Immunocytokine comprising heterodimeric protein complex based on il-15 / il-15ra

Abstract

To provide an IL-15-based molecule that has high stability, an extended in vivo half-life, increased in vivo biological activity, and increased productivity in mammalian cells.SOLUTION: Provided are an immunocytokine comprising a heterodimeric protein complex based on IL-15 / IL-15Rα, and use thereof as a therapeutic agent, in particular as an agent for the treatment of cancer and an autoimmune disease. Provided further are an immunocytokine comprising a heterodimeric protein complex based on an IL-15 / IL-15Rα and an immunomodulatory antibody, and use thereof as a therapeutic agent, in particular as an agent for the treatment of cancer and an autoimmune disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to immunocytokines comprising IL-15 / IL-15Rα-based heterodimeric protein complexes and their use as therapeutic agents, particularly for the treatment of cancer and autoimmune diseases. The present invention further relates to immunocytokines comprising IL-15 / IL-15Rα-based heterodimeric protein complexes and immunomodulatory antibodies and their use as therapeutic agents, particularly for the treatment of cancer and autoimmune diseases. [Background technology]

[0002] Cytokines are a category of signaling proteins and glycoproteins that, like hormones and neurotransmitters, are widely used in cellular communication. While hormones are secreted into the blood by specific organs and neurotransmitters are associated with neural activity, cytokines are a more diverse class of compounds in terms of origin and purpose. They are produced by a wide variety of hematopoietic and nonhematopoietic cells and can affect both nearby cells and the entire organism, sometimes highly dependent on the presence of other chemicals. The cytokine family primarily consists of small, water-soluble proteins and glycoproteins with masses ranging between 8 and 30 kDa. Cytokines are crucial for the development and function of both innate and adaptive immune responses. They are often secreted by immune cells that encounter pathogens, thereby activating and recruiting additional immune cells and increasing the system's response to the pathogen.

[0003] Among cytokines, interleukin-15 (IL-15) is a cytokine with structural similarity to IL-2; the former is secreted by mononuclear phagocytes (and some other cells) after infection with a virus or viruses or after indirect stimulation by cells recognized as "non-self" or compromised. This cytokine induces cell proliferation of natural killer cells, cells of the innate immune system whose primary role is to kill virus-infected cells. The protein encoded by this gene is a cytokine that regulates T cell and natural killer cell activation and proliferation.

[0004] Interleukin-15 (IL-15) is a 14-15 kDa glycoprotein that was simultaneously identified by two groups as a T cell activating factor (Grabstein, (KH et al., Science 1994, 264, 965; Burton, JD et al., Proc. Natl. Acad. Sci. USA 1994, 91, 4935). IL-15 mRNA is widely expressed in different cells and tissues, but its expression is strongly post-transcriptionally regulated at the level of translation and intracellular transport, making it difficult to detect the protein in these cells or in cell supernatants (Bamford RN et al., J. Immunol. 1998, 160:4418-4426; Kurys G et al., J. Biol. Chem. 2000, 275:30653-30659). Furthermore, it has been shown that IL-15 can exist in an active form as a membrane protein (Musso et al., Blood 1999, Vol. 93, No. 10 (May 15): pp 3531-3539), and recently it has been noted that IL-15 functions either as a ligand or as a receptor (Budalgian et al., JBC 2004, vol 279, No 40: pp 42192-42201), inducing the secretion of pro-inflammatory cytokines through this pathway. High expression levels of the soluble protein have been linked to the pathogenesis of autoimmune and inflammatory diseases. IL-15 has been shown to be a key regulator of Crohn's disease (Kirman I., 1996, Am J. 2004). J. Gastroenterol. 91, 1789), psoriasis (Ruckert R. 2000, 165:2240-2250), leukemia (Yamada Y. 1999, Leukemia and Lymphoma, 35(1-2):37-45), and rheumatoid arthritis (RA) (McClnnnes IB 1998, Immunology Today, 19, 75-79). Upon ligand binding to the T cell receptor, expression of IL-15Rα and several activation antigens, such as CD69, CD25, and TNFRII, is induced. Furthermore, IL-15 is a chemoattractant for human blood T lymphocytes (Wilkinson 1995, J. Exp. Med. 181, 1255-1259). All these data suggest that IL-15 expressed by antigen-presenting cells may be important for early T cell activation at inflammatory sites.

[0005] IL-15 is a member of the small four-alpha helix bundle family of cytokines. The biological effects of IL-15 are mediated through binding to a cell membrane receptor composed of three subunits, α, β, and γ. IL-15Rα binds with a very high affinity, Kd of 10 -11 IL-15Ra is a specific subunit of this cytokine that binds to IL-15 at the sushi domain and can be found as a membrane receptor or in a soluble form (Budagian V. et al., JBC 2004, 279, 39:40368-40375; Mortier et al., The Journal of Immunology, 2004, 173:1681-1688). IL-15Ra contains a Sushi domain that can bind to IL-15 and is essential for the biological function of IL-15 after binding.

[0006] IL-15 has limitations related to its low molecular weight, short in vivo half-life, difficulty in controlling repeated dosing, and potential systemic immune side effects. There is an urgent need to discover approaches that can increase the in vivo half-life and promote or enhance the biological activity of IL-15 in vivo.

[0007] Recently, it has been found that the complex formed by IL-15 and its receptor IL-15Rα can significantly enhance the biological activity of IL-15. Studies have shown that the complex formed by IL-15 and the soluble receptor IL-15Rα is significantly more potent than IL-15 alone in stimulating the proliferation of memory CD8+ T lymphocytes and NT / NKT cells. The IL-15 / IL-15Rα complex is more than 10-fold more potent than IL-15 alone in stimulating the proliferation and maintaining the survival of memory CD8+ T cells, and this mechanism may be associated with cis-presentation.

[0008] International Applications WO2007046006 and WO2016095642 disclose fusion proteins intended to stimulate the IL-15R beta / gamma signaling pathway, thereby inducing and / or stimulating the activation and / or proliferation of IL-15R beta / gamma-positive cells, such as NK and / or T cells, characterized by comprising IL-15 indirectly linked by a covalent bond to a polypeptide containing the sushi domain of the extracellular region of IL-15R alpha, and their use in cancer treatment. However, WO2007046006 provides experimental data and characteristics of superagonists only for IL-15 linked to IL-15R alpha via a linker peptide through the N- and C-terminal amino acids, while WO2016095642 provides experimental data and characteristics of superagonists only for IL-15 linked to IL-15R alpha via a chimeric S-S bond formed by Cys mutations in the IL-15 ligand and IL-15R alpha itself. Neither provides any evidence regarding the activity and physicochemical properties of IL15 superagonist variants containing fusion protein domains linked by S-S bond(s) or without S-S bridges.

[0009] These entities have limitations related to their small molecular weight and short in vivo half-life, they have limited activity of interleukin-15, and their production, especially in Chinese hamster ovary cells, is very difficult and associated with low yields.

[0010] International Applications WO 2015103928 and WO 2018071919 disclose mutants aimed at solving the above problems, particularly heterodimeric IL-15 agonist proteins comprising protein (I) and protein (II), where protein (I) is formed by IL-15 fused to a first Fc variant, with the first Fc variant linked to the C-terminus of IL-15, and protein (II) is formed by a second Fc variant or IL-15Rα or a variant thereof fused to the second Fc variant, with the second Fc variant linked to the C-terminus of IL-15Rα. Protein (I) and protein (II) form a stable heterodimeric protein through "knobs-into-holes" interactions between the first Fc variant and the second Fc variant.

[0011] The generation of IL-15-based immunocytokines is of particular interest because it combines the immunomodulatory effects of interleukin-15 with the favorable properties of tumor-specific antibodies that target tumors.

[0012] International Applications WO2015018528 and WO2018071918 disclose an immunocytokine comprising a conjugate and an immunomodulatory antibody or a fragment thereof covalently linked directly or indirectly to the conjugate, wherein the conjugate comprises a polypeptide comprising the amino acid sequence of interleukin-15 or a derivative thereof and the amino acid sequence of the sushi domain of IL-15Rα or a derivative thereof.

[0013] International Application WO 2012175222 discloses an immunocytokine for cancer treatment, comprising a conjugate and an antibody or a fragment thereof selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Facb fragment, a Fd fragment, and an scFv fragment, wherein the conjugate comprises a polypeptide comprising the amino acid sequence of interleukin-15 and a polypeptide comprising the amino acid sequence of the sushi domain of IL-15Rα. The conjugate and the antibody or fragment thereof are covalently linked using a bifunctional coupling agent that provides protein conjugation.

[0014] International application WO2019006472: an IL-15 / IL-15Ra fusion protein comprising an IL-15Ra protein, an IL-15 protein, and a first Fc domain; and an antigen-binding domain monomer that binds to an antigen selected from the group consisting of human CD8, human NKG2A, and human NKG2D, and that comprises a heavy chain comprising a VH-CH1-hinge-CH2-CH3 monomer, wherein VH is a heavy chain variable domain and CH2-CH3 is a second Fc domain, and the light chain comprises a variable light chain (VL) and a light chain constant domain (CL). Including, the first and second Fc domains comprising:

[0015] [ka]

[0016] having a set of amino acid substitutions selected from the group consisting of A bifunctional heterodimeric protein is disclosed. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] WO2007046006 [Patent Document 2] WO2016095642

Patent document 3

Patent document 4

Patent document 5

Patent document 6

Patent document 7

Patent document 8

Non-licensed literature

[0018] [Non-licensed document 1] Grabstein, KHら, Science 1994, 264, 965 [Non-licensed document 2] Burton, J.D., Proc. Natl. Acad. Sci. USA 1994, 91, 4935 [Non-licensed document 3] Bamford RN.ら, J. Immunol. 1998, 160:4418-4426

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Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0019] Despite the above prior art solutions relating to the complex of IL-15 and its receptor IL-15Rα, there remains a need for IL-15-based molecules that have increased stability, extended in vivo half-life, increased in vivo biological activity, and increased productivity in mammalian cells. [Means for solving the problem]

[0020] The novel immunocytokine formats developed by the present inventors, comprising heterodimeric molecules based on IL-15 and IL-15Rα, exhibit higher stability, increased productivity in mammalian cells, extended in vivo half-life, and increased in vivo biological activity. Furthermore, they are universal formats for bispecific immunocytokines with the properties of IL15 activity. The novel immunocytokine formats developed by the present inventors have reduced toxicity, according to the results of preclinical studies.

[0021] In one aspect, the present invention provides an immunocytokine for stimulating the activation and / or proliferation of IL-15R beta / gamma positive cells, comprising: 1) IL-15Rα linked to: a) an antibody light chain constant domain, or b) an antibody heavy chain constant domain, comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and third (CH3) heavy chain constant domain; 2) IL-15 linked to: a) an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; or b) antibody light chain constant domain an IL-15 / IL-15Rα heterodimeric protein complex comprising: the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex may or may not be covalently associated through a native S-S bridge; and When IL-15 or IL-15Rα is linked to an antibody light chain constant domain, the other part of the heterodimeric protein complex selected from IL-15Rα or IL-15 is linked to an antibody heavy chain constant domain. The present invention relates to the immunocytokine.

[0022] In some embodiments, the immunocytokine comprises a first antibody heavy chain constant domain and an antibody light chain constant domain covalently associated through a native S—S bridge in a heterodimeric complex.

[0023] In some embodiments, the immunocytokine comprises a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex that are not covalently associated through a native S—S bridge.

[0024] In some embodiments, the immunocytokine comprises an antibody light chain constant domain selected from CK or CL. In some embodiments, the immunocytokine comprises the amino acid sequence represented by SEQ ID NO:9. The present invention includes IL-15Rα having the amino acid sequence, or any known mutant IL-15Rα variant having similar biological activity.

[0025] In some embodiments, the immunocytokine comprises IL-15 having the amino acid sequence set forth by SEQ ID NO: 10, or any known mutant IL-15 variant having similar biological activity.

[0026] In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody light chain constant domain. In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody light chain constant domain having the amino acid sequence set forth by SEQ ID NO:1 or SEQ ID NO:19.

[0027] In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain. In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain having the amino acid sequence set forth by SEQ ID NO:3 or SEQ ID NO:21.

[0028] In some embodiments, the immunocytokine comprises an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and third (CH3) heavy chain constant domains linked through a hinge.

[0029] In some embodiments, the immunocytokine comprises IL-15 or IL-15Rα linked to antibody heavy chain constant domains arranged in the following order: CH1-hinge-CH2-CH3.

[0030] In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody heavy chain constant domain. In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody heavy chain constant domain having the amino acid sequence set forth by SEQ ID NO:4 or SEQ ID NO:22.

[0031] In some embodiments, the immunocytokine comprises IL-15 linked to an antibody heavy chain constant domain. In some embodiments, the immunocytokine comprises IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence set forth by SEQ ID NO:2 or SEQ ID NO:20.

[0032] In some embodiments, the immunocytokine is: IL-15Rα linked to an antibody light chain constant domain having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 19, and IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:20 Includes.

[0033] In some embodiments, the immunocytokine is: IL-15Rα linked to an antibody heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 22, and An antibody light chain constant having the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 21 Domain-linked IL-15 Includes.

[0034] In some embodiments, the immunocytokine comprises a mutation in an Fc fragment monomer that causes said immunocytokine to have impaired ADCC, CDC, and / or ADCP properties.

[0035] In some embodiments, the immunocytokine comprises an Fc fragment belonging to IgG. In some embodiments, the immunocytokine comprises an Fc fragment selected from the group comprising: human IgG1, IgG2, or IgG4.

[0036] In some embodiments, the immunocytokine comprises two IL-15 / IL-15Rα heterodimeric protein complexes as described above. In some embodiments, the immunocytokines are used as therapeutic agents for the treatment of cancer or autoimmune diseases.

[0037] In some embodiments, the immunocytokines are used as therapeutic agents for the treatment of cancer. In one aspect, the present invention provides an immunocytokine for stimulating the activation and / or proliferation of IL-15R beta / gamma positive cells, the immunocytokine comprising an IL-15 / IL-15R alpha-based heterodimeric protein complex and an immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway; The IL-15 / IL-15Rα heterodimeric protein complex: 1) IL-15Rα linked to: a) an antibody light chain constant domain, or b) an antibody heavy chain constant domain, comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and third (CH3) heavy chain constant domain; 2) IL-15 linked to: a) an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; or b) antibody light chain constant domain Includes; the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex may or may not be covalently associated through a native S-S bridge; and When IL-15 or IL-15Rα is linked to an antibody light chain constant domain, the other part of the heterodimeric protein complex selected from IL-15Rα or IL-15 is linked to an antibody heavy chain constant domain. The present invention relates to the immunocytokine.

[0038] In some embodiments, the immunocytokine comprises a first antibody heavy chain constant domain and an antibody light chain constant domain covalently associated through a native S—S bridge in a heterodimeric complex.

[0039] In some embodiments, the immunocytokine comprises a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex that are not covalently associated through a native S—S bridge.

[0040] In some embodiments, the immunocytokine comprises an antibody light chain constant domain selected from CK or CL. In some embodiments, the immunocytokine comprises the amino acid sequence represented by SEQ ID NO:9. The present invention includes IL-15Rα having the amino acid sequence, or any known mutant IL-15Rα variant having similar biological activity.

[0041] In some embodiments, the immunocytokine comprises IL-15 having the amino acid sequence set forth by SEQ ID NO: 10, or any known mutant IL-15 variant having similar biological activity.

[0042] In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody light chain constant domain. In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody light chain constant domain having the amino acid sequence set forth by SEQ ID NO:1 or SEQ ID NO:19.

[0043] In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain. In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain having the amino acid sequence set forth by SEQ ID NO:3 or SEQ ID NO:21.

[0044] In some embodiments, the immunocytokine comprises an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and third (CH3) heavy chain constant domains linked through a hinge.

[0045] In some embodiments, the immunocytokine comprises IL-15 or IL-15Rα linked to antibody heavy chain constant domains arranged in the following order: CH1-hinge-CH2-CH3.

[0046] In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody heavy chain constant domain. In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody heavy chain constant domain having the amino acid sequence set forth by SEQ ID NO:6 or SEQ ID NO:24.

[0047] In some embodiments, the immunocytokine comprises IL-15 linked to an antibody heavy chain constant domain. In some embodiments, the immunocytokine comprises IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence set forth by SEQ ID NO:5 or SEQ ID NO:23.

[0048] In some embodiments, the immunocytokine comprises an immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway and is an antibody that specifically binds to PD-1.

[0049] In some embodiments, the immunocytokine is: a) a light chain comprising a light chain variable domain and a light chain constant domain; b) a heavy chain comprising an antibody heavy chain constant domain, including a heavy chain variable domain, and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH1) and a third (CH3) heavy chain constant domain; and immunomodulatory antibodies, including

[0050] In some embodiments, the immunocytokine comprises a light chain variable domain comprising LCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) set forth by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[0051] In some embodiments, the immunocytokine comprises a light chain variable domain comprising the amino acid sequence set forth by SEQ ID NO:18. In some embodiments, the immunocytokine comprises a heavy chain variable domain comprising HCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) as set forth by the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively.

[0052] In some embodiments, the immunocytokine comprises a heavy chain variable domain comprising the amino acid sequence set forth by SEQ ID NO:17. In some embodiments, the immunocytokine is: 1) a light chain variable domain comprising LCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) as represented by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; 2) a heavy chain variable domain comprising HCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) as set forth in the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively; Includes.

[0053] In some embodiments, the immunocytokine is: 1) a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 18 2) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 17 Includes.

[0054] In some embodiments, the immunocytokine comprises an immunomodulatory antibody comprising a light chain comprising the amino acid sequence set forth by SEQ ID NO:8. In some embodiments, the immunocytokine comprises an immunomodulatory antibody comprising a heavy chain comprising the amino acid sequence set forth by SEQ ID NO:7.

[0055] In some embodiments, the immunocytokine is: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:7; A light chain comprising the amino acid sequence set forth in SEQ ID NO:8 and immunomodulatory antibodies, including

[0056] In some embodiments, the immunocytokine comprises an immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway, which is an antibody that specifically binds to PD-L1. In some embodiments, the immunocytokine is: a) Based on IL-15 / IL-15Rα: IL-15Rα linked to an antibody light chain constant domain having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 19, and IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:23 a heterodimeric protein complex comprising; b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:7; A light chain comprising the amino acid sequence set forth in SEQ ID NO:8 immunomodulatory antibodies, including Includes.

[0057] In some embodiments, the immunocytokine is: a) IL-15Rα linked to an antibody heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 24, and IL-15 linked to an antibody light chain constant domain having the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:21; b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:7; A light chain comprising the amino acid sequence set forth in SEQ ID NO:8 immunomodulatory antibodies, including Includes.

[0058] In some embodiments, the immunocytokine has a mutation in the antibody constant domain that induces heterodimerization of two distinct moieties, one of which comprises IL-15Rα and IL-15 linked to an antibody constant domain, covalently or non-covalently associated, and the other of which comprises the light and heavy chains of the antibody, covalently or non-covalently associated.

[0059] In some embodiments, the immunocytokine is: A first Fc monomer and a second Fc monomer selected from the following group: where the first Fc monomer is a knob-modified Fc and the second Fc monomer is a hole-modified Fc, or where the second Fc monomer is a knob-modified Fc and the first Fc monomer is a hole-modified Fc. Includes.

[0060] In some embodiments, the immunocytokine comprises a first Fc monomer having amino acid substitutions S354C / T366W and a second Fc monomer having amino acid substitutions Y349C / T366S / L368A / Y407V.

[0061] In some embodiments, the immunocytokine comprises a first Fc monomer having amino acid substitutions Y349C / T366S / L368A / Y407 and a second Fc monomer having amino acid substitutions S354C / T366W.

[0062] In some embodiments, the immunocytokine comprises an Fc fragment belonging to IgG. In some embodiments, the immunocytokine comprises an Fc fragment selected from the group comprising: human IgG1, IgG2, or IgG4.

[0063] In some embodiments, the immunocytokine comprises a mutation in an Fc fragment monomer that causes said immunocytokine to have impaired ADCC, CDC, and / or ADCP properties.

[0064] In some embodiments, the immunocytokine is used for the treatment of a neoplastic or autoimmune disease. In some embodiments, the immunocytokine is used for the treatment of a neoplastic disease.

[0065] In one aspect, the invention relates to an isolated nucleic acid encoding any of the above immunocytokines. In some embodiments, the nucleic acid is DNA.

[0066] In one aspect, the present invention relates to an expression vector comprising the above-described nucleic acid. In one aspect, the present invention relates to a method for producing a host cell for producing the immunocytokine, comprising the step of transforming a cell with the vector described above.

[0067] In one aspect, the present invention relates to a host cell for producing the immunocytokine, which comprises the nucleic acid. In one aspect, the present invention relates to a method for producing a medicament comprising the immunocytokine, comprising culturing the host cell in a medium under conditions sufficient to produce the immunocytokine, and, if necessary, thereafter isolating and purifying the resulting immunocytokine.

[0068] In one aspect, the present invention relates to a pharmaceutical composition for stimulating the activation and / or proliferation of IL-15R beta / gamma positive cells, comprising a therapeutically effective amount of the above immunocytokine in combination with one or more pharmaceutically acceptable excipients.

[0069] In some embodiments, the pharmaceutical composition is used for the treatment of a neoplastic disease or an autoimmune disease. In some embodiments, the pharmaceutical composition is used for the treatment of a neoplastic disease.

[0070] In some embodiments, the pharmaceutical composition is for use in treating: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI It is used for the treatment of neoplastic diseases selected from the group including CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndrome.

[0071] In one aspect, the present invention relates to a method for treating a neoplastic disease, comprising administering to a subject in need of such treatment a therapeutically effective amount of said immunocytokine or said pharmaceutical composition.

[0072] In some embodiments of the methods for treatment, the neoplastic disease is: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI Selected from the group comprising CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndrome.

[0073] In one aspect, the present invention relates to a method for activating the biological activity of a T cell population or an NK cell population in a subject in need thereof, comprising administering to the subject an effective amount of the immunocytokine or the pharmaceutical composition.

[0074] In one aspect, the present invention relates to the use of said immunocytokine or said pharmaceutical composition for the treatment of a neoplastic disease in a subject in need thereof. In some uses, the neoplastic disease is: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI Selected from the group comprising CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndrome. [Brief explanation of the drawings]

[0075] [Figure 1] Immunocytokine form containing the IL-15 / IL-15Rα heterodimeric protein complex (CK_IL15Ra_Hc_IL15CH1FcLALA). [Figure 2] Immunocytokine form containing the IL-15 / IL-15Rα heterodimeric protein complex (CK_IL15_Hc_IL15RaCH1FcLALA). [Figure 3] An immunocytokine format (Fab-CK_IL15_Hc_IL15RaCH1FcKnobLALA) comprising an IL-15 / IL-15Rα heterodimeric protein complex and an immunomodulatory antibody that specifically inhibits the PD-1 pathway. "Knob" is understood to mean a mutation in the CH3 domain that forms a "knob-into-hole" structure between the first Fc variant and the second Fc variant. [Figure 4]An immunocytokine format (Fab-CK_IL15Ra_Hc_IL15CH1FcKnobLALA) comprising an IL-15 / IL-15Rα heterodimeric protein complex and an immunomodulatory antibody that specifically inhibits the PD-1 pathway. "Knob" is understood to mean a mutation in the CH3 domain that forms a "knob-into-hole" structure between the first Fc variant and the second Fc variant. [Figure 5] pEE-IL15Ra-CK vector, where AmpR is a beta-lactamase gene providing ampicillin resistance, pUC origin is the pUC origin of replication in bacteria, CMV-promoter is a cytomegalovirus early gene promoter, leader is a leader sequence, IL15Ra is a gene encoding the interleukin-15 receptor subunit alpha, CK is a gene encoding the light chain constant domain, polyA is a polyadenylation signal sequence to increase mRNA stability, and OriP is a bacterial origin of replication or a high copy number ColE1 / pMB1 / pBR322 / pUC origin of replication in bacterial cells. [Figure 6] pEE-IL15-CH1-Fc-LALA vector, where AmpR is the beta-lactamase gene providing ampicillin resistance, pUC origin is the pUC origin of replication in bacteria, CMV-promoter is the promoter of the cytomegalovirus early gene, leader is a leader sequence, IL15 is the gene encoding interleukin-15, CH1 is the gene encoding the first heavy chain constant domain, Fc-LALA is the gene encoding the Fc fragment carrying the LALA mutation (L234A and L235A mutations in the CH2 constant domain of the Fc fragment), polyA is a polyadenylation signal sequence for increasing mRNA stability, and OriP is the origin of replication in bacteria or the high copy number ColE1 / pMB1 / pBR322 / pUC origin of replication in bacterial cells. [Figure 7]pEE-BCD100-02-VL-CK vector, where AmpR is the beta-lactamase gene providing ampicillin resistance, pUC origin is the pUC origin of replication in bacteria, CMV-promoter is the cytomegalovirus early gene promoter, leader is a leader sequence, aPD1-VL is a gene encoding the light chain of an antibody that specifically binds to PD1, CK is a gene encoding the light chain constant domain, polyA is a polyadenylation signal sequence to increase mRNA stability, and OriP is the origin of replication in bacteria or the high copy number ColE1 / pMB1 / pBR322 / pUC origin of replication in bacterial cells. [Figure 8] pEE-BCD100-02-VH-CH1-Fc-Hole-LALA vector, where AmpR is the beta-lactamase gene providing ampicillin resistance, pUC origin is the pUC origin of replication in bacteria, promoter is the cytomegalovirus early gene promoter, leader is a leader sequence, aPD1-VH is a gene encoding the variable domain of the heavy chain of an antibody that specifically binds to PD1, CH1 is a gene encoding the first heavy chain constant domain, Fc-Hole-LALA is a gene encoding an Fc fragment carrying the "hole"-forming mutation and the LALA mutation (L234A and L235A mutations in the CH2 constant domain of the Fc fragment), stop is a stop codon, and OriP is the origin of replication in bacteria or the high copy number ColE1 / pMB1 / pBR322 / pUC origin of replication in bacterial cells. [Figure 9]pEE-IL15-CH1-Fc-knob-LALA vector, where AmpR is the beta-lactamase gene providing ampicillin resistance, pUC origin is the pUC origin of replication in bacteria, CMV-promoter is the cytomegalovirus early gene promoter, leader is a leader sequence, IL15 is the gene encoding interleukin-15, CH1 is the gene encoding the first heavy chain constant domain, Fc-knob-LALA is the gene encoding the Fc fragment carrying the "knob"-forming mutation and the LALA mutation (L234A and L235A mutations in the CH2 constant domain of the Fc fragment), stop is a stop codon, and OriP is the origin of replication in bacteria or the high copy number ColE1 / pMB1 / pBR322 / pUC origin of replication in bacterial cells. [Figure 10] Figure 10A. SDS gel electrophoresis containing beta-mercaptoethanol. 1. Control antibody 5 μl. 2. Fermentas unstained marker. 3. - 4. Growth medium containing unpurified CK_IL15Ra_Hc_IL15CH1FcLALA 10 μl. 5. Growth medium containing purified CK_IL15Ra_Hc_IL15CH1FcLALA 10 μl. 6. Purified CK_IL15Ra_Hc_IL15CH1FcLALA 10 μl. 7. - 8. Growth medium containing unpurified CK_IL15_Hc_IL15RaCH1FcLALA 10 μl. 9. Growth medium containing purified CK_IL15_Hc_IL15RaCH1FcLALA 10 μl. 10. Purified CK_IL15_Hc_IL15RaCH1FcLALA 10 μl. Figure 10B. SDS gel electrophoresis without beta-mercaptoethanol 1. Purified CK_IL15Ra_Hc_IL15CH1FcLALA 10 μl 2. Purified CK_IL15_Hc_IL15RaCH1FcLALA 10 μl 3. Fermentas unstained marker 4. Control antibody 5 μl [Figure 11]Measurement of proliferation activity. The NK-92 cell line was used in the assay. The assay was performed in a 96-well culture plate. The suspension contained NK-92 cells and the test antibody at the concentrations indicated in the graph. All suspension components were prepared in RPMI-1640 medium supplemented with fetal bovine serum and glutamine. After adding all components, the plate was incubated at 37°C and 5% CO2. Alamar Blue was then added to the wells. After incubation, the fluorescence intensity in the wells was measured. [Figure 12] Measurement of proliferation activity. The assay used isolated natural killer cells isolated from PBMCs of healthy donors by negative selection. The assay was performed in a 96-well culture plate. The suspension contained natural killer cells and the test antibody at the concentrations indicated in the graph. All suspension components were prepared in medium supplemented with autologous human plasma. After adding all components, the plate was incubated at 37°C and 5% CO2. Alamar Blue was then added to the wells. After incubation, the fluorescence intensity in the wells was measured. [Figure 13] Measurement of aPD1-specific activity. For the assay, we used the Jurkat NFAT-FLuc PD-1 cell line, which was generated based on the Jurkat cell line and stably expresses PD-1 on its surface and contains the firefly luciferase-encoding gene under the control of the NFAT promoter; and the Raji PDL1 cell line, which was generated based on the Raji cell line and stably expresses PDL1 on its surface. The assay was performed in a 96-well culture plate. The suspension in each well contained Jurkat NFAT-FLuc PD-1 cells, Raji PDL1 cells, aCD3 / aTAA1 cells at a concentration of 1 ng / ml, and test antibodies at the concentrations indicated in the graph. After adding all components, the plate was incubated at 37°C and 5% CO2, and then the luciferase intensity in the wells was measured using a luminescence assay kit. [Figure 14] Determination of the specific activity of anti-PDL1 / IL15SA antibodies against reporter cell lines. [Figure 15] Determination of the effect of natural killer cells on ADCC. The assay used natural killer cells isolated from PBMCs of healthy donors by negative selection. The assay was performed in a 96-well culture plate. The suspension contained natural killer cells and Raji cells, the effector antibody rituximab, and the test antibody at the concentrations indicated in the graph. All suspension components were prepared in medium supplemented with fetal bovine serum and glutamine. After adding all components, the plate was incubated at 37°C and 5% CO2. The culture medium was then collected, and the lactate dehydrogenase content was measured using an LDH assay kit. [Figure 16] Results of determining the specific activity of immunocytokines compared to the enhanced ADCC potential of rituximab against the TAA-reporter line. [Figure 17] Cytotoxicity study of immunocytokine variants containing the IL-15 / IL-15Rα (IL15SA) heterodimeric protein complex on NK cells. Results show that the candidates have no significant cytotoxic effect, i.e., a 10% or less reduction in the number of immunoreactive cells when compared to the AB negative control (without any exogenous antibody). [Figure 18] Cytotoxicity study of IL15SA variants on B cells. Results show that the candidates have no significant cytotoxic effect, i.e., a <10% reduction in the number of immunoreactive cells when compared to the AB negative control (without any exogenous antibody). [Figure 19] Cytotoxicity study of IL15SA variants on CD3+ cells. Results show that the candidates have no significant cytotoxic effect, i.e., a <10% reduction in the number of immunoreactive cells when compared to the AB negative control (without any exogenous antibody). [Figure 20] Cytotoxicity study of IL15SA variants on CD4+ cells. Results show that the candidates have no significant cytotoxic effect, i.e., a <10% reduction in the number of immunoreactive cells when compared to the AB negative control (without any exogenous antibody). [Figure 21] Cytotoxicity study of IL15SA variants on CD8+ cells. Results show that the candidates have no significant cytotoxic effect, i.e., a <10% reduction in the number of immunoreactive cells when compared to the AB negative control (without any exogenous antibody). [Figure 22] Chromatogram. This chromatogram shows the high homogeneity of the immunocytokines, including the IL-15 / IL-15Rα-based heterodimeric protein complex (IL-15 superagonist) (aggregate content in solution was below 5%). [Figure 23] Experimental scheme for determining the antitumor activity of immunocytokine products in a murine in vivo tumor model, where the immunocytokine comprises an IL-15 / IL-15Rα-based heterodimeric protein complex (BCD-225). [Figure 24] Experimental results (graph) for determining the antitumor activity of immunocytokine product mutants, including the IL-15 / IL-15Rα-based heterodimeric protein complex (BCD-225), in a murine in vivo tumor model. [Figure 25] Experimental results (Figure) to determine the antitumor activity of immunocytokine product variants, including the IL-15 / IL-15Rα-based heterodimeric protein complex (BCD-225), in a murine in vivo tumor model. DETAILED DESCRIPTION OF THE INVENTION

[0076] General definitions and general methods Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0077] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Typically, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid hybridization and chemistry described herein are well known and widely used by those of skill in the art. Enzymatic reactions and purification methods are performed according to manufacturer's instructions, as common in the art or as described herein.

[0078] Unless otherwise specified, the terms "biologically active" and "biological activity" and "biological properties" in reference to a polypeptide of the present invention means having the ability to bind to a biological molecule.

[0079] The term "recombinant protein" means a protein (polypeptide) that is expressed in a cell or cell line that contains a nucleotide sequence(s) encoding said protein, wherein said nucleotide sequence(s) is / are not naturally associated with the cell.

[0080] The term "bispecific antibody" refers to an antibody having antigen-binding domain(s) capable of specifically binding to two distinct epitopes on a single biological molecule or capable of specifically binding to epitopes on two separate biological molecules. Bispecific antibodies are also referred to herein as having "dual specificities" or as being "bispecific" antibodies.

[0081] The term "monoclonal antibody" or "mAb" refers to an antibody synthesized and isolated by a distinct clonal population of cells. The clonal population may be a clonal population of immortalized cells. In some embodiments, the immortalized cells in the clonal population are hybrid cells, hybridomas, typically produced by the fusion of individual B lymphocytes from an immunized animal with individual cells from a lymphoid tumor. Hybridomas are an engineered type of cell and do not exist in nature.

[0082] The term "Ka" as used herein refers to the association (on) rate of a particular antibody-antigen interaction. The term "Kd," as used herein, refers to the dissociation (off) rate of a particular antibody-antigen interaction.

[0083] "Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" refers to the intrinsic (characteristic, true) binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The binding affinity of molecule X is Affinity for binding partner Y can generally be represented by the dissociation constant (Kd). Preferred Kd values ​​are about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or less. Affinity may be measured by common methods known in the art, including those described herein. A variety of methods for measuring binding affinity are known in the art, and any of these methods may be used for purposes of the present invention.

[0084] The term "peptide linker" is intended herein to mean any peptide capable of combining domains, having a length corresponding to the domains to be linked together and containing any amino acid sequence. Preferably, the peptide linker has a length of more than 5 amino acids and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.

[0085] The term "in vitro" refers to a biological entity, process, or reaction under artificial conditions, outside the body. For example, cells grown in vitro shall be understood as cells grown in an environment outside the body, such as in a test tube, culture vial, or microtiter plate.

[0086] As used herein, the words "comprise," "have," "include," or variations such as "comprises," "comprising," "has," "having," "includes," or "including," and all grammatical variations thereof, are understood to indicate the inclusion of a referenced integer or group of integers, but are not intended to exclude any other integer or group of integers.

[0087] Detailed Description of the Invention Immunocytokines In one aspect, the present invention provides an immunocytokine for stimulating the activation and / or proliferation of IL-15R beta / gamma positive cells, comprising: 1) IL-15Rα linked to: a) an antibody light chain constant domain, or b) an antibody heavy chain constant domain, comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and third (CH3) heavy chain constant domain; 2) IL-15 linked to: a) an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; or b) antibody light chain constant domain an IL-15 / IL-15Rα-based heterodimeric protein complex comprising: the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex may or may not be covalently associated through a native S-S bridge; and When IL-15 or IL-15Rα is linked to an antibody light chain constant domain, the other part of the heterodimeric protein complex selected from IL-15Rα or IL-15 is linked to an antibody heavy chain constant domain. The present invention relates to the immunocytokine.

[0088] The formats of the above immunocytokines are shown in FIGS. The term "immunocytokine" refers to a molecule comprising an antibody or a fragment thereof covalently linked directly or indirectly to a cytokine or a derivative thereof. The antibody and cytokine may be linked by a linker peptide.

[0089] The immunocytokine of the present invention is intended to refer to an isolated immunocytokine. The term "isolated," as used to describe the various immunocytokines herein, refers to immunocytokines that have been identified and separated and / or recovered from the cell or cell culture in which they are expressed. Impurities (contaminating components) from the natural environment are substances that would interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the immunocytokines are purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer (Edman sequencer), or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie brilliant blue, or preferably silver staining. Because an isolated immunocytokine will be free of at least one component of the polypeptide's natural environment, it is not considered to be an immunocytokine that is present in a recombinant cell, in vivo, or in vivo. Isolated polypeptides are typically prepared by at least one purification step.

[0090] As used herein, a "heterodimeric protein complex" refers to the protein formed from the combination of IL-15 and IL-15Rα. As used herein, "IL-15" or "IL-15 peptide" or "interleukin-15" refers to any IL-15 (interleukin-15) or mutant thereof, such as human or non-human mammalian IL-15 or non-mammalian IL-15. Exemplary non-human mammalian species include mammals such as pigs, rabbits, monkeys, chimpanzees, and mice; and non-mammalian species include chickens. Preferably, the mature human interleukin-15 molecule is found in the UniProtKB database, accession number P40933, aaa 49-192. The term "IL-15 variant" refers to a variant molecule that has increased or decreased affinity for its receptor or increased or decreased activity in stimulating T cells or NK cells due to one or more amino acid substitutions, additions, or deletions.

[0091] "IL-15Rα" means the interleukin-15 receptor subunit alpha (α), and in accordance with the present invention may be any IL-15Rα compound or functional fragment thereof, such as human IL-15Rα or non-human mammalian IL-15Rα, or non-mammalian IL-15Rα. Exemplary non-human mammals include mammals such as pigs, rabbits, monkeys, chimpanzees, mice, etc.; and non-mammals include chickens, etc. A preferred IL-15Rα molecule is found in the database UniProtKB, accession number Q13261 ( https: / / www.uniprot.org / uniprot / Q13261 ) can be found in

[0092] The fragment crystallizable region of an immunoglobulin ("Fc region, Fc") is the "tail" region of an immunoglobulin molecule that interacts with cell surface Fc receptors as well as several proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the two heavy chains, respectively; in IgM and IgE isotypes, the Fc region contains three heavy chain constant domains (CH2, CH3, and CH4 domains) in each polypeptide chain.

[0093] "Fc fragment monomer" is understood to mean the Fc region derived from the second and third constant domains of one of the two heavy chains (for IgG, IgA and IgD isotypes); for IgM and IgE isotypes, the Fc monomer comprises the three constant domains (CH2, CH3 and CH4 domains) of one of the two heavy chains.

[0094] In some embodiments, the immunocytokine comprises a first antibody heavy chain constant domain and an antibody light chain constant domain in a covalent association through a native S—S bridge in a heterodimeric complex.

[0095] In some embodiments, the immunocytokine comprises a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex that are not covalently associated through a native S—S bridge.

[0096] In some embodiments, the immunocytokine comprises an antibody light chain constant domain selected from CK or CL. In mammals, only two types of light chains are known, designated lambda (λ) and kappa (κ). The constant domain of the lambda light chain is called CL, and the constant domain of the kappa light chain is designated CK.

[0097] In some embodiments, the immunocytokine is

[0098] [ka]

[0099] or any known variant of IL-15Rα containing a mutation that has similar biological activity. Mutant IL-15Rα, an IL-15Rα with similar biological activity, is understood to refer to any IL-15Rα known in the art that contains a mutation and has similar biological activity when compared to the activity of "wild-type" IL-15Rα.

[0100] In some embodiments, the immunocytokine is

[0101] [ka]

[0102] or any known variant of IL-15 containing a mutation that has similar biological activity. Mutant IL-15, an IL-15 with similar biological activity, means any IL-15 known in the art that contains a mutation and has similar biological activity when compared to "wild-type" IL-15.

[0103] In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody light chain constant domain. In some embodiments, the immunocytokine is

[0104] [ka]

[0105] The present invention comprises an IL-15Rα linked to an antibody light chain constant domain having an amino acid sequence represented by: In some embodiments, the immunocytokine is

[0106] [ka]

[0107] The present invention comprises an IL-15Rα linked to an antibody light chain constant domain having an amino acid sequence represented by: The amino acid sequence represented by SEQ ID NO: 19 is modified from the amino acid sequence represented by SEQ ID NO: 1 by a single amino acid substitution of alanine (A) from cysteine ​​(C) at position 216. This substitution makes it possible to produce an immunocytokine comprising a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex without covalent association through the native S—S bridge.

[0108] In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain. In some embodiments, the immunocytokine is

[0109] [ka]

[0110] The antibody comprises IL-15 linked to an antibody light chain constant domain having the amino acid sequence represented by: In some embodiments, the immunocytokine is

[0111] [ka]

[0112] The antibody comprises IL-15 linked to an antibody light chain constant domain having the amino acid sequence represented by: The amino acid sequence represented by SEQ ID NO: 21 is modified from the amino acid sequence represented by SEQ ID NO: 3 by a single amino acid substitution of alanine (A) from cysteine ​​(C) at position 233. This substitution allows for the production of an immunocytokine comprising a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex without covalent association through the native S-S bridge.

[0113] In some embodiments, the immunocytokine comprises an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH2) and third (CH3) heavy chain constant domains linked through a hinge.

[0114] In some embodiments, the immunocytokine comprises IL-15 or IL-15Rα linked to antibody heavy chain constant domains arranged in the following order: CH1-hinge-CH2-CH3.

[0115] In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody heavy chain constant domain. In some embodiments, the immunocytokine is

[0116] [ka]

[0117] The antibody comprises IL-15Rα linked to an antibody heavy chain constant domain having the amino acid sequence represented by: In some embodiments, the immunocytokine is

[0118] [ka]

[0119] The antibody comprises IL-15Rα linked to an antibody heavy chain constant domain having the amino acid sequence represented by: The amino acid sequence represented by SEQ ID NO: 22 is modified from the amino acid sequence represented by SEQ ID NO: 4 by a single amino acid substitution of alanine (A) from cysteine ​​(C) at position 212. This substitution allows for the production of an immunocytokine comprising a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex without covalent association through the native S—S bridge.

[0120] In some embodiments, the immunocytokine comprises IL-15 linked to an antibody heavy chain constant domain. In some embodiments, the immunocytokine is

[0121] [ka]

[0122] The antibody comprises IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence represented by: In some embodiments, the immunocytokine is

[0123] [ka]

[0124] The antibody comprises IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence represented by: The amino acid sequence represented by SEQ ID NO:20 is modified from the amino acid sequence represented by SEQ ID NO:2 by a single amino acid substitution of alanine (A) from cysteine ​​(C) at position 229. This substitution allows for the production of an immunocytokine comprising a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex without covalent association through the native S-S bridge.

[0125] In some embodiments, the immunocytokine is:

[0126] [ka]

[0127] and an IL-15Rα linked to an antibody light chain constant domain having an amino acid sequence represented by

[0128] [ka]

[0129] IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence shown by Includes.

[0130] In some embodiments, the immunocytokine is:

[0131] [ka]

[0132] and an IL-15Rα linked to an antibody light chain constant domain having an amino acid sequence represented by

[0133] [ka]

[0134] IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence shown by Includes.

[0135] In some embodiments, the immunocytokine is:

[0136] [ka]

[0137] and an IL-15Rα linked to an antibody light chain constant domain having an amino acid sequence represented by

[0138] [ka]

[0139] IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence shown by Includes.

[0140] In some embodiments, the immunocytokine is:

[0141] [ka]

[0142] and an IL-15Rα linked to an antibody heavy chain constant domain having an amino acid sequence represented by

[0143] [ka]

[0144] IL-15 linked to an antibody light chain constant domain having the amino acid sequence shown by Includes.

[0145] In some embodiments, the immunocytokine is:

[0146] [ka]

[0147] and an IL-15Rα linked to an antibody heavy chain constant domain having an amino acid sequence represented by

[0148] [ka]

[0149] IL-15 linked to an antibody light chain constant domain having the amino acid sequence shown by Includes.

[0150] In some embodiments, the immunocytokine is:

[0151] [ka]

[0152] and an IL-15Rα linked to an antibody heavy chain constant domain having an amino acid sequence represented by

[0153] [ka]

[0154] IL-15 linked to an antibody light chain constant domain having the amino acid sequence shown by Includes.

[0155] In some embodiments, the immunocytokine comprises, in said immunocytokine: It contains a mutation in the Fc fragment monomer that results in impaired ADCC, CDC, and / or ADCP properties.

[0156] Mutations in the Fc fragment are understood to mean modification(s) of the amino acid sequence of the antibody described herein. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties.

[0157] A variant of modifying the amino acid sequence of an antibody using amino acid substitutions is the replacement of at least one amino acid residue in the antibody molecule with another residue. Conservative substitutions are shown in Table A under "preferred substitutions."

[0158] [Table 1]

[0159] The term "effector functions" of an antibody refers to the biological activities attributable to the Fc region of an antibody (either a native Fc region sequence or an Fc region amino acid variant), and vary with antibody isotype. Examples of antibody effector functions include: C1 q Binding and complement-dependent cytotoxicity; Fc receptors These include binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg, B cell receptor, BCR) and B cell activation.

[0160] "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibody on target cells and subsequently cause lysis or phagocytosis of the target cell. The primary cells mediating ADCC, NK cells, express only FcγRJII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, in In vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0161] "Human effector cells" are leukocytes that express one or more FcRs and perform effector function. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred. Effector cells may be isolated from their native source, for example, from blood or PBMCs, as described herein.

[0162] "Complement-dependent cytotoxicity" and "CDC" refer to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0163] In some embodiments, the immunocytokine comprises an Fc fragment belonging to IgG. In some embodiments, the immunocytokine comprises an Fc fragment selected from the group comprising: human IgG1, IgG2, or IgG4.

[0164] In some embodiments, the immunocytokine comprises the above two heterodimeric protein complexes based on IL-15 / IL-15Rα. The IL15 superagonist of the present invention (also referred to as BCD225 or IL15SA or (IL15SA)2-Fc) is understood to refer to CK_IL15Ra_Hc_IL15CH1FcLALA and CK_IL15_Hc_IL15RaCH1FcLALA.

[0165] The meanings of the names CK, IL15Ra, IL15, CH1FcLALA are provided throughout the text of the present application. CK_IL15Ra_Hc_IL15CH1FcLALA is an immunocytokine comprising two heterodimeric protein complexes based on IL-15 / IL-15Rα, IL15Ra linked to the antibody light chain constant domain CK, and the first (CH1) heavy chain constant domain, and the second (CH2) and third (CH3) heavy chain constant domains. and wherein the Fc comprises a LALA mutation.

[0166] CK_IL15_Hc_IL15RaCH1FcLALA is an immunocytokine comprising two heterodimeric protein complexes based on IL-15 / IL-15Rα, comprising IL15 linked to an antibody light chain constant domain CK, and IL15Ra linked to an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH2) and a third (CH3) heavy chain constant domain, wherein the Fc comprises the LALA mutation.

[0167] The immunocytokine may be produced in either a format in which the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex are covalently associated through a native S-S bridge, or in a format in which the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex are not covalently associated through a native S-S bridge; in certain cases, for the latter purpose, both cysteines are substituted by alanine.

[0168] Experimental studies of the above immunocytokines in both a form in which the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex are covalently associated through a natural S-S bridge, and a form in which the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex are not covalently associated through a natural S-S bridge, showed that there was no significant difference in the activity of the above immunocytokines.

[0169] The formats of the above immunocytokines are shown in FIGS. In some embodiments, the immunocytokines are used as therapeutic agents for the treatment of cancer or autoimmune diseases.

[0170] In some embodiments, the immunocytokines are used as therapeutic agents for the treatment of cancer. In one aspect, the present invention provides an immunocytokine for stimulating the activation and / or proliferation of IL-15R beta / gamma positive cells, the immunocytokine comprising an IL-15 / IL-15R alpha-based heterodimeric protein complex and an immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway; The IL-15 / IL-15Rα-based heterodimeric protein complex: 1) IL-15Rα linked to: a) an antibody light chain constant domain, or b) an antibody heavy chain constant domain, comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and third (CH3) heavy chain constant domain; 2) IL-15 linked to: a) an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; or b) antibody light chain constant domain Includes; the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex may or may not be covalently associated through a native S-S bridge; and When IL-15 or IL-15Rα is linked to an antibody light chain constant domain, the other part of the heterodimeric protein complex selected from IL-15Rα or IL-15 is linked to an antibody heavy chain constant domain. The present invention relates to the immunocytokine.

[0171] The term "antibody" or "immunoglobulin" (Ig) as used herein includes whole antibodies and and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Five types of mammalian Ig heavy chains, designated by the Greek letters: α, δ, ε, γ, and μ, are known. The type of heavy chain present defines the antibody class: these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Different heavy chains vary in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids. Each heavy chain has two regions, a constant region and a variable region. The constant region is identical in all antibodies of the same isotype but differs in antibodies of different isotypes. Heavy chains γ, α, and δ have a constant region consisting of three constant domains, CH1, CH2, and CH3 (in a row), as well as a hinge region for added flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc. 89-99); heavy chains μ and ε have a constant region consisting of four constant domains, CH1, CH2, CH3, and CH4. In mammals, only two types of light chains are known, designated lambda (λ) and kappa (κ). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The approximate length of a light chain is 211-217 amino acids. Preferably, the light chain is a kappa (κ) light chain, and the constant domain CL is preferably Ckappa (κ).

[0172] An "antibody" according to the present invention may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1).

[0173] The VL and VH regions may be further divided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed between more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0174] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH / VHH domain (Ward et al. (1989) Nature 341:544-546); and (vi) extracted complementarity-determining regions (CDRs). Furthermore, the two regions of the Fv fragment, VL and VH, are encoded by separate genes and may be linked using recombinant methods with a synthetic linker that allows for the formation of a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain molecules are also included within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened in the same manner as are intact antibodies.

[0175] Preferably, the CDRs or the entire antigen-binding portion of the antibodies of the invention are derived from mouse, llama or human donor libraries or are substantially of human origin, with specific amino acid residues modified, e.g., substituted with different amino acid residues, to optimize particular antibody properties, e.g., KD, koff, IC50, EC50, ED50. Preferably, the framework regions of the antibodies of the invention are of human origin or substantially human origin (at least 80, 85, 90, 95, 96, 97, 98 or 99% human origin).

[0176] In other embodiments, the antigen-binding portions of the antibodies of the invention may be derived from other non-human species, including, but not limited to, mouse, llama, rabbit, rat, or hamster. Alternatively, the antigen-binding region may be derived from a human species.

[0177] The term "variable" refers to the fact that certain portions of the variable domains vary greatly in sequence among antibodies. The V domain mediates antigen binding and determines the specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed across the 110-amino acid span of the variable domain. Instead, the V region consists of invariant segments of 15-30 amino acids called framework regions (FRs), separated by shorter, highly variable regions called "hypervariable regions" or CDRs. Native heavy and light chain variable domains each contain four FRs, which primarily adopt a beta-sheet configuration and are connected by three hypervariable regions that form loops that connect, and in some cases form part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the antibody. The constant domains are not directly involved in binding the antibody to the antigen but exhibit various effector functions, such as the participation of antibodies in antibody-dependent cellular cytotoxicity (ADCC).

[0178] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity determining regions" or "CDRs" and / or residues from the "hypervariable loops".

[0179] In certain cases, it may also be desirable to modify one or more CDR amino acid residues to improve binding affinity for the target epitope. This is known as "affinity maturation" and may optionally be performed in conjunction with humanization, for example, in situations where antibody humanization leads to a decrease in binding specificity or affinity and backmutation alone is not sufficient to improve binding specificity or affinity. Various affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method described by Wu et al., Proc Natl Acad Sci USA, 95:6037-6042 (1998).

[0180] "Framework regions" (FRs) are residues of the variable domain that are different from the CDR residues. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4. When the CDRs are defined according to Kabat, the light chain FR residues are located at approximately residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues are located at approximately residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 98-107 (LCFR4) in the heavy chain. ), and 103-113 (HCFR4). When a CDR contains amino acid residues from a hypervariable loop, the light chain FR residues are located at approximately residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain, and the heavy chain FR residues are located at approximately residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain. In some cases where a CDR contains amino acids from both a CDR and a hypervariable loop as defined by Kabat, the FR residues will be adjusted accordingly. For example, if CDRH1 contains amino acids H26-H35, the heavy chain FR1 residues are at positions 1-25 and the FR2 residues are at positions 36-49.

[0181] An antibody of the present invention that "binds" to a target antigen refers to an antibody that can bind to the antigen with sufficient affinity so that it can be used as a diagnostic and / or therapeutic agent to target proteins or cells or tissues expressing the antigen, and that only cross-reacts to a small extent with other proteins. In such embodiments, the degree of antibody binding to non-target proteins is less than 10% of the antibody binding to the specific target protein, according to analytical methods: fluorescence-activated cell sorting (FACS), radioimmunoassay (RIA), or ELISA. With respect to antibody binding to a target molecule, the terms "specific binding" or "specifically binds" or "specific for" a particular polypeptide or epitope on a particular polypeptide target refer to binding that is measurably different from non-specific interactions.

[0182] In some embodiments, the immunocytokine comprises a first antibody heavy chain constant domain and an antibody light chain constant domain covalently associated through a native S—S bridge in a heterodimeric complex.

[0183] In some embodiments, the immunocytokine comprises a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex that are not covalently associated through a native S—S bridge.

[0184] In some embodiments, the immunocytokine comprises an antibody light chain constant domain selected from CK or CL. In some embodiments, the immunocytokine is

[0185] [ka]

[0186] or any known variant of IL-15Rα containing a mutation that has similar biological activity. Mutant IL-15Rα, an IL-15Rα with similar biological activity, is understood to refer to any IL-15Rα known in the art that contains a mutation and has similar biological activity when compared to the activity of "wild-type" IL-15Rα.

[0187] In some embodiments, the immunocytokine is

[0188] [ka]

[0189] or any known variant of IL-15 containing a mutation that has similar biological activity. Mutant IL-15, an IL-15 with similar biological activity, means any IL-15 known in the art that contains a mutation and has similar biological activity when compared to "wild-type" IL-15.

[0190] In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody light chain constant domain. In some embodiments, the immunocytokine is

[0191] [ka]

[0192] The present invention comprises an IL-15Rα linked to an antibody light chain constant domain having an amino acid sequence represented by: In some embodiments, the immunocytokine is

[0193] [ka]

[0194] The present invention comprises an IL-15Rα linked to an antibody light chain constant domain having an amino acid sequence represented by: The amino acid sequence represented by SEQ ID NO: 19 is modified from the amino acid sequence represented by SEQ ID NO: 1 by a single amino acid substitution of alanine (A) from cysteine ​​(C) at position 216. This substitution makes it possible to produce an immunocytokine comprising a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex without covalent association through the native S—S bridge.

[0195] In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain. In some embodiments, the immunocytokine is

[0196] [ka]

[0197] The antibody comprises IL-15 linked to an antibody light chain constant domain having the amino acid sequence represented by: In some embodiments, the immunocytokine is

[0198] [ka]

[0199] The antibody comprises IL-15 linked to an antibody light chain constant domain having the amino acid sequence represented by: The amino acid sequence represented by SEQ ID NO: 21 is modified from the amino acid sequence represented by SEQ ID NO: 3 by a single amino acid substitution of alanine (A) from cysteine ​​(C) at position 233. This substitution allows for the production of an immunocytokine comprising a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex without covalent association through the native S-S bridge.

[0200] In some embodiments, the immunocytokine comprises an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH2) and third (CH3) heavy chain constant domains linked through a hinge.

[0201] In some embodiments, the immunocytokine comprises IL-15 or IL-15Rα linked to antibody heavy chain constant domains arranged in the following order: CH1-hinge-CH2-CH3.

[0202] In some embodiments, the immunocytokine comprises IL-15Rα linked to an antibody heavy chain constant domain. In some embodiments, the immunocytokine is

[0203] [ka]

[0204] The antibody comprises IL-15Rα linked to an antibody heavy chain constant domain having the amino acid sequence represented by: In some embodiments, the immunocytokine is

[0205] [ka]

[0206] The antibody comprises IL-15Rα linked to an antibody heavy chain constant domain having the amino acid sequence represented by: The amino acid sequence represented by SEQ ID NO: 24 is modified from the amino acid sequence represented by SEQ ID NO: 6 by a single amino acid substitution of alanine (A) from cysteine ​​(C) at position 212. This substitution allows for the production of an immunocytokine comprising a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex without covalent association through the native S—S bridge.

[0207] In some embodiments, the immunocytokine comprises IL-15 linked to an antibody heavy chain constant domain. In some embodiments, the immunocytokine is

[0208] [ka]

[0209] The antibody comprises IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence represented by: In some embodiments, the immunocytokine is

[0210] [ka]

[0211] The antibody comprises IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence represented by: The amino acid sequence represented by SEQ ID NO: 23 is modified from the amino acid sequence represented by SEQ ID NO: 5 by a single amino acid substitution of alanine (A) from cysteine ​​(C) at position 229. This substitution allows for the production of an immunocytokine comprising a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex without covalent association through the native S—S bridge.

[0212] In some embodiments, the immunocytokine comprises an immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway and is an antibody that specifically binds to PD-1.

[0213] For example, specific binding may be measured by determining binding of a molecule relative to binding of a control molecule. For example, specific binding may be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. As used herein, the terms "specific binding" or "specifically binds" or "to a particular polypeptide or epitope on a particular polypeptide target" are used interchangeably. "Specific" can be described by a molecule having a Kd for a target of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or greater. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantial binding to any other polypeptides or polypeptide epitopes.

[0214] In some embodiments, the immunocytokine is: a) a light chain comprising a light chain variable domain and a light chain constant domain; b) a heavy chain comprising an antibody heavy chain constant domain, comprising a heavy chain variable domain, and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH1) and a third (CH3) heavy chain constant domain; and immunomodulatory antibodies, including

[0215] In some embodiments, the immunocytokine comprises a light chain variable domain comprising LCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) represented by the amino acid sequences GGNNIGSKNVH (SEQ ID NO: 14), RDSNRPS (SEQ ID NO: 15), and CQVWDSSTAV (SEQ ID NO: 16), respectively.

[0216] In some embodiments, the immunocytokine is

[0217] [ka]

[0218] The variable domain comprises a light chain comprising an amino acid sequence represented by: In some embodiments, the immunocytokine comprises a heavy chain variable domain comprising HCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) represented by the amino acid sequences FTFSSYWMY (SEQ ID NO: 11), AIDTGGGRTYYADSVKG (SEQ ID NO: 12), and CARDEGGGTGWGVLKDWPYGLDA (SEQ ID NO: 13), respectively.

[0219] In some embodiments, the immunocytokine is

[0220] [ka]

[0221] The heavy chain variable domain comprises an amino acid sequence represented by: In some embodiments, the immunocytokine is: 1) a light chain variable domain comprising LCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) represented by the amino acid sequences GGNNIGSKNVH (SEQ ID NO: 14), RDSNRPS (SEQ ID NO: 15), and CQVWDSSTAV (SEQ ID NO: 16), respectively; 2) Amino acid sequences FTFSSYWMY (SEQ ID NO: 11) and AIDTGGGR, respectively a heavy chain variable domain comprising HCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) represented by TYYADSVKG (SEQ ID NO: 12), and CARDEGGGTGWGVLKDWPYGLDA (SEQ ID NO: 13); Includes.

[0222] In some embodiments, the immunocytokine is: 1)

[0223] [ka]

[0224] a light chain variable domain comprising the amino acid sequence represented by: 2)

[0225] [ka]

[0226] a heavy chain variable domain comprising the amino acid sequence represented by Includes. In some embodiments, the immunocytokine

[0227] [ka]

[0228] and an immunomodulatory antibody comprising a light chain comprising an amino acid sequence represented by: In some embodiments, the immunocytokine

[0229] [ka]

[0230] and an immunomodulatory antibody comprising a heavy chain comprising an amino acid sequence represented by: In some embodiments, the immunocytokine is:

[0231] [ka]

[0232] a heavy chain comprising the amino acid sequence represented by

[0233] [ka]

[0234] a light chain comprising the amino acid sequence represented by and immunomodulatory antibodies, including In some embodiments, the immunocytokine comprises the immunomodulatory antibody prorugolimab (BCD100, WO2018013017).

[0235] In some embodiments, the immunocytokine comprises an immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway, wherein said antibody or fragment specifically binds to PD-L1.

[0236] In some embodiments, the antibody that specifically binds to PD-L1 is BCD135, disclosed in RU2665790 (WO201894496). In some embodiments, the immunocytokine is: a) Based on IL-15 / IL-15Rα:

[0237] [ka]

[0238] and an IL-15Rα linked to an antibody light chain constant domain having an amino acid sequence represented by

[0239] [ka]

[0240] an IL-14 linked to an antibody heavy chain constant domain having an amino acid sequence represented by 15 a heterodimeric protein complex comprising; b)

[0241] [ka]

[0242] a heavy chain comprising the amino acid sequence represented by

[0243] [ka]

[0244] a light chain comprising the amino acid sequence represented by immunomodulatory antibodies, including Includes.

[0245] In some embodiments, the immunocytokine is: a) Based on IL-15 / IL-15Rα:

[0246] [ka]

[0247] and an IL-15Rα linked to an antibody light chain constant domain having an amino acid sequence represented by

[0248] [ka]

[0249] IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence shown by a heterodimeric protein complex comprising; b)

[0250] [ka]

[0251] a heavy chain comprising the amino acid sequence represented by

[0252] [ka]

[0253] a light chain comprising the amino acid sequence represented by immunomodulatory antibodies, including Includes.

[0254] In some embodiments, the immunocytokine is: a)

[0255] [ka]

[0256] and an IL-15Rα linked to an antibody heavy chain constant domain having an amino acid sequence represented by

[0257] [ka]

[0258] IL-15 linked to an antibody light chain constant domain having the amino acid sequence shown by: b)

[0259] [ka]

[0260] a heavy chain comprising the amino acid sequence represented by

[0261] [ka]

[0262] a light chain comprising the amino acid sequence represented by immunomodulatory antibodies, including Includes.

[0263] In some embodiments, the immunocytokine is: a)

[0264] [ka]

[0265] and an IL-15Rα linked to an antibody heavy chain constant domain having an amino acid sequence represented by

[0266] [ka]

[0267] IL-15 linked to an antibody light chain constant domain having the amino acid sequence shown by: b)

[0268] [ka]

[0269] a heavy chain comprising the amino acid sequence represented by

[0270] [ka]

[0271] a light chain comprising the amino acid sequence represented by immunomodulatory antibodies, including Includes.

[0272] In some embodiments, the immunocytokine has a mutation in the antibody constant domain that induces heterodimerization of two distinct moieties, one of which comprises IL-15Rα and IL-15 linked to an antibody constant domain, covalently or non-covalently associated, and the other of which comprises the light and heavy chains of the antibody, covalently or non-covalently associated.

[0273] In some embodiments, the immunocytokine is: A first Fc monomer and a second Fc monomer selected from the following group: where the first Fc monomer is a knob-modified Fc and the second Fc monomer is a hole-modified Fc, or where the second Fc monomer is a knob-modified Fc and the first Fc monomer is a hole-modified Fc. Includes.

[0274] "Knobs-into-hole" interactions are an approach that can circumvent the problems associated with mispaired by-products. This approach aims to force pairing of two different antibody heavy chains by introducing mutations that modify the contact interface within the CH3 domain. On one chain, bulky amino acids are replaced with amino acids with short side chains to form "holes." Conversely, amino acids with large side chains are introduced into the CH3 domain of the other chain to generate "knobs." By co-expressing these two heavy chains, a high yield of heterodimer formation ("knobs-holes") was observed relative to homodimer formation ("hole-hole" or "knobs-knobs") (Ridgway, JB, Presta, 2014). LG, Carter P; and WO 1996 / 027011). The rate of heterodimerization can be further increased by remodeling the interface between the two CH3 domains using phage display technology and introducing disulfide bridges that stabilize the heterodimer (Merchant, AM et al., Nature Biotech 16 (1998) 677-681; Atwell, S., Ridgway, JB, Wells, JA, Carter, P., J Mol Biol 270 (1997) 26-35).

[0275] In some embodiments, the immunocytokine comprises a first Fc monomer having amino acid substitutions S354C / T366W and a second Fc monomer having amino acid substitutions Y349C / T366S / L368A / Y407V.

[0276] In some embodiments, the immunocytokine comprises a first Fc monomer having amino acid substitutions Y349C / T366S / L368A / Y407 and a second Fc monomer having amino acid substitutions S354C / T366W.

[0277] In some embodiments, the immunocytokine comprises an Fc fragment belonging to IgG. In some embodiments, the immunocytokine comprises an Fc fragment whose Fc fragment isotype is selected from the group comprising: human IgG1, IgG2, or IgG4.

[0278] In some embodiments, the immunocytokine comprises a mutation in an Fc fragment monomer that causes said immunocytokine to have impaired ADCC, CDC, and / or ADCP properties.

[0279] Furthermore, bispecific IL15 superagonist or bispecific IL15 immunocytokine or anti-PD1 / IL15SA immunocytokine (bispecific monoclonal antibody comprising IL-15SA and an anti-PD-1 Fab fragment) is understood to mean: anti-PD-1 Fab-CK_IL15Ra_Hc_IL15CH1Fc knob LALA, Anti-PD-1 Fab-CK_IL15_Hc_IL15RaCH1Fc Knob LALA.

[0280] Furthermore, anti-PD-L1 / IL15SA immunocytokine is understood to mean: anti-PD-L1 Fab-CK_IL15Ra_Hc_IL15CH1Fc knob LALA, Anti-PD-L1 Fab-CK_IL15_Hc_IL15RaCH1Fc Knob LALA.

[0281] The meanings of the names anti-PD-1 Fab, anti-PD-L1 Fab, CK, IL15Ra, IL15, CH1Fc knob LALA are provided throughout the text of this application. Anti-PD-1 Fab-CK_IL15Ra_Hc_IL15CH1Fc knob LALA is an immunocytokine comprising an anti-PD1 antibody, IL15Ra linked to the antibody light chain constant domain CK, and IL15 linked to an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH2) and third (CH3) heavy chain constant domain, wherein the Fc comprises a mutation (e.g., knob) that allows heterodimerization and LALA.

[0282] Anti-PD-1 Fab-CK_IL15_Hc_IL15RaCH1Fc knob LALA is an immunocytokine comprising an anti-PD1 antibody, IL15 linked to the antibody light chain constant domain CK, and IL15Ra linked to an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH2) and third (CH3) heavy chain constant domain, wherein the Fc comprises a mutation (e.g., knob) that allows heterodimerization and LALA.

[0283] Anti-PD-L1 Fab-CK_IL15Ra_Hc_IL15CH1Fc knob LALA is composed of an anti-PD-L1 antibody, IL15Ra linked to the antibody light chain constant domain CK, and the first (CH1) heavy chain constant domain, and the second (CH2) and third (CH3) heavy chain constant domains. IL15 linked to an antibody heavy chain constant domain comprising an Fc fragment monomer comprising a heavy chain constant domain, wherein the Fc comprises a mutation (e.g., knob) that allows heterodimerization and LALA.

[0284] Anti-PD-L1 Fab-CK_IL15_Hc_IL15RaCH1Fc knob LALA is an immunocytokine comprising an anti-PD-L1 antibody, IL15 linked to the antibody light chain constant domain CK, and IL15Ra linked to an antibody heavy chain constant domain comprising a first (CH1) heavy chain constant domain, and an Fc fragment monomer comprising a second (CH2) and third (CH3) heavy chain constant domain, wherein the Fc comprises a mutation (e.g., knob) that allows heterodimerization, and LALA.

[0285] The immunocytokine may be produced in either a format in which the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex are covalently associated through a native S-S bridge, or in a format in which the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex are not covalently associated through a native S-S bridge; in certain cases, for the latter purpose, both cysteines are substituted by alanine.

[0286] Experimental studies of the above immunocytokines in both a format in which the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex are covalently associated through a native S-S bridge, and in a format in which the first antibody heavy chain constant domain and the antibody light chain constant domain in the heterodimeric complex are not covalently associated through a native S-S bridge, showed that there was no significant difference in the activity of the above cytokines.

[0287] "Knob" is understood to mean a mutation that forms a "knob-into-hole" structure between the first Fc variant and the second Fc variant in the CH3 domain, as described above.

[0288] The formats of the immunocytokines are shown in FIGS. In some embodiments, the immunocytokine is used in the treatment of a neoplastic or autoimmune disease.

[0289] In some embodiments, the immunocytokine is used in the treatment of a neoplastic disease. nucleic acid In one aspect, the invention relates to an isolated nucleic acid encoding any of the above immunocytokines.

[0290] The terms "nucleic acid," "nucleic sequence," "nucleic acid sequence," "polynucleotide," "oligonucleotide," "polynucleotide sequence," and "nucleotide sequence" are used interchangeably herein and refer to an exact sequence of nucleotides, modified or unmodified, determining a fragment or region of a nucleic acid, containing or not containing non-naturally occurring nucleotides, and being either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcription product of said DNA.

[0291] It should also be included herein that the present invention is not related to nucleotide sequences in their natural chromosomal environment, i.e., in their natural state. The sequences of the present invention are isolated and / or purified, i.e., they are sampled directly or indirectly, for example, by copying, and their environment is at least partially modified. Thus, isolated nucleic acids obtained by recombinant genetics, for example, by a host cell, or obtained by chemical synthesis, should also be mentioned herein.

[0292] An "isolated" nucleic acid molecule is one that has been identified and separated from at least one nucleic acid molecule impurity with which it is associated in a natural source. An isolated nucleic acid molecule is different from the type or set found under natural conditions. Thus, an isolated nucleic acid molecule is different from the nucleic acid molecule present in a cell under natural conditions. However, an isolated nucleic acid molecule includes a nucleic acid molecule located in a cell in which an immunocytokine is normally expressed, for example, if the nucleic acid molecule has a chromosomal location that is different from its location in the cell under natural conditions.

[0293] In some embodiments, the nucleic acid is DNA. In one embodiment, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. The nucleic acid molecule may also comprise any combination of the above nucleotide sequences.

[0294] As will be recognized by those skilled in the art, due to the degeneracy of the genetic code, a variety of different DNA sequences can encode peptides having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. Generating these alternative DNA sequences that encode the same amino acid sequence is well within the skill of one trained in the art. Such variant DNA sequences are within the scope of the present invention.

[0295] Expression vector In one aspect, the present invention relates to an expression vector comprising the nucleic acid. The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, a vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector, where additional DNA segments can be ligated into the viral genome. In some embodiments, the vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In further embodiments, the vector (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby replicated along with the host genes. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0296] The present invention relates to vectors comprising the above-described nucleic acid molecules encoding any of the amino acid sequences of the above-described immunocytokines or structural portions thereof, as described herein. In some embodiments, the immunocytokines of the present invention are expressed by inserting DNA encoding, in part or in its entirety, the sequences of the first and second immunocytokine domains (e.g., a domain comprising IL15 or IL15Rα linked to antibody constant domain(s), and / or antibody light and heavy chains), and the gene is produced as described above in an expression vector in which the gene is operably linked to desired expression control sequences, e.g., transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. A DNA molecule may be ligated into a vector such that the transcriptional and translational control sequences within the vector perform their intended function of controlling the transcription and translation of the DNA. The expression vector and expression control sequences may be selected to be compatible with the expression host cell used. DNA sequences encoding, in part or in its entirety, the sequences of the first and second binding domains (e.g., heavy and light chain sequences when the binding domains comprise heavy and light chain sequences) may be inserted into an expression vector. The fragments may be introduced into individual vectors. In one embodiment, any combination of the DNA molecules is introduced into the same expression vector. The DNA molecules may be introduced into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if no restriction sites are present).

[0297] Suitable vectors encode functionally complete human CH or CL / CK immunoglobulin sequences with appropriate restriction site manipulations so that any sequence, VH or VL, or IL15 or IL15Rα, can be easily inserted and expressed as described above. The HC and LC-encoding genes in such vectors may contain intron sequences, which stabilize the corresponding mRNA and thereby increase overall antibody protein yield. The intron sequences are flanked by splice donor and splice acceptor sites, which determine where RNA splicing occurs. The intron sequences may be located in either the variable or constant region of the antibody chain, or, if multiple introns are used, in both the variable and constant regions. Polyadenylation and transcription termination may occur at native chromosomal sites downstream of the coding region. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from host cells. The antibody chain gene or the gene for the chain containing IL15 or IL15Rα may be cloned into the vector so that the signal peptide is linked in reading frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

[0298] In addition to the antibody chain genes or genes for IL15- or IL15Rα-containing chains, recombinant expression vectors of the invention may contain regulatory sequences that control the expression of the genes in host cells. Those skilled in the art will understand that the design of the expression vector, including the selection of regulatory sequences, can depend on the choice of host cell to be transformed, the desired protein expression level, and the like. Preferred regulatory sequences for mammalian expression host cells include viral elements that ensure high levels of protein expression in mammalian cells, such as retroviral long terminal repeats (LTRs), cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer), simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the major late promoter adenovirus (AdMLP)), polyoma virus, and strong mammalian promoters, such as the native immunoglobulin promoter or actin promoter. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

[0299] In addition to the above genes and control sequences, the recombinant expression vectors of the present invention may carry additional sequences, such as sequences that control replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically, the selectable marker gene confers resistance to pharmaceutical agents, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells during methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0300] The term "expression control sequence" as used herein refers to polynucleotide sequences necessary to affect the expression and processing of coding sequences to which they are ligated. These sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences will vary depending on the host organism; in prokaryotes, such control sequences generally include a ribosomal binding site, promoter, and transcription termination sequence; in eukaryotes, such control sequences typically include a promoter and transcription termination sequence. The term "control sequences" includes, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is preferred, such as leader sequences and fusion partner sequences.

[0301] The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0302] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous.

[0303] Host cells and methods of production thereof In one aspect, the present invention relates to a method for producing a host cell for producing the immunocytokine, which comprises transforming the cell with the vector described above.

[0304] Nucleic acid molecules encoding the immunocytokines of the present invention and vectors containing these nucleic acid molecules may be used to transfect suitable mammalian or plant, bacterial, or yeast host cells. Transformation may be by any known technique for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules may be introduced into mammalian cells via viral vectors. Methods for transfecting cells are well known in the art. See, e.g., U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0305] In one aspect, the present invention provides a method for producing the immunocytokine, comprising the nucleic acid. The present invention relates to host cells for the purpose of In one aspect, the present invention relates to a method for producing a medicament comprising the immunocytokine, comprising culturing the host cell in a culture medium under conditions sufficient to produce the immunocytokine, followed, if necessary, by isolation and purification of the resulting immunocytokine.

[0306] The term "recombinant host cell" (or simply "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells which may contain, for example, the vectors described in the present invention above. The present invention further relates to host cells which contain, for example, a nucleotide sequence encoding an immunocytokine of the present invention or a portion thereof. It should be understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell but also to the progeny of such a cell. Because modifications may occur in successive generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, although such cells are still included within the scope of the term "host cell" as used herein.

[0307] Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and many other cell lines. Cell lines are selected by determining which cell line has high expression levels and provides the characteristics required for the protein to be produced. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells. When a recombinant expression vector encoding an immunocytokine of the present invention or a portion thereof is introduced into a mammalian host cell, the immunocytokine is produced by culturing the host cell for a period of time sufficient to allow expression of the immunocytokine of the present invention or a portion thereof in the host cell, or more preferably, secretion of the immunocytokine into the culture medium in which the host cell is cultured. The immunocytokine may be isolated from the culture medium using standard protein purification techniques. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0308] Furthermore, many known techniques may be used to enhance the production levels of the immunocytokines described in the present invention from production cell lines. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or in part in connection with EP Nos. 0216846, 0256055, 0323997, and 0338841.

[0309] Immunocytokines of the invention or portions thereof in different cell lines or host cells may have different glycosylation patterns from each other, but the immunocytokines disclosed herein or portions (domains) thereof, including the amino acid sequences provided herein, are part of the invention, regardless of glycosylation of the binding molecule, and generally regardless of the presence or absence of post-translational modifications.

[0310] Pharmaceutical Compositions In one aspect, the present invention relates to a pharmaceutical composition for stimulating the activation and / or proliferation of IL-15R beta / gamma positive cells, comprising a therapeutically effective amount of the above immunocytokine in combination with one or more pharmaceutically acceptable excipients.

[0311] A "pharmaceutical composition" refers to a composition comprising an immunocytokine of the present invention and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, adjuvants, dispersing agents, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickening agents, and sustained-release control agents, the selection and proportions of which depend on the type and route of administration and dosage. The pharmaceutical compositions of the present invention and methods for their preparation will be clearly apparent to those skilled in the art. Pharmaceutical compositions should preferably be manufactured in accordance with Good Manufacturing Practice (GMP) requirements. The composition may also contain a buffer composition, an isotonic agent, a stabilizer, and a solubilizer. The prolonged action of the composition may be achieved by agents that delay absorption of the active pharmaceutical ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents, and delivery agents include water, ethanol, polyalcohols and mixtures thereof, oils, and injectable organic esters.

[0312] A "therapeutically effective amount" refers to that amount of a therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated. A "pharmaceutical product" is a compound or mixture of compounds in the form of tablets, capsules, powders, lyophilisates, injections, infusions, ointments and other ready-to-use pharmaceutical compositions intended for the restoration, improvement or modification of physiological function in humans and animals, for the treatment and prevention of disease, for diagnosis, anesthesia, contraception, cosmetology, etc. Any method accepted in the art for administering peptides, proteins or antibodies may be suitably used with respect to the immunocytokines of the present invention.

[0313] The term "pharmaceutically acceptable" refers to one or more compatible liquid or solid components that are suitable for administration in mammals, preferably humans. The term "excipient" is used herein to describe any component of the present invention other than those mentioned above. These are inorganic or organic substances used in pharmaceutical manufacturing to impart necessary physicochemical properties to the drug product.

[0314] The terms "buffer," "buffer composition," and "buffer" refer to a solution that is capable of resisting changes in pH by the action of its acid-base conjugate components, and that allows the immunocytokine product of the present invention to resist changes in pH. Generally, pharmaceutical compositions preferably have a pH in the range of 4.0 to 8.0. Examples of buffers that may be used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, and the like buffer solutions.

[0315] The terms "isotonicity agent," "osmolyte," or "osmolality adjusting agent" as used herein refer to an excipient that can increase the osmotic pressure of a liquid antibody formulation. An "isotonic" agent is one that has an osmotic pressure equivalent to that of human blood. Isotonic agents typically have an osmotic pressure of about 250-350 mOsm / kg. Isotonicity agents that may be used include, but are not limited to, polyols, sugars and sucrose, amino acids, metal salts such as sodium chloride, etc.

[0316] "Stabilizer" refers to an excipient, or a mixture of two or more excipients, that provides physical and / or chemical stability to an active agent. Stabilizers include amino acids, such as, but not limited to, arginine, histidine, glycine, lysine, glutamine, and proline; surfactants, such as, but not limited to, polysorbate 20 (trade name 10001); Antioxidants include, but are not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfites, and the like; chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, and the like.

[0317] A pharmaceutical composition is "stable" if the active agent retains its physical and / or chemical stability and / or biological activity at a storage temperature, e.g., 2-8°C, for a specified shelf life. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The shelf life is adjusted based on the results of stability testing under accelerated or natural aging conditions.

[0318] The pharmaceutical compositions of the present invention may be manufactured, packaged, or widely sold in the form of a ready-to-use formulation, a single unit dose, or a plurality of single unit doses. The term "single unit dose" as used herein refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is typically equal to the dose of the active ingredient to be administered to a subject, or a suitable fraction of such a dose, such as one-half or one-third of such a dose.

[0319] The pharmaceutical compositions of the present invention are typically suitable for parenteral administration, as sterile formulations intended for administration into the human body via injection, infusion, and implantation, bypassing the gastrointestinal tract and through a breach in the skin or mucosal barrier. For example, parenteral administration is intended to include, inter alia, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intrasynovial, and percutaneous injection or infusion; and kidney dialysis infusion techniques. Intratumoral delivery, such as intratumoral injection, may also be used. Regional perfusion is also provided. Preferred embodiments include intravenous and subcutaneous routes. Any method for administering peptides or proteins accepted in the art may be used appropriately for the immunocytokines of the present invention.

[0320] Injectable formulations may be prepared, packaged, or sold in unit dosage form, for example, without limitation, in ampoules, vials, plastic containers, prefilled syringes, autoinjection devices, etc. Formulations for parenteral administration include suspensions, solutions, emulsions in oily or aqueous bases, pastes, etc., among others.

[0321] In another aspect, the present invention provides compositions for parenteral administration, including pharmaceutical compositions provided in dry (i.e., powder or granule) form for reconstitution with a suitable base (e.g., sterile, pathogen-free water) prior to administration. Such formulations may be prepared, for example, by the process of lyophilization, known in the art as freeze-drying, and involving freezing the product followed by removal of the solvent from the frozen material.

[0322] The immunocytokines according to the invention may also be administered intranasally or by inhalation, either alone or in admixture with suitable pharmaceutically acceptable excipients, from an inhalation device such as a pressurized aerosol container, pump, spray, atomizer or nebulizer, with or without a suitable propellant, or as nasal drops or spray.

[0323] Dosage forms for parenteral administration may be formulated to be immediate or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release. Included.

[0324] In some embodiments, the pharmaceutical composition is used to treat a neoplastic disease or an autoimmune disease. In some embodiments, the pharmaceutical composition is used to treat an autoimmune disease.

[0325] The term "autoimmune disease" as used herein refers to a non-malignant disease or disorder arising from and directed against an individual's own (self) antigens and / or tissues. The definition of "autoimmune disease" includes, but is not limited to, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme osteoarthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, diabetes, thyroiditis, asthma, allergic diseases, psoriasis, atopic dermatitis, scleroderma, "graft versus host" reaction, organ transplant rejection, acute or chronic immune disorders associated with transplantation, sarcoidosis, Kawasaki disease, gray disease, Busu disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schönlein purpura, microscopic renal vasculitis, chronic active hepatitis, uveitis (uvenita), septic shock, toxic shock syndrome, septic syndrome, cachexia, acquired immune deficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, adult (acute) respiratory distress syndrome, alopecia, focal alopecia, seronegative arthropathy, related Arthritis, Reiter's disease, psoriatic arthropathy associated with ulcerative colitis arthropathy, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid disease, linear IgA, autoimmune hemolytic anemia, Coombs-positive hemolytic anemia, pernicious anemia, juvenile pernicious anemia, arthritis, primary sclerosing hepatitis A, idiopathic autoimmune hepatitis, fibrotic lung disease, idiopathic fibrosing alveolitis, postinflammatory interstitial lung disease, interstitial pneumonia, chronic eosinophilic pneumonia, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type I autoimmune hepatitis (old Classic autoimmune hepatitis or lupoid), type II autoimmune hepatitis, osteoarthritis, primary sclerosing cholangitis, type I psoriasis, type II psoriasis, idiopathic leukopenia, autoimmune neutropenia, renal NOS disease, glomerulonephritis, microscopic renal vasculitis, discoid lupus erythematosus, idiopathic or male NOS infertility, autoimmunity against sperm, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, nodular polyarthritisnodosa), pulmonary manifestations, acute rheumatic fever, rheumatoid spondylitis, ankylosing spondylitis, Still's disease, systemic sclerosis, Sjögren's syndrome, Takayasu's disease, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroiditis, hyperthyroidism, Hashimoto's disease, autoimmune atrophic hypothyroidism, primary myxedema, lens-induced uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver disease, allergies, asthma, psychiatric disorders (including depression and schizophrenia), Th2 / T Includes h1-mediated diseases, conjunctivitis, allergic contact dermatitis, allergic rhinitis, alpha-I antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, cystic fibrosis, disorders associated with cytokine therapy, demyelinating diseases, dermatitis, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune premature ovarian failure, and blepharitis.

[0326] In some embodiments, the pharmaceutical composition is used in the treatment of a neoplastic disease. The terms "neoplastic disease," "cancer," and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by uncontrolled cell growth / proliferation. Examples of cancerous diseases include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancerous diseases include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vaginal cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, and various head and neck cancers.

[0327] In some embodiments, the pharmaceutical composition is for use in treating: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI It is used for the treatment of neoplastic diseases selected from the group including CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndrome.

[0328] The term "antiproliferative activity" is intended to refer to stopping or inhibiting the growth of cells, eg, cancer cells. The term “IC 50 "(50% inhibitory concentration)" refers to the concentration of a drug that inhibits a measurable activity or response, e.g., the growth / proliferation of cells, such as tumor cells, by 50%. Using an appropriate dose-response curve and specialized statistical software for curve fitting, the IC 50 The value may be calculated.

[0329] The term GI50 (50% growth inhibition) refers to the concentration of a drug that inhibits the growth of cells, such as tumor cells, by 50%. The term "ED50" (EC50) (50% effective dose / concentration) refers to the drug concentration that produces 50% of the biological effect (which may include cytotoxicity).

[0330] Therapeutic / therapeutic uses In one aspect, the present invention relates to a method for treating a neoplastic disease, comprising administering to a subject in need of such treatment a therapeutically effective amount of said immunocytokine or said pharmaceutical composition.

[0331] "Treating," "treatment," and "therapy" refer to a method of attenuating or eliminating a biological disorder and / or at least one of its associated symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include references to curative, symptomatic, and prophylactic therapy.

[0332] In one aspect, the subject or patient of treatment is a mammal, preferably a human subject. The subject may be male or female of any age. The term "disease" means any condition that would benefit from treatment according to the present invention. The definition of this term includes chronic and acute disorders or diseases.

[0333] In some embodiments of the methods for treatment, the neoplastic disease is: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI Selected from the group comprising CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndrome.

[0334] In one aspect, the present invention provides a method for determining the biological activity of a T cell population or a NK cell population by using the method. The present invention relates to a method for activating such an activation in a subject in need thereof, comprising the step of administering to the subject an effective amount of the immunocytokine or the pharmaceutical composition.

[0335] In one aspect, the present invention relates to the use of said immunocytokine or said pharmaceutical composition for therapy in a subject in need of such treatment of a neoplastic disease. In some uses, the neoplastic disease is: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI Selected from the group comprising CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndrome.

[0336] In the case of tumors (e.g., cancer), a therapeutically effective amount of the immunocytokine of the present invention may reduce the number of cancer cells; reduce initial tumor size; inhibit (i.e., slow to some extent, and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. The immunocytokine of the present invention may prevent to some extent the proliferation of existing cancer cells and / or kill such cells, and may be cytostatic and / or cytotoxic. In cancer therapy, in vivo efficacy may be measured, for example, by assessing survival, time to disease progression (TTP), tumor response rate (RR) to treatment, duration of response, and / or quality of life.

[0337] The immunocytokines of the present invention may be administered without additional therapeutic treatment, i.e., as an independent therapy. Furthermore, treatment with the immunocytokines of the present invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the immunocytokines may be administered together with or combined with another drug / preparation for cancer treatment.

[0338] As used herein, the terms "co-administration," "co-administered," and "in combination with," referring to an immunocytokine of the present invention and one or more other therapeutic agents, are intended to mean, refer to, or include: 1) simultaneous administration of such a combination of an immunocytokine of the present invention and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form that releases said components to said patient substantially simultaneously; 2) simultaneous administration of such a combination of an immunocytokine of the present invention and a therapeutic agent to a patient in need of treatment, wherein such components are formulated separately from one another in separate dosage forms that are ingested by the patient at substantially the same time, whereby the components are released to the patient at substantially the same time; 3) sequential administration of such combinations of immunocytokines and therapeutic agents of the present invention to a patient in need of treatment, wherein such components are formulated separately from one another in separate dosage forms that are taken by the patient at successive times with sufficient time intervals between each administration so that, upon administration, the components are released to the patient at substantially different times; and 4) The use of such a combination of the immunocytokine and therapeutic agent of the present invention in patients in need of treatment. Sequential administration to a patient, where such components are combined together in a single dosage form which releases said components in a controlled manner, such that upon release, they are released to said patient at the same and / or different times, simultaneously, sequentially or overlappingly, and wherein each part may be administered by either the same or different routes.

[0339] The immunocytokines of the present invention may be combined with a therapeutic agent selected from the group comprising: a cytotoxic agent, a chemotherapeutic agent, an antihormonal agent, or another therapeutic antibody. As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term includes radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins of bacterial, fungal, plant or animal origin, such as small molecule toxins or enzymatically active toxins, including fragments and / or variants thereof.

[0340] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (e.g., bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analog topotecan (HYCAMTIN®)); trademark), CPT-11 (including irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluetherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride oxide hydrochloride), melphalan, novembite, fenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, e.g., calicheamicin gamma II and calicheamicin omega II (e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXOL®), liposomal doxorubicin TLC D-99 (MYOCET®), PEGylated liposomal doxorubicin; (including CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenib mexamex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as Ancitabine, azacitidine, 6-azauridine, carmophor, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; Demecolcine; Diaziquone; Elfornithine; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK® Polysaccharide Complex (JHS Natural Products, Eugene, Oregon); Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicone; 2,2',2"-Trichlorotriethylamine;Trichothecenes (e.g., T-2 toxin, veraculin A, roridin A, and anguidine); urethanes; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®), albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin, and carboplatin; vincas, which prevent tubulin polymerization to form microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®), FILDESIN®, and vinorelbine (NAVELBINE®); etoposide (VP16); ifosfamide; mitoxantrone; leucovorin; novantrone; edaravone; trexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), ethilodonate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), amphetamines, benzodiazepines, benzophenone-3, benzodiazepines, benzophenone-4, benzodiazepines, benzophenone-5, benzodiazepines, benzophenone-6, benzophenone-7, benzophenone-8, benzophenone-9, benzophenone-10, benzophenone-11, benzophenone-12, benzophenone-13, benzophenone-14, benzophenone-15, benzophenone-15, benzophenone-16, benzophenone-17, benzophenone-18, benzophenone-19, benzophenone-20, benzophenone-21, benzophenone-22, benzophenone-23, benzophenone-24, benzophenone-25, benzophenone-26, benzophenone-37, benzophenone-38, benzophenone-47, benzophenone-48, benzophenone-49, benzophenone-59, benzophenone-59, benzophenone-59, benzophenone-69, benzophenone-79, benzophenone-89, benzophenone-14, benzophenone-15, benzophenone-16, benzophenone-17, benzophenone-18, benzophenone-19, benzophenone-24, Lendronate (FOSAMAJX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, such as those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R);vaccines, such as the THERATOPE® vaccine and gene therapy vaccines, such as the ALLOVECTIN® vaccine, the LEUVECTIN® vaccine, and the VAXID® vaccine; topoisomers; azeta-1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (RI 1577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, which is an abbreviation for treatment with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™).

[0341] Also included in this definition are antihormonal agents, such as antiestrogens with a mixed agonist / antagonist profile, that act to regulate or inhibit hormone action on tumors, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, trioxifene, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVTSTA®), trioxifene, ketoxifene, and selective estrogen receptor modulators. (SERMs), such as SERM3; pure antiestrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); steroidal aromatase inhibitors, such as formestane and exemestane (AROMASIN®), and non-steroidal aromatase inhibitors, such as anastrazole (AREVI®). Aromatase inhibitors, including IDEX®, letrozole (FEMARA®), and aminoglutethimide, and other aromatase inhibitors, including vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and imidazoles; luteinizing hormone-releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; progestins, such as megestrol acetate and acetaminophen Sex steroids including medroxyprogesterone, estrogens such as diethylstilbestrol and Premarin, and androgens / retinoids such as floxymesterone, all-trans retinoic acid, and fenretinide; onapristone; antiprogesterones; estrogen receptor downregulators (ERDs); antiandrogens such as flutamide, nilutamide, and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0342] Other therapeutic agents that may be used in combination with the immunocytokines of the present invention may be therapeutic antibodies selected from the group including antibodies against PD1 (e.g., prorugolimab, pembrolizumab, or nivolumab), antibodies against PD-L1, antibodies against CTLA4, antibodies against 4-1BB, antibodies against OX40, antibodies against GITR, antibodies against CD20 (e.g., rituximab), antibodies against HER2 (e.g., trastuzumab or pertuzumab), antibodies against VEGF (e.g., bevacizumab), or combinations thereof.

[0343] Other therapeutic agents that may be used in combination with the immunocytokines according to the invention may be therapeutically active antitumor compounds selected from the group of activators of innate or adaptive immunity.

[0344] It will be appreciated that the immunocytokines of the present invention may be used in methods of therapy as described above, in treatments as described above, and / or in the manufacture of medicaments for the therapeutic applications described above.

[0345] Dosage and route of administration The immunocytokines of the present invention will be administered in an amount effective to treat the condition in question, i.e., at dosages and for periods of time necessary to achieve the desired result. A therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether the immunocytokine is administered alone or in combination with one or more additional drugs or therapeutic techniques.

[0346] Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly preferred to formulate parenteral compositions in unit dosage forms. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the patient / subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier. The specification of unit dosage forms of the present invention is typically dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect sought to be achieved, and (b) the limitations of subject sensitivity inherent in the art of compounding such active compounds for treatment.

[0347] Thus, those skilled in the art will recognize, based on the disclosure provided herein, that doses and administration regimens will be adjusted according to methods well known in the therapeutic arts. That is, maximum tolerated doses can be readily established, and effective amounts that provide a detectable therapeutic effect in a patient can also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic effect in a patient. Thus, while specific doses and administration regimens are exemplified herein, these examples do not in any way limit the doses and administration regimens that can be provided to a patient in the practice of embodiments of the present invention.

[0348] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated and may include single or multiple doses. Furthermore, for any particular subject, specific dosage regimens should be adjusted over time according to the individual's needs and the judgment of the medical professional administering or monitoring the administration of the composition, and it should be understood that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. Furthermore, dosage regimens, including compositions of the present invention, may be based on a variety of factors, including the type of disease, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the particular immunocytokine of the present invention used. Thus, dosage regimens may vary widely but are routinely determinable using standard methods. For example, doses may be adjusted based on clinical effects, e.g., toxic effects, and / or pharmacokinetic or pharmacodynamic parameters, which may include laboratory values. Accordingly, the present invention encompasses intrapatient dose escalation, as determined by one of ordinary skill in the art. Methods for determining appropriate dosages and regimens are well known in the art and would be understood by one of ordinary skill in the art once provided with the ideas disclosed herein.

[0349] Examples of suitable dosing regimens are provided above. The appropriate dose of the immunocytokine of the present invention is 0.1 to 200 mg / kg, preferably The immunocytokine may be administered at a dose ranging from 0.1 to 100 mg / kg, including about 0.5 to 50 mg / kg, for example, about 1 to 20 mg / kg. For example, at least 0.25 mg / kg, for example, at least 0.5 mg / kg, including at least 1 mg / kg, for example, at least 1.5 mg / kg, for example, at least 2 mg / kg, for example, at least 3 mg / kg, including at least 4 mg / kg, for example, at least 5 mg / kg; and for example, up to 50 mg / kg, up to 30 mg / kg, for example, up to 20 mg / kg, including up to 15 mg / kg. Administration is typically repeated at appropriate time intervals, for example, once weekly, once every two weeks, once every three weeks, or once every four weeks, and as long as deemed appropriate by the responsible physician, who may increase or decrease the dose in some cases if necessary. [Example]

[0350] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way.

[0351] All publications, patents, and patent applications cited herein are hereby incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art, upon consideration of the description of the invention, that certain changes and modifications may be made without departing from the spirit or scope of the accompanying embodiments.

[0352] Materials and general methods Recombinant DNA Technology DNA manipulations were performed by standard techniques as described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's protocols.

[0353] Gene synthesis The desired gene segments were prepared from oligonucleotides generated by chemical synthesis. Gene segments of 300-4000 bp in length, flanked by unique restriction sites, were assembled by annealing and ligation of PCR-amplified oligonucleotides and subsequently cloned via the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

[0354] DNA sequencing DNA sequences were determined by Sanger sequencing. DNA and protein sequence analysis and sequence data management Infomax's Vector NTI Advance suite of software, version 8.0, and SnapGene Viewer were used for sequence generation, mapping, analysis, annotation, and illustration.

[0355] Example 1 Production of recombinant proteins in mammalian cell suspension cultures To produce the recombinant IL15 superagonist protein, which is shown schematically in Figures 1, 2, 3 and 4, the present inventors synthesized human IL15 ligand ( https: / / www.uniprot.org / uniprot / P40933 ) and IL15Rα( https: / / www.uniprot.org / uniprot / Q13261 ) sequence Constructs containing fragments of both genes were synthesized. array was also synthesized from oligonucleotides using PCR.

[0356] To produce the IL15 superagonist variants as shown by CK_IL15Ra_Hc_IL15CH1FcLALA in Figure 1 and CK_IL15_Hc_IL15RaCH1FcLALA in Figure 2 (hereinafter referred to as IL15SA or BCD225), the gene was cloned into a plasmid as an N-terminal fusion (fusion protein) with the first constant domain of the antibody kappa light chain in the vector pEE-IL15Ra-CK (or pEE-IL15-CK) at the SalI / BsiWI restriction sites (Figure 5) and with the first constant domain of the antibody heavy chain in the vector pEE-IL15-CH1-Fc-LALA (or pEE-IL15Ra-CH1-Fc-LALA)IgG1 at the SalI / NheI restriction sites (Figure 6), and both vectors were combined in a transfection step.

[0357] To generate bispecific IL15 superagonist variants as shown by Fab-CK_IL15Ra_Hc_IL15CH1Fc knob LALA in FIG. 4 (hereinafter referred to as IL15SA / PD1 or anti-PD1 / IL15SA or IL15SA / PDL1 or anti-PDL1 / IL15SA) and Fab-CK_IL15_Hc_IL15RaCH1Fc knob LALA in FIG. 3, IL15 ligand and receptor genes were used. The fragment was cloned into the plasmid as an N-terminal fusion with the first constant domain of the antibody kappa light chain in the vector pEE-IL15Ra-CK (or pEE-IL15-CK) at the SalI / BsiWI restriction sites (Figure 5) and with the first constant domain of the antibody heavy chain in the vector pEE-IL15-CH1-Fc-knob-LALA (or pEE-IL15Ra-CH1-Fc-knob-LALA)IgG1 at the SalI / NheI restriction sites (Figure 9). Furthermore, to form the Fab portions of the asymmetric antibodies shown in Figures 3 and 4, genes for the variable domains of anti-PD1 (prorugolimab) (hereinafter referred to as IL15SA / PD1 or anti-PD1 / IL15SA) or anti-PDL1 (BCD-135) antibodies (hereinafter referred to as IL15SA / PDL1 or anti-PDL1 / IL15SA) were cloned into plasmids as N-terminal fusions with the first constant domain of the antibody kappa light chain in vector pEE-BCD100-02-VL-CK at the SalI / BsiWI restriction sites (Figure 7) and with the first constant domain of the antibody heavy chain in vector pEE-BCD100-02-VH-CH1-Fc-hole-LALA IgG1 at the SalI / NheI restriction sites (Figure 8). As a result, the above four vectors were combined in the transfection process to synthesize four distinct polypeptides in one cell and form a bispecific immunocytokine.

[0358] Required amounts of all the above plasmids were produced in E. coli cells and purified using the Maxiprep Qiagen kit. Recombinant proteins containing IL15SA were transiently cultured in Chinese hamster ovary cell lines (CHO-K1) produced according to published protocols [Biotechnol Bioeng. 2005 Sep 20; 91(6):670-677, Liao Metal., 2004; Biotechnol Lett. 2006 Jun;28(11):843-848; Biotechnol Bioeng. 2003 Nov 5;84(3):332-342]. Cells constitutively expressing the EBNA1 (Epstein-Barr virus nuclear antigen 1) protein gene were used. Suspension culture was performed in flasks on an orbital shaker using serum-free medium from Life Technologies Corporation according to the manufacturer's instructions. For transient expression, linear polyethyleneimine (PEI MAX, Polysciences) was used to induce 2-fold transfection. * 10 6 The cells were transfected at a concentration of 2000 / ml. The DNA / PEI ratio was 1:3 to 1:10. Five to seven days after transfection, 2000 The cell culture was centrifuged at 400 x g for 20 min and filtered through a 0.22 μm filter. The target protein was isolated from the culture medium by affine HPLC.

[0359] The recombinant protein was isolated and purified from the culture medium using a Protein A affinity chromatography column. The clarified culture medium was applied to a 5 ml HiTrap rProtein A Sepharose column equilibrated with phosphate-buffered saline (PBS, pH 7.4). The mixture was passed through a FF column (GE Healthcare). The column was then washed with 5 column volumes of PBS to remove nonspecifically bound components. Bound antigen was eluted using 0.1 M glycine buffer (pH 8). The major protein elution peak was collected and brought to neutral pH with 1 M Tris buffer (pH 8). All steps were performed at a flow rate of 110 cm / h. The protein was then dialyzed into PBS (pH 7.4) using SnakeSkin dialysis tubing technology, filtered (0.22 μm), transferred into tubing, and stored at -70°C.

[0360] The purity of the resulting protein solution was assessed by SDS gel electrophoresis (see example in Figure 10). Example 2 Kinetic assay of (IL15SA)2-Fc and human IL2βγ receptor interaction Affinity constants for the binding of (IL15SA)2-Fc (hereafter referred to as CK_IL15Ra_Hc_IL15CH1FcLALA in Figure 1 and CK_IL15_Hc_IL15RaCH1FcLALA in Figure 2) and human IL2βγ were obtained using an OctetRedBio instrument according to ForteBio's instructions. AR2G biosensors were pre-rehydrated in mQ for 1 hour. After biosensor activation, the (IL15SA)2-Fc product was analyzed at a concentration of 25 μg / ml in acetate buffer, pH 4. The IL15SA / IL2βγ complex was assembled in this solution. The sensor was then immersed in the buffer solution for the subsequent dissociation step. The assay was performed at 30°C using PBS supplemented with 0.1% Tween-20 and 0.1% BSA as the working buffer.

[0361] The resulting sensograms for CK_IL15Ra_Hc_IL15CH1FcLALA and for CK_IL15_Hc_IL15RaCH1FcLALA are shown in Table 1 and were analyzed after subtraction of the reference signal using Octet data analysis software (version 8.0) and a 1:1 interaction model according to standard procedures. The resulting affinity constants are shown in Table 1. Both studied variants showed high affinity and specificity for human IL2βγ.

[0362] Table 1. Affinity constants for (IL15SA)2-Fc immunocytokine interacting with human IL2βγ-Fc.

[0363] [Table 2]

[0364] Example 3 Kinetic assays of the interaction between a bispecific IL-15 superagonist and the human IL-2βγ receptor and other cellular receptors on immune cells The affinity constants for binding of the IL15SA bispecific immunocytokine (hereafter referred to as anti-PD1-Fab-CK_IL15Ra_Hc_IL15CH1Fc knob LALA and anti-PD-L1-Fab-CK_IL15Ra_Hc_IL15CH1Fc knob LALA) to the IL2βγ and human PD-1 / PD-L1 receptors were measured using OctetRed 96 according to ForteBio's instructions. AR2G biosensors were pre-rehydrated in mQ for 1 hour. After biosensor activation, the bispecific immunocytokine was added at a concentration of 25 μg / ml in acetate buffer, pH 4. The bispecific superagonist and the test receptor were nonspecifically immobilized (via NH groups) on the biosensor. The sensor was then immersed in a well containing human CD122 / IL-2RB protein (Fc tag) or PD-1-Fc receptor or PD-L1-Fc receptor (1 and 0.5 μg / ml) in PBS buffer supplemented with 0.1% Tween-20 and 0.1% BSA to allow association of the bispecific superagonist and the test receptor. The sensor was then immersed in a buffer solution for the subsequent dissociation step.

[0365] Instrument data were analyzed after subtracting the reference signal using Octet data analysis software (version 8.0) according to standard procedures and using a 1:1 interaction model.

[0366] The resulting affinity constants are shown in Table 2. Both mutants studied exhibited high affinity and specificity for both human IL2βγ and for the paired receptor, and lacked nonspecific activity toward other receptors.

[0367] Table 2. Affinity constants for bispecific IL15 superagonist antibodies interacting with human IL2βγ-Fc and a second receptor.

[0368] [Table 3]

[0369] Example 4 Cellular assay to determine the proliferative activity of IL-15 superagonists against the NK92 cell line The NK-92 cell line was used in the assay. The assay was performed in a 96-well culture plate. The suspension contained NK-92 cells and the test antibody at the concentrations indicated in the graph. All suspension components were prepared in RPMI-1640 medium supplemented with fetal bovine serum and glutamine. After adding all components, the plate was incubated at 37°C and 5% CO2. Alamar Blue was then added to the wells. After incubation, the fluorescence intensity in the wells was measured. IL15SA product (CK_IL15Ra_Hc_IL15CH1FcLALA), IL15SA / PD1 (anti-PD-1 Fab-CK_IL15Ra_Hc_IL15CH1FcLALA), and IL15SA / PDL1 (anti-PDL-1 Fab-CK_IL15Ra_Hc_IL15CH1FcLALA) were comparatively studied.

[0370] The specific activity determination of the immunocytokines showed high proliferative activity against the NK92 line. The estimated EC50 values ​​for all candidates were identical, and the activation levels, as determined by the upper plateau, were also identical (Figure 11). It should be noted that the valency, and therefore the different avidity, of the IL15SA moiety did not significantly affect this activity.

[0371] Example 5 A cellular assay to determine the proliferative activity of IL-15 superagonists against natural killer strains The assay used isolated natural killer cells (NKCs) isolated from PBMCs of healthy donors by negative selection. The assay was performed in a 96-well culture plate. The suspension contained NKCs and the test antibodies at the concentrations indicated in the graph. All suspension components were prepared in medium supplemented with autologous human plasma. After adding all components, the plate was incubated at 37°C and 5% CO2. Alamar Blue was then added to the wells. After incubation, the fluorescence intensity in the wells was measured. The effects of IL15SA product (CK_IL15Ra_Hc_IL15CH1FcLALA), IL15SA / PD1 (anti-PD-1 Fab-CK_IL15Ra_Hc_IL15CH1FcKnobLALA), and human interleukin-2 (IL2) (Ronleukin, PCI Biotech) on the proliferative activity of NKCs were comparatively studied.

[0372] The results of determining the specific activity of the above immunocytokines showed high proliferation activity against natural killer cells (Figure 12). The estimated EC50 values ​​for all candidates were identical within the error limits and slightly lower than that for the IL2 cytokine. Additionally, the activation level, as defined by the upper plateau, was also completely identical. It is noteworthy that the valency, and therefore the different avidity, of the IL15SA moiety did not significantly affect this activity.

[0373] Example 6 A cellular assay to determine the anti-PD1 antagonist activity of IL-15 superagonists against reporter lines For this assay, we used the Jurkat NFAT-FLuc PD-1 cell line, which was derived from the Jurkat cell line and stably expresses PD-1 on its surface and contains the firefly luciferase gene under the control of the NFAT promoter; and the Raji PDL1 cell line, which was derived from the Raji cell line and stably expresses PDL1 on its surface. We compared the effects of the IL15SA product (CK_IL15Ra_Hc_IL15CH1FcLALA), anti-PD1 / IL15SA (anti-PD-1 Fab-CK_IL15Ra_Hc_IL15CH1FcKnobLALA), and the anti-PD1 antagonist (prorugolimab) on the activation capacity of the reporter cell lines.

[0374] The assay was carried out in a 96-well culture plate. The suspension in each well contained Jurkat cells. The plates contained NFAT-FLuc PD-1 cells, Raji PDL1 cells, aCD3 / aTAA1 cells at a concentration of 1 ng / ml, and test antibodies at the concentrations indicated in the graph. After adding all components, the plates were incubated at 37°C and 5% CO2, and then the luciferase intensity in the wells was measured using a luminescence assay kit.

[0375] The results of the determination of the specific activity of the immunocytokines described above showed an order-of-magnitude lower specific activity EC50 value for anti-PD1 / IL15SA compared to the therapeutically effective anti-PD1 monoclonal antibody prorugolimab (Figure 13). Furthermore, the maximum activity level (upper plateau) was reduced by 30%. The lower specific activity of anti-PD1 / IL15SA compared to prorugolimab can only be explained by the monovalent Fab-bound PD1 in the anti-PD1 / IL15SA molecule compared to the classical antibody. This is also supported by the almost order-of-magnitude difference in affinity for the PD-1 receptor according to the data in Example 3 (Table 2) and the values ​​specified for prorugolimab. It should be noted that the antagonist activity of IL15SA was observed as background, i.e., was virtually absent.

[0376] Example 7 A cellular assay to determine the anti-PDL1 antagonist activity of IL-15 superagonists on reporter lines For the assay, we used the Jurkat NFAT-FLuc PD-1 cell line, which was generated based on the Jurkat cell line and stably expresses PD-1 on its surface and contains the firefly luciferase-encoding gene under the control of the NFAT promoter; and the Raji PDL1 cell line, which was generated based on the Raji cell line and stably expresses PDL1 on its surface. We compared the effects of the IL15SA product (CK_IL15Ra_Hc_IL15CH1FcLALA), anti-PDL1 / IL15SA (anti-PD-L1 Fab-CK_IL15Ra_Hc_IL15CH1FcKnobLALA), and the anti-PDL1 antagonist (atezolizumab) on the activation ability of the reporter cell lines.

[0377] The assay was carried out in a 96-well culture plate. The suspension in each well contained Jurkat cells. The cells contained NFAT-FLuc PD-1 cells, Raji PDL1 cells, aCD3 / aTAA1 cells at a concentration of 1 ng / ml, and test antibodies at the concentrations indicated on the graph. After adding all components, the plate was incubated at 37°C, 5% CO2, and then the luciferase intensity in the wells was measured using a luminescence assay kit.

[0378] The results of the specific activity determination of the above immunocytokines showed a 30-fold lower specific activity EC50 value for anti-PDL1 / IL15SA compared to the therapeutically effective anti-PDL1 monoclonal antibody atezolizumab (Figure 14). However, the maximum activity level (upper plateau) was only reduced by 10%. The lower specific activity of anti-PDL1 / IL15SA compared to that of atezolizumab can only be explained by the monovalent Fab binding PD-L1 within the anti-PD1 / IL15SA molecule compared to classical antibodies. Therefore, no significant antagonist activity of IL15SA was observed.

[0379] Example 8 A cellular assay to determine the effect of IL-15 superagonists on antibody-dependent cellular cytotoxicity (ADCC) of natural killer cells against tumor-associated antigen (TAA) reporter lines in the presence of TAA-specific antibodies The assay used natural killer cells isolated from PBMCs of healthy donors by negative selection. The assay was performed in a 96-well culture plate. The suspension contained natural killer cells and Raji cells, the effector antibody rituximab, and the test antibody at the concentrations indicated on the graph. All suspension components were prepared in medium supplemented with fetal bovine serum and glutamine. After adding all components, the plate was incubated at 37°C and 5% CO2. The culture medium was then collected, and the lactate dehydrogenase content was measured using an LDH assay kit. The ADCC potential of rituximab in a reporter cell line was compared between the IL15SA product (CK_IL15Ra_Hc_IL15CH1FcLALA) and the anti-PD1 / IL15SA (anti-PD-1 Fab-CK_IL15Ra_Hc_IL15CH1FcKnobLALA).

[0380] The results of determining the specific activity of the above immunocytokines showed enhanced activity compared to the enhancement of the ADCC ability of rituximab against the TAA reporter strain (Figures 15 and 16). The efficacy of rituximab-dependent ADCC, as measured by the percentage of lysed Raji cells with overexpressed CD20 receptors on their surface, increased by 30% in the presence of each candidate studied compared to rituximab alone. This effect appears to be explained by the proliferation of activated natural killer cells under the influence of IL-15SA and anti-PD1 / IL15SA in the assay, according to the results provided in Examples 4 and 5. The estimated EC50 values ​​for all candidates were identical within the error limits. It should be noted that the valency and therefore different avidity of the IL15SA moiety did not significantly affect this activity, a fact further supported by Examples 4 and 5.

[0381] Example 9 A cellular assay to determine the cytotoxicity of IL-15 superagonists against PBMCs derived from whole blood of healthy donors PBMCs were isolated from whole blood from healthy donors by Ficoll density gradient centrifugation.

[0382] Assays were performed in 96-well culture plates. The suspensions contained freshly isolated PBMCs (per well) and antibodies at a concentration of 0.1 μg / ml as indicated on the graph. After mixing the PBMCs and antibodies, the plates were incubated at 37°C and 5% CO for 16 hours. The proportions of CD56+, CD19+, CD3+, CD4+, and CD8+ subpopulations of PBMCs in the suspensions were then measured by directly staining the suspensions with fluorescently labeled antibodies against the corresponding CDs and further analyzing the cells using a flow cytofluorometer. For CD56+, CD19+, and CD3+ cells, the graphs show the percentages of CD56+, CD19+, and CD3+ cells in the test suspensions. The graph shows the percentage of CD4+ and CD8+ cells relative to the percentage of CD3+ cells. The cytotoxic effects of IL15SA product (CK_IL15Ra_Hc_IL15CH1FcLALA), anti-PD1 / IL15SA (anti-PD-1 Fab-CK_IL15Ra_Hc_IL15CH1FcKnobLALA), anti-PD1 (prorugolimab), a mixture of anti-PD1 (prorugolimab) + IL15SA, and anti-CD20 GA101 antibody on human PBMCs were compared.

[0383] The results, as presented in Figures 17-21, demonstrate that the cytotoxicity of the candidates is insignificant, i.e., a reduction of no more than 10% in the number of immunoreactive cells when compared to the AB negative control (i.e., the absence of any exogenous antibody). The control anti-CD20 antibody thereby demonstrated a consistent and complete depletion of the CD20+ B cell population. Thus, all of the candidates in question do not exhibit any significant nonspecific in vitro cytotoxicity against human blood cells.

[0384] Example 10 Determining the aggregation stability of IL-15 superagonists under heat stress conditions The IL15 superagonist product at a concentration of 9 mg / ml in PBS buffer was heated at 50°C for 12 hours. Aggregation after heat stress was determined by high-performance gel filtration chromatography. Chromatography was performed on an HPLC system (Agilent) on a Tosoh TSK-Gel G3000SWXL column, 7.8 mm x 30 cm, order number 08541, with a Tosoh TSKgel Guard SWXL precolumn, 6.0 mm x 4.0 cm, 7 μm particle diameter, order number 08543. Elution was performed isocratically with a mobile phase of 50 mM NaFb, 0.3 M NaCl, pH 7.0, at a flow rate of 0.5 ml / min. Detection was performed at wavelengths of 214 and 280 nm. The antibody sample was diluted to a concentration of ~1 mg / ml with PBS buffer, pH 7.5. The injection volume was 10 microliters. A gel filtration standard calibration mixture (Bio-Rad), order number 151-1901, was prechromatographed. Figure 22 shows the chromatogram showing the high homogeneity of the IL15 superagonist (aggregate content in solution did not exceed 5%).

[0385] Example 11 Evaluation of the antitumor activity of IL-15 superagonist products in a syngeneic B16F10 melanoma model Efficacy was evaluated using immunocompetent C57BL / 6 mice inoculated with the B16F10 tumor line. 5 Tumor cells were injected subcutaneously into the right flank of each animal in each group. On the seventh day after cell inoculation (the first day of the experiment), the animals were divided into groups as shown in Table 3. Efficacy was evaluated using one dose of the following products: BCD-225, IL15SA / a-mPD-1 (a bispecific monoclonal antibody containing IL-15SA and a Fab fragment against mouse PD-1, a derivative of the mouse RPM1-14 antibody). The sequence of the variable domain of the RMP1-14 antibody was kindly provided by Dr. Hideo Yagita of Juntendo University School of Medicine. The gene and variable domain of the RMP1-14 antibody product were synthesized according to Example 1. Furthermore, the present inventors have used the reference product ALT-803 [ Han KP et al., Cytokine.2011 Dec;56(3):804-10.] (positive control) and placebo (negative control) were synthesized. The antitumor activity of the product in combination with anti-mouse PD-1 antibody (a-mPD-1) was also evaluated.

[0386] Table 3. Animal groups in antitumor activity studies of IL-15 superagonists

[0387] [Table 4]

[0388] The BCD-225 (IL15SA), IL15SA / a-mPD-1, and ALT-803 products were injected intraperitoneally in a volume of 0.2 ml on experimental days 1, 4, 8, and 11. The anti-mPD-1 product was injected intraperitoneally in a volume of 0.2 ml on experimental days 2, 5, 9, and 12. The negative control group was injected with sodium acetate buffer in a volume of 0.2 ml on experimental days 1, 2, 4, 5, 8, 9, 11, and 12 (Figure 23).

[0389] Mouse body weights and tumor linear dimensions were measured throughout the experiment. Tumor volume was calculated using the following formula: V=LxWxWxπ / 6. The following equation:

[0390] [ka]

[0391] A negative control group (V c ) and product group (V t The efficacy of the test products was evaluated by the Tumor Growth Inhibition Index (TGI), calculated taking into account the median tumor volume in the 100% POI (n = 100) group.

[0392] The experimental results are shown in Figures 24 and 25. Data from day 11 of the experiment are provided because nearly all animals in the experimental groups died from tumor burden, making it impossible to reliably assess the antitumor activity of the products on day 15.

[0393] According to the study results, the BCD-225 product and the reference ALT-803 product showed comparable antitumor activity, with a TGI of 52% in the BCD-225 group and 53% in the ALT-803 group. The inventors observed an increased trend in the TGI value of the IL15SA / anti-mPD-1 bispecific antibody group when compared to the TGI values ​​of the BCD-225 and ALT-803 groups. The TGI of the IL15SA / anti-mPD-1 group was 58% on day 11 of the experiment.

[0394] The TGI values ​​for the product combination groups BCD-225 + anti-mPD-1 and ALT-803 + anti-mPD-1 were 72% and for the product combination groups IL15SA / a-mPD-1 and anti-mPD-1 the TGI value was 69% on experiment day 11. The resulting TGI values ​​indicate a more pronounced antitumor activity of the combination of the test products and anti-mPD-1 compared to the activity of these products in monotherapy.

[0395] Example 12 Evaluation and analysis of comparative mortality in mice with IL15 superagonist and ALT803 product in a syngeneic B16F10 melanoma model A comparative evaluation of mortality in mice following the use of IL15 superagonist products was performed using models and animals as described in Example 11. Survival was assessed by counting animals that died or were euthanized in distress.

[0396] The results are shown in Table 4. As can be seen from the resulting data, the mortality rates of the IL15SA / a-mPD-1 and IL15SA / a-mPD-1 + anti-mPD-1 product groups were, on average, lower in terms of number of mice and survival time when compared with those of IL15SA (BCD225), IL15SA + anti-mPD-1, ALT-803, and ALT-803 + anti-mPD-1. The resulting mortality values ​​likely indicate less pronounced toxicity of the IL15SA / a-mPD-1 bispecific immunocytokine when compared with those of the monospecific bivalent IL15SA and ALT-803. The presence or absence of anti-PD1 antibodies did not have a statistically significant effect on mortality. Furthermore, examination of the organs of dead mice from the IL15SA and ALT-803 batches revealed hepatocellular necrosis, the presence of hydrodystrophic livers with sinusoidal compression; lymphohistiocytic infiltration in the parenchyma; and lymphocyte accumulation in the sinusoids. These findings are consistent with previously reported literature data on the ability of IL15-based superagonists to cause liver injury in mice.

[0397] Table 4. Animal groups in mortality studies following IL15 superagonist and ALT803 superagonist products and their combination with anti-PD1 antibodies

[0398] [Table 5]

Claims

1. 1. An immunocytokine for stimulating the activation and / or proliferation of IL-15Rbeta / gamma positive cells, comprising: 1) IL-15Rα linked to: a) an antibody light chain constant domain, or b) antibody heavy chain constant domains, including an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and third (CH3) heavy chain constant domain; 2) IL-15 linked to: a) an antibody heavy chain constant domain, comprising an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and third (CH3) heavy chain constant domain; or b) Antibody Light Chain Constant Domain IL-15 / IL-15Rα-based heterodimeric protein complexes comprising: a first antibody heavy chain constant domain and an antibody light chain constant domain in the heterodimeric complex, which may or may not be covalently associated through a native S—S bridge; and When IL-15 or IL-15Rα is linked to an antibody light chain constant domain, the other part of the heterodimeric protein complex selected from IL-15Rα or IL-15 is linked to an antibody heavy chain constant domain. The immunocytokine.

2. The immunocytokine of claim 1, wherein the antibody light chain constant domain is selected from CK or CL.

3. 2. The immunocytokine of claim 1, wherein the IL-15Rα has the amino acid sequence set forth by SEQ ID NO: 9, or any known mutant IL-15Rα variant having similar biological activity.

4. 2. The immunocytokine of claim 1, wherein the IL-15 has the amino acid sequence set forth by SEQ ID NO: 10, or any known mutant IL-15 variant having similar biological activity.

5. The immunocytokine of claim 1, wherein IL-15Rα is linked to an antibody light chain constant domain.

6. The immunocytokine of claim 5, wherein the IL-15Rα linked to the antibody light chain constant domain has the amino acid sequence shown by SEQ ID NO:1 or SEQ ID NO:

19.

7. The immunocytokine of claim 1, wherein IL-15 is linked to an antibody light chain constant domain.

8. 8. The immunocytokine of claim 7, wherein the IL-15 linked to the antibody light chain constant domain has the amino acid sequence shown by SEQ ID NO:3 or SEQ ID NO:

21.

9. 2. The immunocytokine of claim 1, wherein the Fc fragment monomer comprises a first (CH1) heavy chain constant domain, and a second (CH2) and a third (CH3) heavy chain constant domain, linked through a hinge.

10. 2. The immunocytokine of claim 1, wherein IL-15 or IL-15Rα is linked to antibody heavy chain constant domains arranged in the following order: CH1-hinge-CH2-CH3.

11. The immunocytokine of claim 10, wherein IL-15Rα is linked to an antibody heavy chain constant domain.

12. The immunocytokine of claim 11, wherein the IL-15Rα linked to the antibody heavy chain constant domain has the amino acid sequence shown by SEQ ID NO:4 or SEQ ID NO:

22.

13. The immunocytokine of claim 10, wherein IL-15 is linked to an antibody heavy chain constant domain.

14. 14. The immunocytokine of claim 13, wherein the IL-15 linked to the antibody heavy chain constant domain has the amino acid sequence shown by SEQ ID NO:2 or SEQ ID NO:

20.

15. the IL-15Rα linked to the antibody light chain constant domain has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:19; and IL-15 linked to an antibody heavy chain constant domain has the amino acid sequence shown by SEQ ID NO:2 or SEQ ID NO:20 The immunocytokine of claim 1.

16. the IL-15Rα linked to the antibody heavy chain constant domain has the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:22; and IL-15 linked to an antibody light chain constant domain has the amino acid sequence shown by SEQ ID NO:3 or SEQ ID NO:21 The immunocytokine of claim 1.

17. 2. The immunocytokine of claim 1, comprising a mutation in an Fc fragment monomer that causes the immunocytokine to have impaired ADCC, CDC, and / or ADCP properties.

18. The immunocytokine of claim 1, wherein the Fc fragment belongs to IgG.

19. 19. The immunocytokine of claim 18, wherein the Fc fragment isotype is selected from the group comprising: human IgG1, IgG2, or IgG4.

20. The immunocytokine of any one of claims 1 to 19, comprising the two heterodimeric protein complexes based on IL-15 / IL-15Rα.

21. 21. The immunocytokine of claim 20 for use as a therapeutic agent for the treatment of cancer or autoimmune disease.

22. An immunocytokine for stimulating the activation and / or proliferation of IL-15Rbeta / gamma positive cells, comprising an IL-15 / IL-15Rα-based heterodimeric protein complex and an immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway; The IL-15 / IL-15Rα-based heterodimeric protein complex: 1) IL-15Rα linked to: a) an antibody light chain constant domain, or b) antibody heavy chain constant domains, including an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and third (CH3) heavy chain constant domain; 2) IL-15 linked to: a) a first (CH1) heavy chain constant domain and a second (CH2) and third (CH 3) an antibody heavy chain constant domain, comprising an Fc fragment monomer comprising a heavy chain constant domain; or b) Antibody Light Chain Constant Domain Including; a first antibody heavy chain constant domain and an antibody light chain constant domain in the heterodimeric complex, which may or may not be covalently associated through a native S—S bridge; and When IL-15 or IL-15Rα is linked to an antibody light chain constant domain, the other part of the heterodimeric protein complex selected from IL-15Rα or IL-15 is linked to an antibody heavy chain constant domain. The immunocytokine.

23. 23. The immunocytokine of claim 22, wherein the antibody light chain constant domain is selected from CK or CL.

24. 23. The immunocytokine of claim 22, wherein the IL-15Rα has the amino acid sequence set forth by SEQ ID NO: 9, or any known mutant IL-15Rα variant having similar biological activity.

25. 23. The immunocytokine of claim 22, wherein the IL-15 has the amino acid sequence set forth by SEQ ID NO: 10, or any known mutant IL-15 variant having similar biological activity.

26. The immunocytokine of claim 22, wherein the IL-15Rα is linked to an antibody light chain constant domain.

27. 27. The immunocytokine of claim 26, wherein the IL-15Rα linked to the antibody light chain constant domain has the amino acid sequence shown by SEQ ID NO:1 or SEQ ID NO:

19.

28. 23. The immunocytokine of claim 22, wherein IL-15 is linked to an antibody light chain constant domain.

29. 29. The immunocytokine of claim 28, wherein the IL-15 linked to the antibody light chain constant domain has the amino acid sequence set forth by SEQ ID NO:3 or SEQ ID NO:

21.

30. 23. The immunocytokine of claim 22, wherein the Fc fragment monomer comprises a first (CH1) heavy chain constant domain, and a second (CH2) and third (CH3) heavy chain constant domain, linked through a hinge.

31. 23. The immunocytokine of claim 22, wherein IL-15 or IL-15Rα is linked to antibody heavy chain constant domains arranged in the following order: CH1-hinge-CH2-CH3.

32. The immunocytokine of claim 31, wherein the IL-15Rα is linked to an antibody heavy chain constant domain.

33. The immunocytokine of claim 32, wherein the IL-15Rα linked to the antibody heavy chain constant domain has the amino acid sequence shown by SEQ ID NO:6 or SEQ ID NO:

24.

34. The immunocytokine of claim 31, wherein IL-15 is linked to an antibody heavy chain constant domain.

35. IL-15 linked to an antibody heavy chain constant domain is SEQ ID NO:5 or SEQ ID NO:23 The immunocytokine of claim 34 having an amino acid sequence represented by:

36. The immunocytokine of claim 22, wherein the immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway is an antibody that specifically binds to PD-1.

37. Immunomodulatory antibodies: a) a light chain comprising a light chain variable domain and a light chain constant domain; b) a heavy chain comprising an antibody heavy chain constant domain, including a heavy chain variable domain, and an Fc fragment monomer comprising a first (CH1) heavy chain constant domain, and a second (CH1) and a third (CH3) heavy chain constant domain; The immunocytokine of claim 22, comprising:

38. 38. The immunocytokine of claim 37, wherein the light chain variable domain comprises LCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) set forth by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

39. 39. The immunocytokine of claim 38, wherein the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:

18.

40. 38. The immunocytokine of claim 37, wherein the heavy chain variable domain comprises HCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) represented by the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively.

41. 41. The immunocytokine of claim 40, wherein the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:

17.

42. 1) the light chain variable domain comprises LCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) represented by the amino acid sequences of SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; 2) the heavy chain variable domain comprises HCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3) represented by the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively; The immunocytokine of claim 37.

43. 1) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 18; 2) the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 17 43. The immunocytokine of claim 42.

44. 38. The immunocytokine of claim 37, wherein the immunomodulatory antibody comprises a light chain comprising the amino acid sequence set forth by SEQ ID NO:

8.

45. 38. The immunocytokine of claim 37, wherein the immunomodulatory antibody comprises a heavy chain comprising the amino acid sequence set forth by SEQ ID NO:

7.

46. Immunomodulatory antibodies: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:7; A light chain comprising the amino acid sequence set forth in SEQ ID NO:8 38. The immunocytokine of claim 37, comprising:

47. The immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway is PD-L1 The immunocytokine of claim 22, which is an antibody that specifically binds to

48. a) A heterodimeric protein complex based on IL-15 / IL-15Rα: IL-15Rα linked to an antibody light chain constant domain having the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:19, and IL-15 linked to an antibody heavy chain constant domain having the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:23 Including; b) an immunomodulatory antibody: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:7; A light chain comprising the amino acid sequence set forth in SEQ ID NO:8 The immunocytokine of claim 22, comprising:

49. a) the IL-15Rα linked to the antibody heavy chain constant domain has the amino acid sequence shown by SEQ ID NO:6 or SEQ ID NO:24; and the IL-15 linked to the antibody light chain constant domain has the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:21; b) an immunomodulatory antibody: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:7; A light chain comprising the amino acid sequence set forth in SEQ ID NO:8 The immunocytokine of claim 22, comprising:

50. 38. The immunocytokine of claim 22 or 37, having a mutation in the antibody constant domain that induces heterodimerization of two distinct moieties, one of which comprises IL-15Rα linked to an antibody constant domain and IL-15 linked to an antibody constant domain, covalently or non-covalently associated, and the other moiety comprises the light and heavy chains of an antibody, covalently or non-covalently associated.

51. 38. The immunocytokine of claim 22 or 37, wherein the first Fc monomer and the second Fc monomer are selected from the following group: the first Fc monomer is a knob-modified Fc and the second Fc monomer is a hole-modified Fc, or the second Fc monomer is a knob-modified Fc and the first Fc monomer is a hole-modified Fc.

52. a) a first Fc monomer has amino acid substitutions S354C / T366W and a second Fc monomer has amino acid substitutions Y349C / T366S / L368A / Y407V; b) a first Fc monomer has the amino acid substitutions Y349C / T366S / L368A / Y407 and a second Fc monomer has the amino acid substitutions S354C / T366W The immunocytokine of claim 51.

53. 38. The immunocytokine of claim 22 or 37, wherein the Fc fragment belongs to IgG.

54. 54. The immunocytokine of claim 53, wherein the Fc fragment isotype is selected from the group comprising: human IgG1, IgG2, or IgG4.

55. 38. The immunocytokine of claim 22 or 37, having a mutation in an Fc fragment monomer that causes the immunocytokine to have impaired ADCC, CDC, and / or ADCP properties.

56. An immunocytokine according to any one of claims 22 to 55 for the treatment of a tumor disease or an autoimmune disease.

57. An isolated nucleic acid encoding the immunocytokine of any of claims 1-20 or 22-55.

58. 58. The nucleic acid of claim 57, wherein the nucleic acid is DNA.

59. An expression vector comprising the nucleic acid of any one of claims 57 to 58.

60. A method for producing a host cell for producing the immunocytokine of any of claims 1 to 20 or 22 to 55, comprising the step of transforming the cell with the vector of claim 59.

61. A host cell for producing the immunocytokine of any one of claims 1 to 20 or 22 to 55, comprising the nucleic acid of any one of claims 57 to 58.

62. 62. A method for producing a product comprising an immunocytokine of any of claims 1-20 or 22-55, comprising culturing a host cell of claim 61 in a culture medium under conditions sufficient to produce said immunocytokine, and, if necessary, thereafter isolating and purifying the produced immunocytokine.

63. A pharmaceutical composition for stimulating the activation and / or proliferation of IL-15Rbeta / gamma positive cells, comprising a therapeutically effective amount of an immunocytokine of any of claims 1 to 20 or 22 to 55 in combination with one or more pharmaceutically acceptable excipients.

64. 64. The pharmaceutical composition of claim 63 for the treatment of a neoplastic disease or an autoimmune disease.

65. Neoplastic diseases include: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, MSI 65. The pharmaceutical composition of claim 64, wherein the cancer is selected from the group comprising CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndrome.

66. A method for treating a neoplastic disease, comprising administering to a subject in need of such treatment a therapeutically effective amount of an immunocytokine of any of claims 1 to 20 or 22 to 55, or a pharmaceutical composition of claim 63.

67. Neoplastic diseases include: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, MSI 56. The method of claim 55, wherein the cancer is selected from the group comprising CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndrome.

68. 63. A method for activating the biological activity of a T cell population or an NK cell population in a subject in need thereof, comprising administering to the subject an effective amount of an immunocytokine of any of claims 1-20 or 22-55, or a pharmaceutical composition of claim 63.

69. Use of an immunocytokine according to any of claims 1 to 20 or 22 to 55, or a pharmaceutical composition according to claim 63, for the treatment of a neoplastic disease in a subject in need thereof.

70. Neoplastic diseases include: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple-negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, MSI 70. The use of claim 69, wherein the cancer is selected from the group comprising CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndromes.

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