13a R 13b )、-OC(O)N(R 13 R 13a ) and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different;

[0438] -R 12 , -R 12a , -R 13 , -R 13a , -R 13b are each independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different;

[0439] Optionally, -R 1 / -R 1a , -R 2 / -R 2a , -R 3 / -R 3a , -R 6 / -R 6a , -R 7 / -R 7a One or more of the pairs together with the atoms to which they are attached form C 3-10 Cycloalkyl or 3- to 10-membered heterocyclic group;

[0440] Optionally, -R 1 / -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 , -R3 / -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 the pairs together with the atoms to which they are attached form ring A;

[0441] A is selected from phenyl; naphthyl; indenyl; indanyl; tetrahydronaphthyl; C 3-10 cycloalkyl; 3- to 10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl;

[0442] Where -L 1 - by at least one -L 2 -Z replaces and where -L 1 - is optionally further substituted.

[0443] -L of formula (III) 1 The optional further substituents of - are preferably as described above.

[0444] Preferably, -L of formula (III) 1 -Being a-L 2 -Z partial replacement.

[0445] In one embodiment, -L of formula (III) 1 - has not been further replaced.

[0446] In another embodiment, -L 1 - having a structure as disclosed in EP1536334B1, WO2009 / 009712A1, WO2008 / 034122A1, WO2009 / 143412A2, WO2011 / 082368A2 and US8618124B2, which are incorporated herein by reference.

[0447] In another embodiment, -L 1 - has a structure as disclosed in US8946405B2 and US8754190B2, which are incorporated herein by reference. Therefore, in another embodiment, -L 1 - having formula (IV):

[0448]

[0449] in

[0450] The dashed line indicates that the functional group of -D is connected to -D through the functional group of -D selected from -OH, -SH and -NH2;

[0451] m is 0 or 1;

[0452] -R 1 and -R 2 At least one or two of them are independently selected from -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 ,

[0453] -R 1 and -R 2 one and only one of the group consisting of -H, optionally substituted alkyl, optionally substituted arylalkyl, and optionally substituted heteroarylalkyl;

[0454] -R 3 is selected from -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 9 and -N(R 9 )2;

[0455] -R 4 is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

[0456] -R 5 each independently selected from -H, optionally substituted alkyl, optionally substituted alkenylalkyl, optionally substituted alkynylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

[0457] -R 9 is selected from -H and optionally substituted alkyl;

[0458] -Y- is absent and -X- is -O- or -S-; or

[0459] -Y- is -N(Q)CH2- and -X- is -O-;

[0460] Q is selected from optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

[0461] Optionally, -R 1 and -R 2 may together form a 3- to 8-membered ring; and

[0462] Optionally, two -R 9 together with the nitrogen to which they are attached form a heterocycle;

[0463] wherein -L 1 - is replaced by -L 2 -Z and wherein -L 1 - is optionally further substituted.

[0464] Only in the context of formula (IV), the terms used have the following meanings:

[0465] As used herein, the term "alkyl" includes straight-chain, branched-chain, or cyclic saturated hydrocarbon groups having 1 to 8 carbon atoms or in some embodiments having 1 to 6 or 1 to 4 carbon atoms.

[0466] The term "alkoxy" includes an alkyl group bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and the like.

[0467] The term "alkenyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon double bond.

[0468] The term "alkynyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon triple bond.

[0469] The term "aryl" includes aromatic hydrocarbon groups having 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 containing at least one N, O, or S atom and having 3 to 15 carbon atoms, preferably aromatic rings containing at least one N, O, or S atom and having 3 to 7 carbon atoms, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, indenyl, and the like.

[0470] In some instances, an alkenyl, alkynyl, aryl, or heteroaryl moiety may be linked to the remainder of the molecule via an alkylene linker. In those cases, the substituent will be referred to as alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl, indicating that the alkylene moiety is between the alkenyl, alkynyl, aryl, or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl, or heteroaryl is attached.

[0471] The term "halogen" includes bromine, fluorine, chlorine, and iodine.

[0472] The term "heterocycle" refers to a 4-8 membered aromatic or non-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".

[0473] When the ring system is optionally substituted, suitable substituents are selected from alkyl, alkenyl, alkynyl or another ring, each optionally further substituted. Any group, including the optional substituents on any group above, include halogen, nitro, cyano, -OR, -SR, -NR2, -OCOR, -NRCOR, -COOR, -CONR2, -SOR, -SO2R, -SONR2, -SO2NR2, wherein R is independently alkyl, alkenyl, alkynyl, aryl or heteroaryl, or two R groups together with the atoms to which they are attached form a ring.

[0474] Preferably, -L of formula (IV) 1 -Being a-L 2 -Z partial replacement.

[0475] In another embodiment, -L 1 - has a structure as disclosed in WO2013 / 036857A1, which is incorporated herein by reference. Therefore, in another embodiment, -L 1 - having formula (V):

[0476]

[0477] in

[0478] The dashed line indicates the connection to -D via the amine function of -D;

[0479] -R 1 is selected from optionally substituted C1-C6 straight chain, branched or cyclic alkyl; optionally substituted aryl; optionally substituted heteroaryl; alkoxy; and -NR 5 2;

[0480] -R 2 is selected from -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

[0481] -R 3 is selected from -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

[0482] -R 4 is selected from -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

[0483] -R5 each independently selected from -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl; or when taken together two -R 5 can be cycloalkyl or cycloheteroalkyl;

[0484] wherein -L 1 - is substituted by -L 2 -Z and wherein -L 1 - is optionally further substituted.

[0485] Only in the context of formula (V), the terms used have the following meanings:

[0486] "Alkyl", "alkenyl" and "alkynyl" include straight-chain, branched-chain or cyclic hydrocarbon groups having 1-8 carbon atoms or 1-6 carbon atoms or 1-4 carbon atoms, wherein alkyl is a saturated hydrocarbon, alkenyl includes one or more carbon-carbon double bonds, and alkynyl includes one or more carbon-carbon triple bonds. Unless otherwise specified, they contain 1-6 C.

[0487] "Aryl" includes aromatic hydrocarbon groups having 6-18 carbon atoms, preferably 6-10 carbon atoms, including groups such as phenyl, naphthyl and anthracene. "Heteroaryl" includes aromatic rings containing at least one N, O or S atom and having 3-15 carbon atoms, preferably aromatic rings containing at least one N, O or S atom and having 3-7 carbon atoms, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl (thiszolyl), isothiazolyl, quinolinyl, indolyl, indenyl, etc.

[0488] The term "substituted" means that an alkyl, alkenyl, alkynyl, aryl or heteroaryl group includes one or more substituent groups replacing one or more hydrogen atoms. Substituents can generally be selected from halogen, including F, Cl, Br and I; lower alkyl, including straight-chain, branched-chain and cyclic; lower haloalkyl, including fluoroalkyl, chloroalkyl, bromoalkyl and iodoalkyl; OH; lower alkoxy, including straight-chain, branched-chain and cyclic; SH; lower alkylthio, including straight-chain, branched-chain and cyclic; amino, alkylamino, dialkylamino, silyl, including alkylsilyl, alkoxysilyl and arylsilyl; nitro; cyano; carbonyl; carboxylic acid, carboxylic acid ester, carboxylic acid amide, aminocarbonyl; aminoacyl; carbamate; urea; thiocarbamate; thiourea; ketone; sulfone; sulfonamide; aryl, including phenyl, naphthyl and anthracenyl; heteroaryl, including 5-membered heteroaryl, including pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole and tetrazole, 6-membered heteroaryl, including pyridine, pyrimidine, pyrazine, and fused heteroaryl, including benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole and benzisothiazole.

[0489] Preferably, -L of formula (V) 1 - is replaced by a -L 2 -Z moiety.

[0490] In another embodiment, -L 1 - has the structure disclosed in US7585837B2, which is incorporated herein by reference. Thus, in another embodiment, -L 1 - has formula (VI):

[0491]

[0492] wherein

[0493] the dashed line indicates connection via the amine functionality of -D to -D;

[0494] R 1 and R 2 are independently selected from hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, -SO3H, -SO2NHR 5 、amino, ammonium, carboxyl, PO3H2 and OPO3H2;

[0495] R 3 、R 4 and R 5 are independently selected from hydrogen, alkyl and aryl;

[0496] wherein -L 1 - is replaced by -L 2 -Z and wherein -L 1 - is optionally further substituted.

[0497] Suitable substituents for formula (VI) are alkyl (e.g., C 1-6 alkyl), alkenyl (e.g., C 2-6 alkenyl), alkynyl (e.g., C 2-6 alkynyl), aryl (e.g., phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl (e.g., aromatic 4-7 membered heterocycle) or halogen moieties.

[0498] Only in the context of formula (VI), the terms used have the following meanings:

[0499] The terms "alkyl", "alkoxy", "alkoxyalkyl", "aryl", "alkaryl" and "aralkyl" refer to alkyl groups having 1-8, preferably 1-4 carbon atoms, such as methyl, ethyl, propyl, isopropyl and butyl, and aryl groups having 6-10 carbon atoms, such as phenyl and naphthyl. The term "halogen" includes bromine, fluorine, chlorine and iodine.

[0500] Preferably, -L of formula (VI) 1 - is replaced by a -L2 -The -Z part is substituted.

[0501] In another embodiment, -L 1 -Has the structure disclosed in WO2002 / 089789A1, which is incorporated herein by reference. Thus, in another embodiment, -L 1 -Has the formula (VII):

[0502]

[0503] Wherein

[0504] The dashed line indicates connection via the amine functional group of -D to -D;

[0505] L1 is a bifunctional linking group,

[0506] Y1 and Y2 are independently O, S or NR 7 ;

[0507] R 2 , R 3 , R 4 , R 5 , R 6 and R 7 Are independently selected from hydrogen, C 1-6 alkyl, C 3-12 branched alkyl, C 3-8 cycloalkyl, C 1-6 substituted 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 heteroalkoxy;

[0508] Ar is a moiety that forms a polysubstituted aromatic hydrocarbon or a polysubstituted heterocyclic group when included in formula (VII);

[0509] X is a chemical bond or a moiety that is actively transported into the target cell, a hydrophobic moiety, or a combination thereof,

[0510] y is 0 or 1;

[0511] Wherein -L 1 -Is substituted by -L 2 -Z and wherein -L 1 -Is optionally further substituted.

[0512] Only in the context of formula (VII), the terms used have the following meanings:

[0513] The term "alkyl" should be understood to include, for example, straight-chain, branched-chain, substituted C 1-12 alkyl, including alkoxy, C 3-8 cycloalkyl or substituted cycloalkyl, etc.

[0514] The term "substituted" should be understood to include adding one or more atoms within a functional group or compound or replacing one or more atoms with one or more different atoms.

[0515] Substituted alkyl includes carboxyalkyl, aminoalkyl, dialkylamino, hydroxyalkyl, and mercaptoalkyl; substituted cycloalkyl includes moieties such as 4-chlorocyclohexyl; aryl includes moieties such as naphthyl; substituted aryl includes moieties such as 3-bromo-phenyl; aralkyl includes moieties such as tolyl; heteroalkyl includes moieties such as ethylthiophene; substituted heteroalkyl includes moieties such as 3-methoxythiophene; alkoxy includes moieties such as methoxy; and phenoxy includes moieties such as 3-nitrophenoxy. Halogen should be understood to include fluorine, chlorine, iodine, and bromine.

[0516] Preferably, -L of formula (VII) 1 - is substituted by one -L 2 -Z moiety.

[0517] In another embodiment, -L 1 - includes the substructure of formula (VIII)

[0518]

[0519] wherein

[0520] The dotted line marked with an asterisk refers to connection to the nitrogen of -D by forming an amide bond;

[0521] The unmarked dotted line refers to connection to the remainder of -L 1 -; and

[0522] where -L 1 - is substituted by -L 2 -Z and where -L 1 - is optionally further substituted.

[0523] Preferably, -L of formula (VIII) 1 - is substituted by one -L 2 -Z moiety.

[0524] In one embodiment, -L of formula (VIII) 1 - is not further substituted.

[0525] In another embodiment, -L 1 - includes the substructure of formula (IX)

[0526]

[0527] wherein

[0528] The dotted line marked with an asterisk refers to connection to the nitrogen of -D through formation of a carbamate bond;

[0529] The unmarked dotted line refers to connection to the remainder of -L 1 -; and

[0530] wherein -L 1 - is replaced by -L 2 -Z and wherein -L 1 - is optionally further substituted.

[0531] Preferably, -L 1 - of formula (IX) is replaced by one -L 2 -Z moiety.

[0532] In one embodiment, -L 1 - of formula (IX) is not further substituted.

[0533] In one embodiment, -L 1 - has formula (IX-a):

[0534]

[0535] wherein

[0536] The dotted line marked with an asterisk refers to connection to the nitrogen of -D and the unmarked dotted line refers to connection to -L 2 -Z;

[0537] n is 0, 1, 2, 3 or 4;

[0538] =Y1 is selected from =O and =S;

[0539] -Y2- is selected from -O- and -S-;

[0540] -Y3- is selected from -O- and -S-;

[0541] -Y4- is selected from -O-, -NR 5 -, and -C(R 6 R 6a )-;

[0542] =Y5 is selected from =O and =S;

[0543] -R 3 -, -R 5 -, -R 6 -, -R 6aare independently selected from -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;

[0544] -R 4 Selected from 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;

[0545] -W-Selected from C 1-20 Alkyl, optionally substituted with one or more selected from C 3-10 Cycloalkyl, 8- to 30-membered carbon polycyclic group, 3- to 10-membered heterocyclic group, -C(O)-, -C(O)N(R 7 )-, -O-, -S- and -N(R 7 )-groups;

[0546] -Nu is a nucleophilic group selected from -N(R 7 R 7a )、-N(R 7 OH), -N(R 7 )-N(R 7a R 7b )、-S(R 7 ), -COOH,

[0547]

[0548] -Ar- is selected from

[0549]

[0550] in

[0551] The dotted line refers to -L 1 - the rest of the connection,

[0552] -Z 1 - is selected from -O-, -S- and -N(R 7 )-,and

[0553] -Z 2 -Yes-N(R 7 )-;and

[0554] -R 7 , -R 7a , -R 7b are independently selected from -H, C1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl;

[0555] Where -L 1 - is optionally further substituted.

[0556] In one embodiment, -L of formula (IX-a) 1 - has not been further replaced.

[0557] In another embodiment, -L 1 - having the formula (IX-b):

[0558]

[0559] in

[0560] The dashed line marked with an asterisk refers to the nitrogen bond to -D and the unmarked dashed line refers to the nitrogen bond to -L 2 -Z connection;

[0561] n is 0, 1, 2, 3, or 4;

[0562] =Y1 is selected from =O and =S;

[0563] -Y2- is selected from -O- and -S-;

[0564] -Y3- is selected from -O- and -S-;

[0565] -Y4- is selected from -O-, -NR 5 -and-C(R 6 R 6a )-;

[0566] =Y5 is selected from =O and =S;

[0567] -R 2 , -R 3 , -R 5 , -R 6 , -R 6a are independently selected from -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;

[0568] -R 4Selected from 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;

[0569] -W-Selected from C 1-20 Alkyl, optionally substituted with one or more selected from C 3-10 Cycloalkyl, 8- to 30-membered carbon polycyclic group, 3- to 10-membered heterocyclic group, -C(O)-, -C(O)N(R 7 )-, -O-, -S- and -N(R 7 )-groups;

[0570] -Nu is a nucleophilic group selected from -N(R 7 R 7a )、-N(R 7 OH), -N(R 7 )-N(R 7a R 7b )、-S(R 7 ), -COOH,

[0571]

[0572] -Ar- is selected from

[0573]

[0574] in

[0575] The dotted line refers to -L 1 - the rest of the connection,

[0576] -Z 1 - is selected from -O-, -S- and -N(R 7 )-,and

[0577] -Z 2 -Yes-N(R 7 )-;and

[0578] -R 7 , -R 7a , -R 7b are independently selected from -H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl;

[0579] Where -L 1 - is optionally further substituted.

[0580] In one embodiment, -L of formula (IX-b)1 - has not been further replaced.

[0581] In some embodiments, =Y of formula (IX-a) and (IX-b) 1 Yes = O.

[0582] In some embodiments, -Y of Formula (IX-a) and (IX-b) 2 -Yes-O-.

[0583] In some embodiments, -Y of Formula (IX-a) and (IX-b) 3 -Yes-O-.

[0584] In some embodiments, -Y of Formula (IX-a) and (IX-b) 4 -Yes-NR 5 -.

[0585] In some embodiments, =Y of formula (IX-a) and (IX-b) 5 Yes = O.

[0586] In some embodiments, n of Formula (IX-a) and (IX-b) is 0 or 1. In some embodiments, n of Formula (IX-a) and (IX-b) is 0. In some embodiments, n of Formula (IX-a) and (IX-b) is 1.

[0587] In some embodiments, -R of formula (IX-b) 2 is selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. 2 is selected from -H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, -R of formula (IX-b) 2 In some embodiments, -R of formula (IX-b) is selected from -H, methyl and ethyl. 2 Yes -H.

[0588] In some embodiments, -R of Formula (IX-a) and (IX-b) 3 is selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In some embodiments, -R of formula (IX-a) and (IX-b) 3 is selected from -H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, -R of formula (IX-a) and (IX-b) 3 In some embodiments, -R of formula (IX-a) and (IX-b) is selected from -H, methyl and ethyl. 3 Yes -H.

[0589] In a preferred embodiment, -R of formula (IX-a) and (IX-b) 4 Each is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In some embodiments, -R of formula (IX-a) and (IX-b) 4 is selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In some embodiments, -R of formula (IX-a) and (IX-b) 4 Selected from methyl and ethyl.

[0590] In some embodiments, -R of Formula (IX-a) and (IX-b) 5 is selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In some embodiments, -R of formula (IX-a) and (IX-b) 5 is selected from -H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, -R of formula (IX-a) and (IX-b) 5 In some embodiments, -R of formula (IX-a) and (IX-b) is selected from methyl and ethyl. 5 It's methyl.

[0591] In some embodiments, -R of Formula (IX-a) and (IX-b) 6 and -R 6a is independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In some embodiments, -R of formula (IX-a) and (IX-b) 6 and -R 6a is independently selected from -H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, -R of formula (IX-a) and (IX-b) 6 and -R 6a In some embodiments, -R of formula (IX-a) and (IX-b) is independently selected from -H, methyl and ethyl. 6 and -R 6a All are -H.

[0592] In some embodiments, Ar of formula (IX-a) and (IX-b) is phenyl. In some embodiments, Ar of formula (IX-a) and (IX-b) is

[0593]

[0594] The dashed lines indicate the connection to the remainder of the moieties (IX-a) and (IX-b).

[0595] In some embodiments, W of formula (IX-a) and (IX-b) is C 1-20Alkyl, optionally C 3-10 Cycloalkyl, -C(O)-, -C(O)N(R 7 )-, -O-, -S- and -N(R 7 In some embodiments, W of formula (IX-a) and (IX-b) is C 1-10 Alkyl, optionally C 3-10 Cycloalkyl, -C(O)-, -C(O)N(R 7 )-, -O-, -S- and -N(R 7 In some embodiments, W of formula (IX-a) and (IX-b) is C 1-6 Alkyl, optionally C 3-10 Cycloalkyl, -C(O)-, -C(O)N(R 7 )-, -O-, -S- and -N(R 7 In some embodiments, W of formula (IX-a) and (IX-b) is

[0596]

[0597] Therein, the dashed line indicates the connection to the remainder of the moiety of formula (IX-a) or (IX-b), respectively.

[0598] In some embodiments, -Nu of Formula (IX-a) and (IX-b) is -N(R 7 R 7a ).

[0599] In some embodiments, -R of Formula (IX-a) and (IX-b) 7 , -R 7a and -R 7b are independently selected from -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In some embodiments, -R of formula (IX-a) and (IX-b) 7 , -R 7a and -R 7b are independently selected from -H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, -R of formula (IX-a) and (IX-b) 7 , -R 7a and -R 7b are independently selected from methyl or ethyl. In some embodiments, -R in formula (IX-a) and (IX-b) 7 , -R 7a and -R 7b All are methyl.

[0600] In some embodiments, -L 1 - having formula (IX-c)

[0601]

[0602] in

[0603] The dashed line marked with an asterisk indicates the nitrogen connection to -D;

[0604] Unmarked dashed lines refer to -L 2 -Z connection; and

[0605] s1 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0606] In some embodiments, s1 of formula (IX-c) is an integer selected from 1, 2, 3, 4, and 5. In some embodiments, s1 of formula (IX-c) is 1. In some embodiments, s1 of formula (IX-c) is 2. In some embodiments, s1 of formula (IX-c) is 3. In some embodiments, s1 of formula (IX-c) is 4. In some embodiments, s1 of formula (IX-c) is 5.

[0607] In some embodiments, -L 1 - having formula (IX-d)

[0608]

[0609] in

[0610] The dashed line marked with an asterisk indicates the attachment to the nitrogen of -D; and

[0611] Unmarked dashed lines refer to -L 2 -Z connection.

[0612] -L 1 The - moiety may be connected to -D via any type of linkage, provided that it is reversible. Preferably, -L 1 - is linked to -D via a linkage selected from the group consisting of amide, ester, carbamate, acetal, aminal, imine, oxime, hydrazone, disulfide and acylguanidine. Even more preferably, -L 1 - is linked to -D via a linkage selected from amides, esters, carbamates and acylguanidines. It will be appreciated that these linkages may not themselves be reversible, but reversibility may be -L 1 -The effect of some atomic groups or parts present in it.

[0613] In one embodiment, -L 1 - is linked to -D via an ester linkage.

[0614] In another embodiment, -L 1 - is connected to -D via a carbamate linkage.

[0615] In another embodiment, -L 1 - is linked to -D via an acylguanidine.

[0616] In a preferred embodiment, -L 1 - is linked to -D via an amide linkage.

[0617] In some 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. In some 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 between -D and -L 1 - is a carbamate. Embodiments for such lysine residues are as described above.

[0618] In one embodiment, -L 2 - is a chemical bond.

[0619] In another embodiment, -L 2 - is the spacer moiety.

[0620] In some embodiments, -L 2 - is selected from -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 Alkynyl; wherein -T-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more identical or different -R y2 Substitute, and where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y3 )-、-S(O)2N(Ry3 )-、-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 )-groups;

[0621] -R y1 and -R y1a are independently selected from -H, -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more identical or different -R y2 Substitute, and where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more selected from -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 )-groups;

[0622] T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbocyclic group and 8- to 30-membered heteropolycyclic group; wherein each T is independently optionally replaced by one or more identical or different -R y2 replace;

[0623] -R y2are independently selected from halogen, -CN, oxo (=O), -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 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0624] -R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b are each independently selected from -H and C 1-6 Alkyl, where C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

[0625] In some embodiments, -L 2 - is selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-、-S(O)2N(R y1 )-、-S(O)N(Ry1 )-、-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 Alkynyl; wherein -T-, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 The alkynyl group is optionally substituted with one or more identical or different -R y2 Substitute, and where C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 The alkynyl group is optionally substituted with one or more selected from -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 )-groups;

[0626] -R y1 and -R y1a are independently selected from -H, -T, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl; wherein -T, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 The alkynyl group is optionally substituted with one or more identical or different -R y2 Substitute, and where C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 The alkynyl group is optionally substituted with one or more selected from -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 )-groups;

[0627] T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbocyclic group and 8- to 30-membered heteropolycyclic group; wherein each T is independently optionally replaced by one or more identical or different -R y2 replace-;

[0628] -R y2 Selected from halogen, -CN, oxo (=O), -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 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0629] -R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b Each independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

[0630] In some embodiments, -L 2 - is selected from -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 Alkynyl; wherein -T-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more identical or different -R y2 Substitute, and where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more selected from -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(Ry3 )-、-OC(OR y3 )(R y3a )-、-N(R y3 )C(O)N(R y3a )-and-OC(O)N(R y3 )-groups;

[0631] -R y1 and -R y1a Independently selected from -H, -T, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl;

[0632] T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclic group, 8- to 11-membered heterobicyclic group, 8- to 30-membered carbopolycyclic group, and 8- to 30-membered heteropolycyclic group;

[0633] -R y2 are each independently selected from halogen and C 1-6 alkyl; and

[0634] -R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b Each independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

[0635] In some embodiments, -L 2 - Yes C 1-20 an alkyl chain, which is optionally substituted by one or more independently selected from -O-, -T- and -C(O)N(R y1 )-group; and said C 1-20 The alkyl chain is optionally substituted with one or more independently selected from -OH, -T and -C(O)N(R y6 R y6a ) is substituted by a group; wherein -R y1 , -R y6 , -R y6a Independently selected from H and C 1-4 wherein T is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclic group, 8- to 11-membered heterobicyclic group, 8- to 30-membered carbopolycyclic group and 8- to 30-membered heteropolycyclic group.

[0636] In some embodiments, -L 2 - has a molecular weight of 14 to 750 g / mol.

[0637] In some embodiments, -L 2 - contains a part selected from the following

[0638]

[0639]

[0640] in

[0641] The dotted lines refer to -L 1 -、-L 2 - or -Z; and

[0642] -R and -R a are independently selected from -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0643] In some embodiments, -L 2 - having formula (IX-e)

[0644]

[0645] in

[0646] The dashed line marked with an asterisk refers to -L 1 -connect;

[0647] The unlabeled dashed line indicates the connection to -Z; and

[0648] 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.

[0649] In some embodiments, s2 of formula (IX-e) is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, s2 of formula (IX-e) is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8. In some embodiments, s2 of formula (IX-e) is 1. In some embodiments, s2 of formula (IX-e) is 2. In some embodiments, s2 of formula (IX-e) is 3. In some embodiments, s2 of formula (IX-e) is 4. In some embodiments, s2 of formula (IX-e) is 5. In some embodiments, s2 of formula (IX-e) is 6. In some embodiments, s2 of formula (IX-e) is 7. In some embodiments, s2 of formula (IX-e) is 8.

[0650] In some embodiments, -L 1 -L 2 - a portion having the formula (IX-f)

[0651]

[0652] in

[0653] The dashed line marked with an asterisk indicates the nitrogen connection to -D;

[0654] Unlabeled dashed lines refer to connections with -Z;

[0655] s1 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and

[0656] 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.

[0657] Therefore, the -L formed in the compound of formula (IX-f) 1 The link between the - and -D parts is the carbamate.

[0658] In some embodiments, s1 of formula (IX-f) is an integer selected from 1, 2, 3, 4, and 5. In some embodiments, s1 of formula (IX-f) is 1. In some embodiments, s1 of formula (IX-f) is 2. In some embodiments, s1 of formula (IX-f) is 3. In some embodiments, s1 of formula (IX-f) is 4. In some embodiments, s1 of formula (IX-f) is 5.

[0659] In some embodiments, s2 of formula (IX-f) is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, s2 of formula (IX-f) is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8. In some embodiments, s2 of formula (IX-f) is 1. In some embodiments, s2 of formula (IX-f) is 2. In some embodiments, s2 of formula (IX-f) is 3. In some embodiments, s2 of formula (IX-e) is 4. In some embodiments, s2 of formula (IX-f) is 5. In some embodiments, s2 of formula (IX-e) is 6. In some embodiments, s2 of formula (IX-f) is 7. In some embodiments, s2 of formula (IX-f) is 8.

[0660] In some embodiments, s1 of formula (IX-f) is 3 and s2 of formula (IX-f) is 3.

[0661] In one embodiment, the IL-2 conjugate of the invention has 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.

[0662] In another embodiment, the IL-2 conjugate of the invention has formula (Ib). In one embodiment, y is 2. In another embodiment, y is 3. In another embodiment, y is 4.

[0663] Another aspect of the invention is a pharmaceutical composition comprising at least one IL-2 conjugate of the invention and at least one excipient.

[0664] Preferably, the pharmaceutical composition comprising at least one IL-2 conjugate of the invention has a pH between pH 3 and pH 8, inclusive.

[0665] In one embodiment, the pharmaceutical composition comprising at least one IL-2 conjugate of the invention and at least one excipient is a liquid formulation.

[0666] In another embodiment, the pharmaceutical composition comprising at least one IL-2 conjugate of the present invention and at least one excipient is a dry formulation.

[0667] Such liquid or dry pharmaceutical compositions contain at least one excipient. Excipients for parenteral formulations can be classified, for example, as buffers, isotonic modifiers, preservatives, stabilizers, anti-adsorbents, oxidative protectants, tackifiers / viscosity enhancers or other adjuvants. However, in some cases, an excipient may have dual or triple functions. Preferably, the at least one excipient contained in the pharmaceutical composition of the present invention is selected from:

[0668] (i) Buffers: Physiologically tolerated buffers to maintain the pH in the desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate, chloride, pyruvate; antacids such as Mg(OH)2 or ZnCO3 may also be used;

[0669] (ii) Isotonic modifiers: minimize pain due to cellular damage caused by osmotic pressure differences at the injection site; effective concentrations can be determined by osmometric methods using an assumed osmotic pressure of 285-315 mOsmol / kg for serum;

[0670] (iii) Preservatives and / or antimicrobial agents: Multiple-dose parenteral formulations require the addition of preservatives in sufficient concentrations to minimize the risk of infection to the patient during injection, and regulatory requirements have been established; typical preservatives include m-cresol, phenol, methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosal, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and benzalkonium chloride;

[0671] (iv) Stabilizers: Stabilization is achieved by enhancing protein stability, by destabilizing the denatured state, or by directly binding the excipient to the protein; stabilizers can be amino acids, such as alanine, arginine, aspartic acid, glycine, histidine, lysine, proline, sugars, such as glucose, sucrose, trehalose, polyols, such as glycerol, mannitol, sorbitol, salts, such as potassium phosphate, sodium sulfate, chelating agents, such as EDTA, hexaphosphate, ligands, such as divalent metal ions (zinc, calcium, etc.), other salts or organic molecules, such as phenol derivatives; in addition, oligomers or polymers, such as cyclodextrins, dextran, dendrimers, PEG or PVP or protamine or HSA can be used;

[0672] (v) Anti-adsorption agents: primarily ionic or nonionic surfactants or other proteins or soluble polymers used to coat or competitively adsorb to the inner surface of the dosage container; 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 excipient concentration and type chosen depends on the effects to be avoided, but usually a monolayer of surfactant is formed at the interface just above the CMC value;

[0673] (vi) Oxidative protectants: Antioxidants such as ascorbic acid, ectoine, 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.

[0674] (vii) Viscosifiers or viscosity enhancers: delay the settling of particles in vials and syringes, serve to facilitate mixing and re-suspension of particles and serve to make the suspension easier to inject (i.e., less force on the syringe plunger); suitable viscosifiers or viscosity enhancers are, for example, carbomer viscosifiers, such as Carbomer 940, Carbopol Ultrez 10, cellulose derivatives, such as hydroxypropyl methylcellulose (hydroxypropyl methylcellulose, HPMC) or diethylaminoethylcellulose (DEAE or DEAE-C), colloidal magnesium silicate (Veegum) or sodium silicate, hydroxyapatite gel, tricalcium phosphate gel, xanthan gum, carrageenan such as Satiagum UTC 30, aliphatic polyhydroxy acids, such as poly (D, L- or L-lactic acid) (PLA) and polyglycolic acid (PGA) and their copolymers (PLGA), terpolymers of D, L-lactide, glycolide and caprolactone, poloxamers, hydrophilic poly (oxyethylene) blocks and hydrophobic poly (oxypropylene) blocks to form triblocks of poly (oxyethylene)-poly (oxypropylene)-poly (oxyethylene) (e.g. ), polyetherester copolymers such as polyethylene terephthalate / polybutylene terephthalate copolymers, sucrose acetate isobutyrate (SAIB), dextran or its derivatives, a combination of dextran and PEG, polydimethylsiloxane, collagen, chitosan, polyvinyl alcohol (PVA) and derivatives, polyalkylene diimide, poly(acrylamide-co-diallyldimethylammonium (DADMA)), polyvinylpyrrolidone (PVP), glycosaminoglycans (GAGs) such as dermatan sulfate, chondroitin sulfate , keratan sulfate, heparin, heparan sulfate, hyaluronan, ABA triblock or AB block copolymers composed of a hydrophobic A block such as polylactide (PLA) or poly(lactide-co-glycolide) and a hydrophilic B block such as polyethylene glycol (PEG) or polyvinylpyrrolidone; such block copolymers as well as the poloxamers described above can exhibit opposite thermogelation behavior (fluid state at room temperature to facilitate administration, gel state above the solution-gel transition temperature at body temperature after injection);

[0675] (viii) dispersants or spreading agents: alter the permeability of connective tissue by hydrolyzing components of the extracellular matrix of the interstitial spaces, such as, but not limited to, hyaluronic acid, a polysaccharide found in the intercellular spaces of connective tissue; dispersants such as, but not limited to, hyaluronidase temporarily reduce the viscosity of the extracellular matrix and facilitate the diffusion of injected drugs; and

[0676] (ix) Other adjuvants: such as wetting agents, viscosity modifiers, antibiotics, hyaluronidase; acids and bases, such as hydrochloric acid and sodium hydroxide, are adjuvants required for adjusting pH during production.

[0677] Another aspect of the present invention is an IL-2 conjugate or a pharmaceutically acceptable salt thereof for use as a medicament or a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention.

[0678] Another aspect of the present invention is an IL-2 conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention for use in treating a disease treatable with IL-2.

[0679] Preferably, the disease is cancer. Even 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 carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell cancer, hepatoma, bile duct cancer, choriocarcinoma, seminoma, embryonal 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, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia and leukemia. Even more preferred is a disease 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.

[0680] Preferred sarcoma types include fibrosarcoma, myxosarcoma, leiomyosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, gastrointestinal stromal tumors, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, and rhabdomyosarcoma.

[0681] In one embodiment, an IL-2 conjugate or a pharmaceutically acceptable salt thereof for treating a disease treatable with IL-2, or a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention, is administered to a patient before, simultaneously with, or after administration of one or more additional drugs, wherein the one or more additional drugs are preferably selected from: PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, such as tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; Toll-like receptor agonists (TLR), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8 and TLR9; and substances that agonize immune activating receptors such as 41BB (CD137), OX40, ICOS, CD40, CD28, NKG2D, NKp30, NKp44, NKp46, LFA1, CD16, CD64, CD32A and CD3. In one embodiment, the IL-2 conjugate or its pharmaceutically acceptable salt for treating a disease that can be treated with IL-2 or a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention is administered to a patient before, simultaneously with, or after the administration of one or more additional drugs, wherein the one or more additional drugs are preferably selected from: PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, such as tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; and Toll-like receptor agonists (TLR), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8, and TLR9. The administration of the IL-2 conjugate or its pharmaceutically acceptable salt or a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention and one or more additional drugs can be a single event or can be repeated multiple times.

[0682] In one embodiment, the PD-1 inhibitor is pembrolizumab. In another embodiment, the PD-1 inhibitor is nivolumab.

[0683] In one embodiment, the PD-L1 inhibitor is atuzumab. In another embodiment, the PD-L1 inhibitor is avelumab. In another embodiment, the PD-L1 inhibitor is durvalumab.

[0684] In one embodiment, the CTLA-4 inhibitor is ipilimumab. In another embodiment, the CTLA-4 inhibitor is tremelimumab.

[0685] In another embodiment, the IL-2 conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention is administered to the patient before, simultaneously with, or after CAR-T therapy. The administration of an IL-2 conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention in combination with CAR-T therapy may be a single event or may be repeated multiple times.

[0686] Preferably, the IL-2 conjugate or a pharmaceutically acceptable salt thereof for use in therapy or a pharmaceutical composition comprising at least one IL-2 conjugate of the invention is administered to a mammalian patient, preferably a human patient.

[0687] Another aspect of the present invention is the use of an IL-2 conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention for the preparation of a medicament for the treatment of a disease treatable with IL-2.

[0688] Preferably, the disease is cancer. Even 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 carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell cancer, hepatoma, bile duct cancer, choriocarcinoma, seminoma, embryonal 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, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia and leukemia. Even more preferred is a disease 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.

[0689] Preferred sarcoma types include fibrosarcoma, myxosarcoma, leiomyosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, gastrointestinal stromal tumors, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, and rhabdomyosarcoma.

[0690] In one embodiment, the agent is administered to the patient before, simultaneously with, or after one or more other drugs are administered, and the one or more other drugs are preferably selected from: PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; Cancer vaccines, such as 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 substances that excite immune activation receptors such as 41BB (CD137), OX40, ICOS, CD40, CD28, NKG2D, NKp30, NKp44, NKp46, LFA1, CD16, CD64, CD32A, and CD3. The administration of the agent with one or more other drugs can be a single event or can be repeated multiple times. In one embodiment, the agent is administered to the patient before, simultaneously with, or after the administration of one or more additional drugs, preferably selected from: PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, such as tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; and Toll-like receptor agonists (TLR), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8, and TLR9. The administration of the agent with one or more additional drugs can be a single event or can be repeated multiple times.

[0691] In one embodiment, the PD-1 inhibitor is pembrolizumab. In another embodiment, the PD-1 inhibitor is nivolumab.

[0692] In one embodiment, the PD-L1 inhibitor is atuzumab. In another embodiment, the PD-L1 inhibitor is avelumab. In another embodiment, the PD-L1 inhibitor is durvalumab.

[0693] In one embodiment, the CTLA-4 inhibitor is ipilimumab. In another embodiment, the CTLA-4 inhibitor is tesimumab.

[0694] In another embodiment, the agent is administered to the patient before, simultaneously with, or after the CAR-T therapy. The administration of the agent in combination with the CAR-T therapy can be a single event or can be repeated multiple times.

[0695] Preferably, the medicament is administered to a mammalian patient, more preferably a human patient.

[0696] Another aspect of the present invention is a method for treating, controlling, delaying or preventing one or more diseases treatable with IL-2 in a mammalian patient, preferably a human patient, in need of such treatment, the method comprising administering to said patient in need thereof a therapeutically effective amount of an IL-2 conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the IL-2 conjugate of the present invention.

[0697] Preferably, the one or more diseases treatable with IL-2 are cancers. Even 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 carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell cancer, hepatoma, bile duct cancer, choriocarcinoma, seminoma, embryonal 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, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia and leukemia. Even more preferred is a disease 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.

[0698] Preferred sarcoma types include fibrosarcoma, myxosarcoma, leiomyosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, gastrointestinal stromal tumors, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, and rhabdomyosarcoma.

[0699] In one embodiment, the IL-2 conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the IL-2 conjugate of the present invention is administered to a patient before, simultaneously with, or after administration of one or more additional drugs, wherein the one or more additional drugs are preferably selected from: PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, such as tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; Toll-like receptor agonists (TLR), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8 and TLR9; and substances that agonize immune activation receptors such as 41BB (CD137), OX40, ICOS, CD40, CD28, NKG2D, NKp30, NKp44, NKp46, LFA1, CD16, CD64, CD32A and CD3. The administration of IL-2 conjugates or pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising at least one IL-2 conjugate of the present invention with one or more additional drugs may be a single event or may be repeated multiple times. In one embodiment, IL-2 conjugates or pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising at least one IL-2 conjugate of the present invention are administered to a patient before, simultaneously with, or after the administration of one or more additional drugs, wherein the one or more additional drugs are preferably selected from: PD-1 inhibitors; PD-L1 inhibitors; CTLA-4 inhibitors; cancer vaccines, such as tumor cell vaccines, antigen vaccines, dendritic cell vaccines, vector-based vaccines; and Toll-like receptor agonists (TLR), including agonists targeting TLR2, TLR3, TLR2 / 4, TLR4, TLR5, TLR7 / 8, and TLR9. The administration of IL-2 conjugates or pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising at least one IL-2 conjugate of the present invention with one or more additional drugs may be a single event or may be repeated multiple times.

[0700] In one embodiment, the PD-1 inhibitor is pembrolizumab. In another embodiment, the PD-1 inhibitor is nivolumab.

[0701] In one embodiment, the PD-L1 inhibitor is atuzumab. In another embodiment, the PD-L1 inhibitor is avelumab. In another embodiment, the PD-L1 inhibitor is durvalumab.

[0702] In one embodiment, the CTLA-4 inhibitor is ipilimumab. In another embodiment, the CTLA-4 inhibitor is tesimumab.

[0703] In another embodiment, the IL-2 conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention is administered to the patient before, simultaneously with, or after CAR-T therapy. The administration of an IL-2 conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one IL-2 conjugate of the present invention in combination with CAR-T therapy may be a single event or may be repeated multiple times.

[0704] Another method of the present invention is a method of administering an IL-2 conjugate, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention, wherein the method comprises the step of administering an IL-2 conjugate, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention via topical, enteral or parenteral administration, and by methods of external application, injection or infusion, including intra-articular, peri-articular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, intracapsular, intraorbital, intravitreal, intratympanic, intravesical, intracardiac, transtracheal, subcutaneous, subcapsular, subarachnoid, intravertebral, intraventricular, intrasternal injection and infusion, direct delivery to the brain (e.g., Ommaya Reservoir) via an implanted device that allows delivery of the present invention, etc. to brain tissue or brain fluid, direct intraventricular injection or infusion, injection or infusion into the brain or brain-related areas, injection into the subchoroidal space, retro-orbital injection, and eye drops, preferably via subcutaneous injection.

[0705] In one embodiment, the present invention relates to an IL-2 conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present invention for use in treating one or more diseases treatable with 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 of the present invention for use in treating one or more diseases treatable with IL-2 via subcutaneous injection. DETAILED DESCRIPTION

[0706] Material

[0707] 0.7 kDa PEG maleimide (MeO-dPEG(12)-mal, maleimido-N-(2,5,8,11,14,17,20,23,26,29,32,35-dodecanoic acid heptatriacont-37-yl)propionamide, CAS 724722-89-8) was obtained from IrisBiotech GmbH, Marktredwitz, Germany. 2 kDa PEG-maleimide (Sunbright ME-020-MA, CAS 883993-35-9), 5 kDa PEG-maleimide (Sunbright ME-050MA, CAS 883993-35-9), 10 kDa PEG-maleimide (Sunbright ME-100MA, CAS 883993-35-9), and 20 kDa PEG-maleimide (Sunbright ME-200MA0B, CAS 883993-35-9) were obtained from NOF Europe NV, Grobbendonk, Belgium.

[0708] 10 kDa PEG amine (Sunbright ME-100EA, CAS 80506-64-5) is available from NOF Europe NV, Grobbendonk, Belgium.

[0709] Recombinant human IL-2 (Aldesleukin, catalog number AF-200-02) was obtained from Peprotech, Rocky Hill, NJ, USA.

[0710] Microbial transglutaminase (Cat. No. T001) and MTG blocker (Cat. No. C102) were obtained from Zedira GmbH, Darmstadt, Germany.

[0711] 10 kDa Mal-PEG-NH2 (Cat. No. PHB-943) can be obtained, for example, from Creative PEGWorks, Chapell Hill, NC, USA.

[0712] method

[0713] Example 1: Preparation of IL-2 variants

[0714] IL-2 variants (mutants) were custom made and obtained from external suppliers, where protein expression was performed from E. coli, followed by standard purification strategies known to those skilled in the art. The following proteins were prepared:

[0715] 1a-·SEQ ID NO:3:PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTCMLTFKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADETATIVEFLNRW ITFSQSIISTLT

[0716] 1b-SEQ ID NO:4:PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT CKFYMPKKATELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADETATIVEFLNRWITFSQSIIST LT

[0717] 1c-SEQ ID NO:5:PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFCMPKKATELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRWITFSQSIIST LT

[0718] 1d-SEQ ID NO:6:PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKATELKHLQCLEE CLKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRWITFSQSIISTLT

[0719] Example 2: Preparation of 0.7 kDa PEG-IL-2 mutant protein conjugate 2

[0720] About 2 mL of 0.2 mg / mL IL-2 mutant protein 1a formulated in 50 mM acetic acid pH 3 was concentrated via a centrifugal filter to a final volume of 0.47 mL with a concentration of 0.85 mg / mL as determined by UV (A280). Conjugation was performed at about pH 7.5 as follows. 0.385 mg 1a (0.455 mL, 0.85 mg / mL) in 50 mM acetic acid pH 3 was mixed with 0.36 vol. eq. (164 μ L) 0.5M sodium phosphate pH 8, 16 μL 37mM acetic acid, 132mM sodium phosphate pH7 and 3mol eq. (5 μ The mixture was stirred for 15 minutes at room temperature for 15 minutes. The 1a was purified by MS. The conjugates were separated 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 a GE Healthcare Superdex 200 Increase 10 / 300 GL (24 mL volume) column. The system was used, the mobile phase was 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, and the flow rate was 0.75 mL / min. A fraction containing the conjugate was concentrated using a centrifugal filter to obtain 2, which was 0.45 mL of protein solution with a protein content of 0.22 mg / mL and a purity of approximately 79%, as determined by SEC.

[0721] Example 3: Preparation of 2kDa PEG-IL-2 mutant protein conjugate 3

[0722] About 2 mL of 0.2 mg / mL IL-2 mutant protein 1a formulated in 50 mM acetic acid pH 3 was concentrated via a centrifugal filter to a final volume of 0.34 mL at a concentration of 1.16 mg / mL as determined by A280. Conjugation was performed at about pH 7 as follows. 0.38 mg 1a (0.33 mL, 1.16 mg / mL) in 50 mM acetic acid pH 3 was mixed with 0.36 vol. eq. (118 μL) of 0.5 M sodium phosphate pH 8 and 20 mol eq. (20 μL) of 24.8mM 2kDa PEG-Mal in water. The solution was carefully shaken and incubated at ambient temperature for 15 minutes. 0.82mL of a thiol-functionalized hydrogel suspension as described in Example 29 on page 159 of WO2014056923 (hydrogel content of 21.8mg / mL and thiol content of 3μmol / mL, equivalent to 5moleq. thiol for 2kDa PEG maleimide) was transferred to a 2mL PP reactor equipped with a PE sintered funnel. The reaction mixture was transferred to a reaction vessel and incubated at ambient temperature for 30 minutes under gentle stirring to allow excess PEG-maleimide to bind to the hydrogel. The protein-containing solution was discharged and the protein conjugate was separated from the conjugated mixture by size exclusion chromatography (SEC) using a GE Healthcare Superdex 75Increase10 / 300GL (24mL volume) column connected to a 2mL PP reactor. The system was used, the mobile phase was 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, and the flow rate was 0.75 mL / min. The fractions containing the conjugate were concentrated using a centrifugal filter to obtain 0.37 mL of 2 kDa PEG IL-2 mutant protein conjugate 3, with a protein concentration of 0.27 mg / mL and a purity of 78% according to SEC.

[0723] Example 4: Preparation of 5 kDa PEG-IL-2 mutant protein conjugate 4

[0724] About 2mL 0.2mg / mL IL-2 mutant protein 1a prepared in 50mM acetic acid pH3 is concentrated to a final volume of 0.36mL via a centrifugal filter, and a concentration of 1.01mg / mL is measured by A280. It is conjugated as follows at about pH 7.5. 0.35mg protein (0.35mL, 1.01mg / mL) in 50mM acetic acid pH3 is mixed with 0.36vol.eq. (125μL) of 0.5M sodium phosphate pH 8 and 3mol eq. (9.8μL, activity correction) of 7.49mM 5kDa PEG-maleimide in water. The solution is carefully shaken and incubated at ambient temperature for 15 minutes. In order to quench excess PEG-maleimide, 6mol eq. of 13.8mM L-cysteine ​​aqueous solution is added relative to 1a (10μL) in the conjugated mixture. The solution was shaken gently and incubated at ambient temperature for 2.5 hours. The conjugate was separated 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 System, mobile phase is 10mM HEPES, 150mM sodium chloride, 0.05% Tween 20, pH 7.4, flow rate is 0.75mL / min. The fractions containing the majority of the conjugates were pooled and concentrated using a centrifugal filter to obtain 0.42mL of 5kDa PEG IL-2 mutant protein conjugate 4 with a protein concentration of 0.30mg / mL. The separated conjugates were analyzed by SEC and RP-HPLC. Quantitative removal of impurities could not be obtained by the purification steps, as shown by RP-HPLC. According to SEC analysis at 215nm, the approximate purity of conjugate 4 was determined to be 68%.

[0725] Example 5: Preparation of 10 kDa PEG-IL-2 mutant protein conjugate 5

[0726] About 2 mL of 0.2 mg / mL IL-2 mutant protein 1a prepared in 50 mM acetic acid pH 3 was concentrated to a final volume of 0.35 mL via a centrifugal filter, with a concentration of 1.03 mg / mL, as determined by A280. Conjugation was performed at about pH 7.5 as follows. 0.34 mg of protein (0.33 mL, 1.03 mg / mL) in 50 mM acetic acid pH 3 was mixed with 0.36 vol. eq. (120 μL) of 0.5 M sodium phosphate pH 8 and 3 mol eq. (5 μL) of 13.54 mM 10 kDa PEG-maleimide in water. The solution was carefully shaken and incubated at ambient temperature for 15 minutes. The conjugate was separated from the conjugated mixture via size exclusion chromatography (SEC) using a GE Healthcare Superdex200Increase10 / 300GL (24 mL volume) column, which was connected to a 400 μL column. System, mobile phase is 10mM HEPES, 150mM sodium chloride, 0.05% Tween 20, pH 7.4, flow rate is 0.75mL / min. The fractions containing the conjugate were pooled and concentrated using a centrifugal filter to obtain 0.55mL 10kDa PEG IL-2 mutant protein conjugate 5 with a protein concentration of 0.22mg / mL. The separated conjugate was analyzed by SEC and RP-HPLC. Quantitative removal of impurities could not be obtained by the purification steps. According to SEC analysis at 215nm, the approximate purity of conjugate 5 was determined to be 57%, and the main impurity was excess PEG-Mal.

[0727] Example 6: Preparation of 20 kDa PEG-IL-2 mutant protein conjugate 6

[0728] About 2mL 0.2mg / mL IL-2 mutant protein 1a prepared in 50mM acetic acid pH3 is concentrated to a final volume of 0.33mL via a centrifugal filter, with a concentration of 1.11mg / mL, as determined by A280. Conjugation is performed as follows at about pH 7.5. 0.35mg protein (0.32mL, 1.11mg / mL) in 50mM acetic acid pH3 is mixed with 0.36vol.eq. (115μL) of 0.5M sodium phosphate pH 8, 6μL of 37mM acetic acid, 132mM sodium phosphate pH 7 and 3mol eq. (10μL) of 6.96mM 20kDa PEG-maleimide in water. The solution is carefully shaken and incubated at ambient temperature for 15 minutes. The conjugate was separated 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 System, mobile phase is 10mM HEPES, 150mM sodium chloride, 0.05% Tween 20, pH7.4, flow rate is 0.75mL / min. The fractions containing the conjugate were pooled and concentrated using a centrifugal filter to obtain 0.67mL 20kDa PEG IL-2 mutant protein conjugate 6 with a protein concentration of 0.18mg / mL. The separated conjugate was analyzed by SEC and RP-HPLC. Quantitative removal of impurities could not be obtained by the purification steps. According to RP-HPLC analysis at 215nm, the approximate purity of conjugate 6 was determined to be 44%, and the main impurity was excess PEG-Mal.

[0729] Example 7: Preparation of 5 kDa PEG-IL-2 mutant protein conjugate 7

[0730] 1.1 mL 0.66 mg / mL IL-2 mutant protein 1c prepared in 50 mM acetic acid pH 3 was mixed with 0.15 vol. eq. (165 μL) 0.5 M sodium phosphate pH 8 and 141 μL 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 centrifugal filter to obtain a final volume of 0.67 mL with a concentration of 0.99 mg / mL, as determined by A280. Conjugation was performed as follows. 0.65 mg protein (0.66 mL, 0.99 mg / mL) in 58 mM sodium phosphate, about 57 mM hydroxylamine hydrochloride, 39 mM acetic acid pH 6.5 was mixed with 6 mol eq. (320 μL) 81 mM 5kDa PEG-maleimide in water. The resulting reaction mixture was incubated at ambient temperature for 2.25 hours. Subsequently, the buffer was exchanged to 37 mM acetic acid, 132 mM phosphate pH 7 using a centrifugal filter to obtain a final volume of 0.75 mL. 1.5 mol eq. (80 μL) of 81 mM 5 kDa PEG-maleimide in water was added to the reaction solution, followed by 3 mol eq. (160 μL) of 81 mM 5 kDa PEG-maleimide in water 3.5 hours later. The resulting reaction mixture was incubated overnight at ambient temperature. The conjugate was separated from the conjugated mixture via size exclusion chromatography (SEC) using a GE Healthcare Superdex 75 Increase 10 / 300 GL (24 mL volume) column connected to a 400 μL column. System, mobile phase is 10mM HEPES, 150mM sodium chloride, 0.05% Tween20, pH 7.4, flow rate is 0.75mL / min. The collected fractions containing conjugate are concentrated using a centrifugal filter to obtain 0.45mL 5kDa PEG IL-2 mutant protein 7, which is 0.25mg / mL. The isolated conjugate is analyzed by SEC and RP-HPLC. The removal of impurities cannot be obtained through the purification steps, resulting in their presence in the conjugate sample, and the approximate purity of conjugate 7 is 60% according to SEC.

[0731] Example 8: Preparation of 10 kDa PEG-IL-2 mutant protein conjugate 8

[0732] 1.1 mL of 0.66 mg / mL IL-2 mutant protein 1c prepared in 50 mM acetic acid pH 3 was mixed with 0.15 vol. eq. (165 μL) 0.5 M sodium phosphate pH 8 and 141 μL 0.5 M hydroxylamine hydrochloride, 0.5 M sodium phosphate pH 7. The resulting reaction mixture was incubated overnight at 25 ° C. The solution was concentrated using a centrifugal filter to a final volume of 0.48 mL with a concentration of 1.42 mg / mL, as determined by A280. Conjugation was performed as follows. 0.678 mg of protein (0.48 mL, 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 3 mol eq. (18 μL) 7.42 mM 10 kDa PEG10-maleimide in water. The resulting reaction mixture was shaken carefully and incubated at ambient temperature for 15 minutes. An additional 3 mol eq. (17.9 μL) of 7.42 mM PEG-maleimide in water was added to the conjugation mixture and then incubated at ambient temperature for 15 minutes. The conjugate was separated 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 a System, mobile phase is 10mM HEPES, 150mM sodium chloride, 0.05% Tween 20, pH 7.4, flow rate is 0.75mL / min. The collected fractions are analyzed by RP-HPLC, and the fractions containing the conjugate are pooled and concentrated using a centrifugal filter to obtain 0.57mL 10kDa PEG IL-2 mutant protein 8, which is 0.50mg / mL. The separated conjugate is analyzed by SEC and RP-HPLC. Quantitative removal of impurities from the raw material cannot be achieved through the purification steps, resulting in their presence in the conjugate sample, as shown by RP-HPLC. The approximate purity of conjugate 8 is 25% according to RP-HPLC at 280nm, and the main impurity is excess PEG-Mal.

[0733] Example 9: SPR analysis of PEG IL-2 mutein conjugates

[0734] The binding kinetics of aldesleukin and IL-2-biased compounds were evaluated using surface plasmon resonance (SPR) spectroscopy with a Biacore instrument (T200, GE Healthcare) using immobilized IL-2 receptor subunit extracellular domains. Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare) were activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Immobilization of monoclonal mouse anti-human IgG (Fc) antibodies was also performed according to the supplier's instructions (GE Healthcare, instruction number BR-1008-39).

[0735] For determination of binding kinetics to IL-2Rα, the following chip preparation was used: human IL-2 receptor α, 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) and fixed at a flow rate of 10 μL / min for 60 s to obtain 80-100 response units (RU).

[0736] For determination of binding kinetics to IL-2Rβ, the following chip preparation was used: human IL-2 receptor β, 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) and fixed at a flow rate of 10 μL / min for 60 s to obtain 140-180 response units (RU).

[0737] For determination of binding kinetics to the IL-2Rαβ complex, the following chip preparation was used: human IL-2 receptor α, Fc-Tag (Symansis, New Zealand) and human IL-2 receptor β, Fc-Tag (Symansis, New Zealand) were mixed, diluted in HBS-EP running buffer (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20, pH 7.4) to approximately 1.33 μg / mL each, fixed at a flow rate of 10 μL / min for 60 s, and 360-440 response units (RU) were obtained.

[0738] For kinetic assays on all three receptor subunits, aldesleukin or IL-2-biased compounds were analyzed with multicycle kinetics, thus injecting at least five different concentrations in HBS-EP running buffer at 25°C (30 μL / min flow rate, 120 sec contact time, 600 sec dissociation time). Double-referenced data (data subtracted from reference flow cell and buffer-only samples) were analyzed kinetically (Biacore T200 Evaluation Software, version 3.1) to determine K if possible via a 1:1 binding model or via steady-state analysis. D and if possible k a and k d Regeneration after each cycle was performed using 3M MgCl2 at a flow rate of 30 μL / min for 30 seconds.

[0739] The obtained data are summarized in Table 1.

[0740] Table 1: Summary of SPR binding data to IL-2 receptor subunits

[0741]

[0742] Example 10: Calculation of bias ratio

[0743] As used herein, the term "biased IL-2" refers to a region wherein the K of IL-2 is biased toward IL-2Rαβ D The bias of IL-2 on IL-2Rβ D The ratio is greater than the K of aldesleukin to IL-2Rαβ D Compared with aldesleukin, the K D This is described by the following formula:

[0744]

[0745] in

[0746]

[0747] in

[0748] “K D "Biased IL-2 towards IL-2Rαβ" is the K biased IL-2 towards IL-2Rαβ D ,

[0749] “K D The bias of IL-2 towards IL-2Rαβ is the K bias of IL-2 towards IL-2Rβ D ,

[0750] “K D"Aldesleukin to IL-2Rαβ" is the K of aldesleukin to IL-2Rαβ D ,and

[0751] “K D "Aldesleukin for IL-2Rβ" is the K of aldesleukin for IL-2Rβ D .

[0752] The following ratios were calculated using the above formula and are summarized in Table 2.

[0753] Table 2: Ratios for different IL-2 mutein conjugates 偏倚IL-2 / Proportion 阿地白介素

[0754] Compound <![CDATA[Ratio 偏倚IL-2 / Ratio 阿地白介素 > 6 >100 5 >120 4 >140 3 >340 8 nd 7 >140

[0755] Example 11: Preparation of 10 kDa PEG-IL-2 conjugate 9

[0756] Connect to The recombinant human IL-2 was buffer exchanged into 0.2M Tris / HCl, pH 7.5 using the HiTrap desalting column of the system. Subsequently, IL-2 was enzymatically PEGylated using microbial transglutaminase (MTG) based on Sato et al., Bioconjugate Chem 2001, 12 (5), 701-710. IL-2 at a concentration of 5 μM was incubated in 0.2M Tris / HCl, pH 7.5 for 12 hours at 25°C in the presence of 1.25mM 10kDa PEG amine (10kDa methoxy-PEG-(CH2)2-NH2) and 0.2 units / mL MTG. The 10kDa PEG IL-2 conjugate (9) was purified from the reaction mixture by size exclusion chromatography using a GE Healthcare Superdex200Increase 10 / 300GL column connected to a 100 μM column. The system was used with a mobile phase of 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, and a flow rate of 0.75 mL / min.

[0757] Example 12: Preparation of 10 kDa PEG-IL-2 conjugate 10

[0758] Recombinant human IL-2 (2.7 mg / mL in 50 mM acetic acid, pH 3.0) was mixed with one volume equivalent of 140 mM HEPES, 300 mM NaCl, 6 mM sodium EDTA, 0.1% Tween 20, pH 8.2 to yield 1.35 mg / mL 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 for 4 hours at 37°C 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 PEG amine (10 kDa methoxy-PEG-(CH2)2-NH2) and 1 U / mL MTG. Afterwards, MTG blocker was added to a final concentration of 0.5 mM and the reaction mixture was incubated for another 30 minutes. The 10 kDa 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 a 400 μL column. The system was used with a mobile phase of 10 mM HEPES, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4, and a flow rate of 0.75 mL / min.

[0759] Example 13: Preparation of Biased IL-2 Mutant Protein Polymer Prodrug 11

[0760] The PEG IL-2 mutant protein was buffer exchanged into 100mM sodium borate pH 9 and concentrated to a concentration of about 2.5mg / mL IL-2eq. A 40kDa mPEG-linker reagent (as described in Example 2 of patent WO 2016079114) with a 5-fold molar excess relative to the amount of IL-2 mutant protein was dissolved in an equal volume of water compared to the protein solution to be used for the conjugation reaction. The protein solution (2.5mg / mL IL-2eq.) and the 40kDa mPEG-linker solution (32g / L) were mixed and incubated at 14-16°C for 2 hours. The biased IL-2 mutant protein polymer prodrug 11 was separated by CIEX and analyzed by SEC.

[0761] Example 14: Preparation of novel conjugate 12

[0762] The 40kDa mPEG-linker reagent (as described in Example 2 of patent WO 2016079114) was dissolved in water to produce a 32g / L solution. 10kDa Mal-PEG-NH2 was dissolved in 0.1M sodium phosphate, 6mM sodium EDTA, pH 7.4 to a final concentration of 1mM. The two solutions were mixed in a volume ratio of 1:1 and incubated at ambient temperature for 2 hours. Afterwards, 0.5 volume equivalents (in terms of the volume of the reaction mixture of 40kDa mPEG-linker reagent and 10kDa Mal-PEG-NH2) of IL-2 mutant protein 1a in 50mM sodium phosphate, 3mM sodium EDTA, pH 7.4 was added to the reaction mixture and incubated at ambient temperature for 1 hour. 40+10kDa PEG IL-2 mutant protein conjugate 12 was separated from the reaction mixture by cation exchange chromatography and analyzed by size exclusion chromatography.

[0763] Abbreviations

[0764] A280 Absorption at 280nm

[0765] AcOH Acetic acid

[0766] CIEX Cation Exchange

[0767] E.coli Escherichia coli

[0768] EDC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

[0769] EDTA Ethylenediaminetetraacetic acid

[0770] eq. equivalent

[0771] HBS-EP 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Surfactant P20, pH7.4

[0773] HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

[0774] IL-2 Interleukin-2

[0775] IL-2R Interleukin-2 receptor

[0776] Mal Maleimide

[0777] mol.eq. molecular equivalent

[0778] mPEG Methoxy PEG

[0779] MS mass spectrometry

[0780] MTG Microbial Transglutaminase

[0781] NaCl Sodium chloride

[0782] NHS N-Hydroxysuccinimide

[0783] PEG Polyethylene glycol

[0784] PTFE Polytetrafluoroethylene

[0785] PyBOP Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

[0786] RP-HPLC Reversed-phase high performance liquid chromatography

[0787] SEC Size Exclusion Chromatography

[0788] SPR Surface Plasmon Resonance

[0789] Tween 20 Sorbitan Monolaurate

Claims

1. An IL-2 conjugate of formula (Ia) or (Ib) or a pharmaceutically acceptable salt thereof, in -D is a biased IL-2 portion, the biased IL-2 portion comprising an IL-2 portion, and for the biased IL-2 portion, the biased IL-2 has a K for IL-2Rαβ D The bias of IL-2 on IL-2Rβ D The ratio is higher than that of aldesleukin for IL-2Rαβ D Compared with aldesleukin, the K D wherein the biased IL-2 portion comprises at least one amino acid mutation; -L 1 - is a linker moiety that is covalently and reversibly attached to -D; -L 2 - 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; and y is an integer selected from 2, 3, 4 and 5.

2. The IL-2 conjugate of claim 1 , or a pharmaceutically acceptable salt thereof, wherein the IL-2 portion 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, or at the corresponding position of a homolog or variant thereof.

3. The IL-2 conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the IL-2 portion comprises at least one amino acid mutation at position R37 or F41 based on SEQ ID NO: 2, or the corresponding position of a homologue or variant thereof.

4. The IL-2 conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the IL-2 portion comprises at least one amino acid mutation at position R37 based on SEQ ID NO: 2 or a corresponding position of a homologue or variant thereof.

5. The IL-2 conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the at least one mutation is a replacement 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.

6. The IL-2 conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the at least one mutation is a replacement of a naturally occurring amino acid with a non-proteinogenic amino acid.

7. The IL-2 conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the IL-2 portion comprises at least one amino acid mutation selected from R37A, R37C, R37G, R37S, R37T, R37Q, R37E, R37N, R37D, R37H, R37W, R37Y and R37K based on SEQ ID NO: 2 or the corresponding positions thereof.

8. The IL-2 conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the at least one mutation is a replacement of a naturally occurring amino acid with cysteine.

9. The IL-2 conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the IL-2 portion comprises an R37C mutation.

10. The IL-2 conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the IL-2 conjugate has formula (Ia), wherein x is 1.

11. The IL-2 conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein -Z is a polymeric moiety.

12. The IL-2 conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein -Z comprises a moiety of formula (A) in -BP 1 <, -BP 2 <, -BP 3 <each independently selected from -N< and -C(R 8 )<; R 8 Selected from H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl; -P 1 , -P 2 , -P 3 , -P 4 are, independently of one another, PEG-based chains comprising at least 40% PEG and having a molecular weight of 3 to 40 kDa; -C 1 -、-C 2 - independently selected from C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more identical or different R 9 Substitute, and where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more selected from -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 )-groups; T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbocyclic group and 8- to 30-membered heteropolycyclic group; wherein each T is independently optionally replaced by one or more identical or different R 9 replace; R 9 are independently selected from halogen, -CN, oxo (=O), -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 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and R 10 , R 10a , R 11 , R 11a and R 11b are each independently selected from -H and C 1-6 Alkyl, where C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

13. The IL-2 conjugate according to claim 12 or a pharmaceutically acceptable salt thereof, wherein C of formula (A) 1 and C 2 With formula (Aa) in The dashed line marked with an asterisk refers to BP 1 connect; Unlabeled dashed lines indicate BP 2 or BP 3 connect; q1 is selected from 1, 2, 3, 4, 5, 6, 7 and 8; q2 is selected from 1, 2, 3, 4 and 5; q3 is selected from 1, 2, 3, 4, 5, 6, 7 and 8; and q4 is selected from 1, 2 and 3.

14. The IL-2 conjugate according to claim 12 or a pharmaceutically acceptable salt thereof, wherein P of formula (A) 1 , P 2 , P 3 and P 4 Each independently is of formula (Ab) in The dashed line indicates the connection to the rest of -Z; m is 0 or 1; p is an integer from 70 to 900; and q is selected from 1, 2, 3, 4, 5 and 6.

15. The IL-2 conjugate according to claim 12 or a pharmaceutically acceptable salt thereof, wherein BP of formula (A) 1 Yes -N<.

16. The IL-2 conjugate according to claim 12 or a pharmaceutically acceptable salt thereof, wherein BP of formula (A) 2 and BP 2 All are -CH<.

17. The IL-2 conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein -Z comprises a moiety of formula (Ac): in p1, p2, p3, and p4 are each independently an integer of 70 to 900.

18. The IL-2 conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein -L 1 - the amino acid residue linked to the IL-2 moiety.

19. The IL-2 conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein -L 1 - an amino acid residue linked to the IL-2 moiety, said amino acid residue being selected from the group consisting of cysteine, methionine, histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, glutamine and arginine.

20. The IL-2 conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein L 1 - linked to a lysine residue selected from K7, K8, K31, K34, K42, K47, K48, K53, K63, K75 and K96 based on SEQ ID NO:

2.

21. The IL-2 conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein -L 1 - having the formula (IX-a): in The dashed line marked with an asterisk indicates the nitrogen bond to -D, and the unmarked dashed line indicates the nitrogen bond to -L. 2 -Z connection; n is 0, 1, 2, 3, or 4; =Y1 is selected from =O and =S; -Y2- is selected from -O- and -S-; -Y3-, -Y5- are independently selected from -O- and -S-; -Y4- is selected from -O-, -NR 5 -and-C(R 6 R 6a )-; -R 3 , -R 5 , -R 6 , -R 6a are independently selected from -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 Selected from 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 optionally selected from one or more selected from C 3-10 Cycloalkyl, 8- to 30-membered carbon polycyclic group, 3- to 10-membered heterocyclic group, -C(O)-, -C(O)N(R 7 )-, -O-, -S- and -N(R 7 )-intervening C 1-20 alkyl; -Nu is a nucleophilic group selected from -N(R 7 R 7a )、-N(R 7 OH), -N(R 7 )-N(R 7a R 7b )、-S(R 7 ), -COOH, -Ar- is selected from in The dotted line refers to -L 1 - the rest of the connection, -Z 1 - is selected from -O-, -S- and -N(R 7 )-,and -Z 2 -Yes-N(R 7 )-;and -R 7 , -R 7a , -R 7b are independently selected from -H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl; Where -L 1 - is optionally further substituted.

22. The IL-2 conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein -L 1 - having formula (IX-c) in The dashed line marked with an asterisk indicates the nitrogen bond to -D; the unmarked dashed line indicates the nitrogen bond to -L 2 -Z connection; and s1 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

23. The IL-2 conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein -L 2 - is selected from -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 Alkynyl; wherein -T-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more identical or different -R y2 Substitute, and where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more selected from -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 )-groups; -R y1 and -R y1a are independently selected from -H, -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally substituted with one or more identical or different -R y2 Substitute, and where C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -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 )-; T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbocyclic group and 8- to 30-membered heteropolycyclic group; wherein each T is independently optionally replaced by one or more identical or different -R y2 replace; -R y2 are independently selected from halogen, -CN, oxo (=O), -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 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and -R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b are each independently selected from -H and C 1-6 Alkyl, where C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

24. The IL-2 conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein -L 2 - having formula (IX-e) in The dashed line marked with an asterisk refers to -L 1 -connect; The unlabeled dashed line indicates the connection to -Z; and 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.

25. A pharmaceutical composition comprising at least one IL-2 conjugate according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof and at least one excipient.

26. The IL-2 conjugate according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 25 for use as a medicament.

27. Use of the IL-2 conjugate according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 25 in the preparation of a medicament for treating a disease treatable with IL-2.

28. The method of claim 27, wherein the disease treatable with IL-2 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 carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, uterine carcinoma, Neck 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, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia and leukemia.

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