EA043937B1 - IMMUNOCYTOKINE, INCLUDING A HETERODIMERIC PROTEIN COMPLEX BASED ON IL-15 AND IL-15Rα, AND ITS APPLICATION - Google Patents

Description

Field of technology

The invention relates to an immunocytokine comprising a heterodimeric protein complex based on IL-15/IL-15Ra, and its use as a therapeutic agent, in particular as a therapy for cancer and autoimmune diseases. The present invention also relates to an immunocytokine comprising a heterodimeric protein complex based on IL-15/IL-15Ra and an immunomodulatory antibody, and its use as a therapeutic agent, in particular as an agent for the therapy of cancer and autoimmune disease.

Prior Art

Cytokines belong to the category of signaling proteins and glycoproteins, which, like hormones and neurotransmitters, are actively used in cellular communication. While hormones are secreted into the blood by certain organs, and neurotransmitters are related to neuronal activity, cytokines are a more diverse class of compounds in terms of origin and purpose. They are produced by a wide range of hematopoietic and non-hematopoietic cells and can affect both nearby cells and the entire body, sometimes showing a strong dependence on the presence of other chemical compounds. The cytokine family consists primarily of small water-soluble proteins and glycoproteins ranging from 8 to 30 kDa. Cytokines play a key role in the implementation of innate and acquired immune responses. They are often secreted by immune system cells that have encountered a pathogen to activate and mobilize more immune system cells and enhance the immune system's response to the pathogen.

Among the cytokines, interleukin 15 (IL-15) is a cytokine with structural similarity to IL-2, secreted by mononuclear phagocytes (and some other cells) in response to infection by virus(s) or indirect stimulation by cells that are recognized as non-self or weakened. This cytokine induces the proliferation of natural killer cells; cells that provide innate immunity, the main role of which is to destroy cells infected with the virus. The protein encoded by this gene is a cytokine that regulates the activation and proliferation of T cells and natural killer cells.

Interleukin 15 (IL-15), is a 14-15 kDa glycoprotein that simultaneously belongs to two groups as a T-cell activating factor (Grabstein K.H. et al., Science 1994, 264, 965; Burton J.D. et al., Proc. Natl Acad Sci USA 1994, 91, 4935). IL-15 mRNA is widely expressed in various cells and tissues, but detection of the protein in these cells or in the cell supernatant is difficult due to the strong post-transcriptional control of its expression at the level of translation and intracellular transport (Bamford RN. et al., J. Immunol. 1998, 160 : 4418-4426; Kurys G. et al., J. Biol. Chem. 2000, 275: 30653-30659). In addition, 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 it has recently been noted that that it can function as either a ligand or 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 levels of soluble protein expression have been associated with the pathogenesis of autoimmune and inflammatory diseases. IL-15 has been found in several diseases, including Crohn's disease (Kirman L. 1996, Am. 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 (PA), (Mclnnes I.B. 1998, Immunology Today, 19, 75-79). Ligand binding to the T cell receptor induces the expression of IL-15Ra and the expression of several activation antigens such as CD69, CD25 and TNFRII. In addition, IL-15 is a chemoattractant for T lymphocytes in human blood (Wilkinson 1995, J. Exp. Med. 181, 12551259). Taken together, these data indicate that IL-15, expressed by antigen presenting cells, may play an important role in the early activation of T cells at the site of inflammation.

IL-15 is a member of a small family of cytokines that have a four-helix bundle structure. The biological effects of IL-15 are mediated through its binding to a cell membrane receptor consisting of three subunits α, β, and γ. IL-15Ra is a subunit specific for this cytokine, to which it binds with very high affinity Kd 10 ^-11 , and can be found as a membrane receptor, or in 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 is capable of binding to IL-15 and is essential for the biological functions of IL-15 upon binding.

IL-15 has the disadvantages of a small molecular weight, short half-life in vivo, poorly controlled repeated dosing, and is likely to cause systemic immune side effects. There is an urgent need to find an approach that could increase the half-life in vivo and stimulate or enhance the biological activity of IL-15 in vivo.

Recently, it was discovered that the complex formed by IL-15 and its receptor IL-15Ra can significantly enhance the biological activity of IL-15. Studies have shown that the complex formed by IL-15 and the soluble receptor IL-15Ra is significantly superior to individual IL-15 in stimulating the proliferation of CD8+ memory T lymphocytes and NT/NKT cells. The IL-15/IL-15Ra complex is more than 10 times more potent than IL-15 alone in stimulating the proliferation of memory CD8+ T lymphocytes and supporting their survival, and the mechanism of this action may be due to cis-presentation.

International applications WO 2007046006, WO 2016095642 describe a fusion protein designed to stimulate the IL-15Rbeta/gamma signaling pathway to induce and/or stimulate the activation and/or proliferation of IL-15Rbeta/gamma positive cells such as NK and/or T cells characterized in that it contains IL-15 indirectly covalently linked to a polypeptide containing the sushi domain of the extracellular region of the alpha subunit of IL-15R, and for its use in the treatment of cancer. In this case, the experimental data and characteristics of the superagonist in the application WO 2007046006 are presented only for IL-15 connected by a linker peptide through the N- and Terminal amino acids to alpha IL-15R. Whereas the experimental data and characteristics of the superagonist in the application WO 2016095642 are presented only for those connected by a chimeric S-S bond formed by Cys mutations in the IL-15 ligand and alpha IL-15R themselves. Both options do not provide evidence of activity and physicochemical characteristics of IL15 superagonist variants containing fusion protein domains connected by S-S bond(s) or without S-S bridges.

These objects have disadvantages due to their low molecular weight and short half-life in vivo. They have limited interleukin 15 activity, and their production is very difficult, in particular in Chinese hamster ovary cells, and is accompanied by low yield.

International applications WO 2015103928, WO 2018071919 describe options for solving the above problems, namely a heterodimeric IL-15 agonist protein consisting of a protein (I) and a protein (II), where the specified protein (I) is formed by IL-15 fused with the first option Fc, wherein the first Fc variant is associated with the C-terminus of IL-15, and the specified protein (II) is a second Fc variant or formed by IL-15Ra or a variant thereof fused to a second Fc variant, and the second Fc variant is associated with the C-terminus IL15Ra. Protein (I) and protein (II) form a stable heterodimeric protein through interaction to form a Knob-to-Hole structure between the first Fc variant and the second Fc variant.

The development of IL-15-based immunocytokines is of particular interest for combining the beneficial properties of tumor-specific antibodies that target tumors with the immunomodulatory effects of interleukin 15.

International applications WO 2015018528, WO2018071918 describe an immunocytokine containing a conjugate and an immunomodulatory antibody or fragment thereof, directly or indirectly linked by a covalent bond to the specified conjugate, where the specified conjugate contains a polypeptide containing the amino acid sequence of interleukin-15 or its derivatives, and a polypeptide containing the amino acid sequence sushi domain IL-15Ra or its derivatives.

International application WO 2012175222 describes an immunocytokine for the treatment of cancer, which contains a conjugate and an antibody or fragment thereof, which is selected from the group consisting of a Fab fragment, Fab' fragment, F(ab') ₂ fragment, Facb, Fd, and scFv, wherein specified the conjugate includes a polypeptide containing the amino acid sequence of interleukin 15 and a polypeptide containing the amino acid sequence of the sushi domain of IL-15Ra. The conjugate and the antibody or its fragment are covalently linked using bifunctional cross-linking agents that ensure protein attachment.

International application WO 2019006472 describes a bifunctional heterodimer protein containing an IL-15/IL-15Ra fusion protein, which includes an IL-15Ra protein, an IL-15 protein and a first Fc domain; and an antigen binding domain monomer that binds antigen selected from the group consisting of human CD8, human NKG2A and human NKG2D, comprising 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 constant light chain (CL), wherein said first and said second Fc domains have a set of amino acid substitutions selected from the group consisting of

S267K/L368D/K370S:S267K/S364K/E357Q;

S364K/E357Q:L368D/K370S;

L368D/K370S:S364K;

L368E/K370S:S364K;

Т411T/K360E/Q362E:D401K;

L368D/K370S:S364K/E357L and

K370S:S364K/E357Q.

Despite the above-mentioned prior art solutions for the complex formed by IL-15 and its receptor IL-15Ra, there is still a need for an IL15-based molecule that will have high stability, prolonged half-life in vivo, increased biological

- 2 043937 in vivo activity and increased productivity in mammalian cells.

The new immunocytokine format developed by the present inventors, which includes heterodimeric molecules based on IL-15 and IL-15Ra, exhibits higher stability, increased productivity in mammalian cells, prolonged half-life in vivo and increased biological activity in vivo. In addition, they represent a universal format for a bispecific immunocytokine with IL15 activity. The new immunocytokine format developed by the authors of this invention has reduced toxicity according to the results of preclinical studies.

Brief description of the invention

In one aspect, the present invention relates to an immunocytokine for promoting the activation and/or proliferation of IL-15Rbeta/gamma positive cells, which includes a heterodimeric protein complex based on IL-15 and IL-15Ra, containing:

1) IL-15Ra, which is associated with:

a) the constant domain of the antibody light chain; or

b) constant domains of the antibody heavy chain, including the first (CH1) constant domain of the heavy chain and an Fc fragment monomer containing the second (CH2) and third (CH3) constant domains of the heavy chain;

2) IL-15, which is associated with:

a) antibody heavy chain constant domains, including the first (CH1) heavy chain constant domain and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains, or

b) the constant domain of the antibody light chain;

wherein the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain in the heterodimer complex have or do not have a covalent association through a natural S-S bridge and, where, if IL-15 or IL-15Ra is associated with the constant domain of the antibody light chain, then the other a portion of the heterodimeric protein complex selected from IL-15Ra or IL-15 is associated with the constant domains of the antibody heavy chain.

In some embodiments, the immunocytokine includes a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex that are covalently associated through a natural S-S bridge.

In some embodiments, the immunocytokine includes 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 natural S-S bridge.

In some embodiments, the immunocytokine includes an antibody light chain constant domain selected from CK or CL.

In some embodiments, the immunocytokine includes IL-15Ra with the amino acid sequence represented by SEQ ID NO: 9 or any known variant of IL-15Ra containing mutations with similar biological activity.

In some embodiments, the immunocytokine includes IL-15 that has the amino acid sequence represented by SEQ ID NO: 10 or any known variant of IL-15 containing mutations with similar biological activity.

In some embodiments, the immunocytokine includes IL-15Ra, which is bound to the light chain constant domain of the antibody.

In some embodiments, the immunocytokine comprises IL-15Ra linked to an antibody light chain constant domain that has the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19.

In some embodiments, the immunocytokine includes IL-15, which is bound to the light chain constant domain of the antibody.

In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain that has the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 21.

In some embodiments, the immunocytokine includes a first (CH1) heavy chain constant domain and an Fc moiety monomer comprising a second (CH2) and third (CH3) heavy chain constant domain that are connected via a hinge.

In some embodiments, the immunocytokine includes IL-15 or IL-15Ra, which is associated with antibody heavy chain constant domains in the order CH1-hinge-CH2-CH3.

In some embodiments, the immunocytokine includes IL-15Ra, which is associated with the constant domains of the antibody heavy chain.

In some embodiments, the immunocytokine comprises IL-15Ra linked to antibody heavy chain constant domains that have the amino acid sequence set forth in SEQ

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ID NO: 4 or SEQ ID NO: 22.

In some embodiments, the immunocytokine includes IL-15, which is bound to the constant domains of the antibody heavy chain.

In some embodiments, the immunocytokine comprises IL-15 linked to antibody heavy chain constant domains that have the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 20.

In some embodiments, the immunocytokine includes

IL-15Ra linked to an antibody light chain constant domain having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19, and

IL-15 linked to antibody heavy chain constant domains having the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 20.

In some embodiments, the immunocytokine includes:

IL-15Ra linked to antibody heavy chain constant domains having the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 22, and

IL-15 linked to an antibody light chain constant domain having the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 21.

In some embodiments, the immunocytokine has mutations in the monomer of the Fc fragment, resulting in the absence of ADCC, CDC and/or ADCP properties of the specified immunocytokine.

In some embodiments, the immunocytokine includes an Fc moiety that is classified as IgG.

In some embodiments, the immunocytokine includes an Fc fragment that is selected from the group: human IgG1, IgG2, or IgG4.

In some embodiments, the immunocytokine includes the above two heterodimeric protein complexes based on IL-15 and IL-15Ra.

In some embodiments, the immunocytokine is used as a therapeutic agent for the treatment of cancer or an autoimmune disease.

In some embodiments, the immunocytokine is used as a therapeutic agent for cancer therapy.

In one aspect, the present invention relates to an immunocytokine for promoting the activation and/or proliferation of IL-15Rbeta/gamma positive cells, which includes a heterodimeric protein complex based on IL-15 and IL-15Ra and an immunomodulatory antibody or antigen binding fragment thereof that specifically inhibits PD-1 pathway, where a heterodimeric protein complex based on IL-15 and IL-15Ra contains:

1) IL-15Ra, which is associated with:

a) the constant domain of the antibody light chain; or

b) constant domains of the antibody heavy chain, including the first (CH1) constant domain of the heavy chain and an Fc fragment monomer containing the second (CH2) and third (CH3) constant domains of the heavy chain;

2) IL-15, which is associated with:

a) antibody heavy chain constant domains, including the first (CH1) heavy chain constant domain and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains, or

b) the constant domain of the light chain of the antibody, wherein the first constant domain of the heavy chain of the antibody and the constant domain of the light chain of the antibody as part of the heterodimer complex have or do not have a covalent association through a natural S-S bridge and, where, if IL-15 or IL-15Ra is associated with the antibody light chain constant domain, then another part of the heterodimeric protein complex selected from IL-15Ra or IL-15 is associated with the antibody heavy chain constant domains.

In some embodiments, the immunocytokine includes a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex that are covalently associated through a natural S-S bridge.

In some embodiments, the immunocytokine includes 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 natural S-S bridge.

In some embodiments, the immunocytokine includes an antibody light chain constant domain selected from CK or CL.

In some embodiments, the immunocytokine includes IL-15Ra that has the amino acid sequence represented by SEQ ID NO: 9 or any known mutation-containing variant of IL-15Ra with similar biological activity.

In some embodiments, the immunocytokine comprises IL-15, which has the amino acid sequence represented by SEQ ID NO: 10, or any known variant of IL-15 containing mutations, with similar biological activity.

In some embodiments, the immunocytokine includes IL-15Ra, which is bound to the light chain constant domain of the antibody.

In some embodiments, the immunocytokine comprises IL-15Ra linked to an antibody light chain constant domain that has the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19.

In some embodiments, the immunocytokine includes IL-15, which is bound to the light chain constant domain of the antibody.

In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain that has the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 21.

In some embodiments, the immunocytokine includes a first (CH1) heavy chain constant domain and an Fc moiety monomer comprising a second (CH2) and third (CH3) heavy chain constant domain that are connected via a hinge.

In some embodiments, the immunocytokine includes IL-15 or IL-15Ra, which is associated with antibody heavy chain constant domains in the order CH1-hinge-CH2-CH3.

In some embodiments, the immunocytokine includes IL-15Ra, which is associated with the constant domains of the antibody heavy chain.

In some embodiments, the immunocytokine comprises IL-15Ra linked to antibody heavy chain constant domains that have the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 24.

In some embodiments, the immunocytokine includes IL-15, which is bound to the constant domains of the antibody heavy chain.

In some embodiments, the immunocytokine comprises IL-15 linked to antibody heavy chain constant domains that have the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 23.

In some embodiments, the immunocytokine includes 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.

In some embodiments, the immunocytokine includes an immunomodulatory antibody that includes:

a) a light chain containing a light chain variable domain and a light chain constant domain;

b) a heavy chain containing a heavy chain variable domain and constant domains of the antibody heavy chain, including the first (CH1) constant domain of the heavy chain and an Fc fragment monomer containing the second (CH2) and third (CH3) constant domains of the heavy chain.

In some embodiments, the immunocytokine includes a light chain variable domain that includes LCDRs 1, 2, and 3 (hypervariable regions 1, 2, and 3), which are respectively represented by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO : 16.

In some embodiments, the immunocytokine includes a light chain variable domain that includes the amino acid sequence represented by SEQ ID NO: 18.

In some embodiments, the immunocytokine includes a heavy chain variable domain that includes HCDR 1, 2, and 3 (hypervariable regions 1, 2, and 3), which are respectively represented by the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO : 13.

In some embodiments, the immunocytokine includes a heavy chain variable domain that includes the amino acid sequence represented by SEQ ID NO: 17.

In some embodiments, the immunocytokine includes:

1) a light chain variable domain, which includes LCDR 1, 2 and 3 (hypervariable regions 1, 2 and 3), which are respectively represented by the amino acid sequences SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16;

2) a heavy chain variable domain, which includes HCDR 1, 2 and 3 (hypervariable regions 1, 2 and 3), which are respectively represented by the amino acid sequences SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

In some embodiments, the immunocytokine includes:

1) a light chain variable domain, which includes the amino acid sequence represented by SEQ ID NO: 18;

2) a heavy chain variable domain, which includes the amino acid sequence represented by SEQ ID NO: 17.

In some embodiments, the immunocytokine includes an immunomodulatory antibody that includes a light chain comprising the amino acid sequence represented by SEQ ID NO: 8.

In some embodiments, the immunocytokine includes an immunomodulatory antibody that includes a heavy chain comprising an amino acid sequence as represented by SEQ ID

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NO: 7.

In some embodiments, the immunocytokine includes an immunomodulatory antibody that includes a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 7;

a light chain containing the amino acid sequence represented by SEQ ID NO: 8.

In some embodiments, the immunocytokine includes an immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway, which is an antibody that specifically binds PD-L1.

In some embodiments, the immunocytokine includes:

a) a heterodimeric protein complex based on IL-15 and IL-15Ra, which contains

IL-15Ra linked to an antibody light chain constant domain having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19, and

IL-15 linked to antibody heavy chain constant domains having the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 23;

b) an immunomodulatory antibody that includes a heavy chain containing the amino acid sequence represented by SEQ ID NO: 7;

a light chain containing the amino acid sequence represented by SEQ ID NO: 8.

In some embodiments, the immunocytokine includes:

a) IL-15Ra linked to antibody heavy chain constant domains that have the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 24, and IL-15 linked to antibody light chain constant domains that have the amino acid sequence which is represented by SEQ ID NO: 3 or SEQ ID NO: 21;

b) an immunomodulatory antibody that includes a heavy chain containing the amino acid sequence represented by SEQ ID NO: 7;

a light chain containing the amino acid sequence represented by SEQ ID NO: 8.

In some embodiments, the immunocytokine has mutations in the antibody constant domains causing heterodimerization of two different parts, one of which includes covalently or non-covalently associated IL-15Ra bound to the antibody constant domains and IL-15 bound to the antibody constant domain, and the other includes covalently associated or non-covalently associated light and heavy chains of an antibody.

In some embodiments, the immunocytokine comprises a first Fc monomer and a second Fc monomer that are selected from the group: the first Fc monomer is an Fc modified to form a Knob and the second Fc monomer is an Fc modified to form a Hole, or where the second Fc monomer is Fc modified to form Knob, and the first Fc monomer is Fc modified to form Hole.

In some embodiments, the immunocytokine includes a first Fc monomer that has the amino acid substitutions S354C/T366W and a second Fc monomer that has the amino acid substitutions Y349C/T366S/L368A/Y407V.

In some embodiments, the immunocytokine includes a first Fc monomer that has the amino acid substitutions Y349C/T366S/L368A/Y407 and a second Fc monomer that has the amino acid substitutions S354C/T366W.

In some embodiments, the immunocytokine includes an Fc moiety that is classified as IgG.

In some embodiments, an immunocytokine, wherein the Fc fragment isotype is selected from the group: human IgG1, IgG2, or IgG4.

In some embodiments, the immunocytokine includes mutations in the Fc fragment monomer resulting in the absence of ADCC, CDC and/or ADCP properties of the immunocytokine.

In some embodiments, the immunocytokine is used to treat cancer or an autoimmune disease.

In some embodiments, the immunocytokine is used to treat cancer.

In one aspect, the present invention provides an isolated nucleic acid that encodes any of the above immunocytokines.

In some embodiments, the nucleic acid is DNA.

In one aspect, the present invention relates to an expression vector that contains the above nucleic acid.

In one aspect, the present invention relates to a method of obtaining a host cell for producing the above immunocytokine, which includes transforming the cell with the above vector.

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In one aspect, the present invention relates to a host cell for producing the above immunocytokine, which contains the above nucleic acid.

In one aspect, the present invention relates to a method for producing a preparation containing the above immunocytokine, which consists of culturing the above host cells in a culture medium under conditions sufficient to obtain the specified immunocytokine, if necessary, followed by the isolation and purification of the resulting immunocytokine.

In one aspect, the present invention relates to a pharmaceutical composition for promoting the activation and/or proliferation of IL-15Rbeta/gamma positive cells, which contains the above immunocytokine in a therapeutically effective amount, in combination with one or more pharmaceutically acceptable excipients.

In some embodiments, the pharmaceutical composition is used to treat cancer or an autoimmune disease.

In some embodiments, the pharmaceutical composition is used to treat cancer.

In some embodiments, the pharmaceutical composition is used to treat a cancer selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer without an identified primary source, 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 CRC (colorectal cancer with high microsatellite instability), leukemia (acute or myeloblastic), lymphoma, multiple myeloma, melanoma , breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory B-cell non-Hodgkin lymphoma , follicular lymphoma, marginal zone B-cell lymphoma, large B-cell diffuse lymphoma, squamous cell carcinoma of the head and neck, pancreatic cancer, ovarian cancer, acute myeloid leukemia and high-risk myelodysplastic syndrome.

In one aspect, the invention relates to a method of treating cancer, which includes administering to a subject in need of such treatment, the above immunocytokine or the above pharmaceutical composition in a therapeutically effective amount.

In some embodiments of the treatment method, the cancer is selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer without an identified primary source, 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 CRC (colorectal cancer with high microsatellite instability), leukemia (acute or myeloblastic), lymphoma, multiple myeloma, melanoma, breast cancer , colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory B-cell non-Hodgkin lymphoma, follicular lymphoma, Marginal zone B-cell lymphoma, large cell diffuse lymphoma, squamous cell carcinoma of the head and neck, pancreatic cancer, ovarian cancer, acute myeloid leukemia and high-risk myelodysplastic syndrome.

In one aspect, the present invention relates to a method of activating the biological activity of a T cell population or NK cell population in a subject in need of such activation, which comprises administering to the subject an effective amount of the above immunocytokine or the above pharmaceutical composition.

In one aspect, the present invention relates to the use of the above immunocytokine or the above pharmaceutical composition for the treatment of a cancer in a subject in need of such treatment.

In some embodiments, the cancer is selected from the group of: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer without an identified primary source, 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 CRC (microsatellite instability-high colorectal cancer), leukemia (acute or myeloblastic), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory B-cell non-Hodgkin lymphoma, follicular lymphoma, B- marginal zone cell lymphoma, large cell diffuse lymphoma, squamous cell carcinoma of the head and neck, pancreatic cancer, ovarian cancer, acute myeloid leukemia and high-risk myelodysplastic syndrome.

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Brief description of the figures

Fig. 1. Immunocytokine format, including a heterodimeric protein complex based on IL15 and IL-15Ra (CK_IL15Ra_Hc_IL15CH1FcLALA).

Fig. 2. Immunocytokine format, including a heterodimeric protein complex based on IL15 and IL-15Ra (CK_IL15_Hc_IL15RaCH1FcLALA).

Fig. 3. An immunocytokine format that includes a heterodimeric protein complex based on IL15 and IL-15Ra and an immunomodulatory antibody that specifically inhibits the PD-1 pathway (FabCK_IL15_Hc_IL15RaCH1FcknobLALA). Knob refers to mutations that produce a Knob-to-Hole structure between the first Fc variant and the second Fc variant in the CH3 domain.

Fig. 4. An immunocytokine format that includes a heterodimeric protein complex based on IL15 and IL-15Ra and an immunomodulatory antibody that specifically inhibits the PD-1 pathway (FabCK_IL15Ra_Hc_IL15CH1FcknobLALA). Knob refers to mutations that produce a Knob-to-Hole structure between the first Fc variant and the second Fc variant in the CH3 domain.

Fig. 5. Vector pEE-IL15Ra-CK.

AmpR - beta-lactamase gene that provides resistance to ampicillin, pUC origin - pUC origin of replication in bacteria,

CMV-Promoter - promoter of early genes of cytomegalovirus, leader-leader sequence,

IL15Ra - gene encoding subunit a of the interleukin 15 receptor,

CK - gene encoding the light chain constant domain,

Poly A - polyadenylation signal sequence to increase mRNA stability,

OriP is the origin of replication in bacteria or the high-copy ColE1/pMB1/pBR322/pUC origin of replication in bacterial cells.

Fig. 6. Vector pEE-IL15-CH1-Fc-LALA.

AmpR - beta-lactamase gene that provides resistance to ampicillin, pUC origin - pUC origin of replication in bacteria,

CMV-Promoter - promoter of early genes of cytomegalovirus, leader-leader sequence,

IL15 - gene encoding interleukin 15,

CH1 - gene encoding the first constant domain of the heavy chain,

Fc-LALA - a gene encoding an Fc fragment with LALA mutations (mutations L234A and L235A in the CH2 constant domain of the Fc fragment),

Poly A - polyadenylation signal sequence to increase mRNA stability,

OriP is the origin of replication in bacteria or the high-copy ColE1/pMB1/pBR322/pUC origin of replication in bacterial cells.

Fig. 7. Vector pEE-BCD100-02-VL-CK.

AmpR - beta-lactamase gene that provides resistance to ampicillin, pUC origin - pUC origin of replication in bacteria,

CMV-Promoter - promoter of early genes of cytomegalovirus, leader-leader sequence, aPD1-VL - gene encoding the light chain of an antibody that specifically binds to PD1,

CK - gene encoding the light chain constant domain,

Poly A - polyadenylation signal sequence to increase mRNA stability,

OriP is the origin of replication in bacteria or the high-copy ColE1/pMB1/pBR322/pUC origin of replication in bacterial cells.

Fig. 8. Vector pEE-BCD100-02-VH-CH1-Fc-hole-LALA.

AmpR - beta-lactamase gene that provides resistance to ampicillin, pUC origin - pUC origin of replication in bacteria, promoter - promoter of early genes of cytomegalovirus, leader - leader sequence, aPD1-VH - gene encoding the variable domain of the heavy chain of an antibody that specifically binds to PD1,

CH1 - gene encoding the first constant domain of the heavy chain,

Fc-hole-LALA - a gene encoding an Fc fragment with mutations for the formation of hole and LALA (mutations L234A and L235A in the CH2 constant domain of the Fc fragment),

STOP - stop codon,

OriP is an origin of replication in bacteria or a high-copy ColE1/pMB1/pBR322/pUC origin of replication in bacterial cells.

Fig. 9. Vector pEE-IL15-CH1-Fc-knob-LALA.

AmpR - beta-lactamase gene that provides resistance to ampicillin, pUC origin - pUC origin of replication in bacteria,

- 8 043937

CMV-Promoter - promoter of early genes of cytomegalovirus,

Leader - leader sequence,

IL15 - gene encoding interleukin 15,

CH1 - gene encoding the first constant domain of the heavy chain,

Fc-knob-LALA - a gene encoding an Fc fragment with mutations for the formation of knob and LALA (mutations L234A and L235A in the CH2 constant domain of the Fc fragment),

STOP - stop codon,

OriP is an origin of replication in bacteria or a high-copy ColE1/pMB1/pBR322/pUC origin of replication in bacterial cells.

Fig. 10A. SDS gel electrophoresis with beta-mercaptoethanol.

1. Control antibody 5 µl.

2. Fermentas unstained marker.

3. -

4. Growth medium with CKIL15Ra_Hc_IL15CH1FcLALA before purification 10 µl.

5. Growth medium with CKIL15Ra_Hc_IL15CH1FcLALA after purification 10 µl.

6. Purified CKIL15Ra_Hc_IL15CH1FcLALA 10 µl.

7. -

8. Growth medium with CK_IL15_Hc_IL15RaCH1FcLALA before purification 10 µl.

9. Growth medium with CK_IL15_Hc_IL15RaCH1FcLALA after purification 10 µl.

10. Purified CK_IL15_Hc_IL15RaCH1FcLALA 10 µl.

Fig. 10B. SDS gel electrophoresis without beta-mercaptoethanol.

1. Purified CKIL15Ra_Hc_IL 15CH1FcLALA 10 µl.

2. Purified CK_IL15_Hc_IL15RaCH1FcLALA 10 µl.

3. Fermentas unstained marker

4. Control antibody 5 µl.

Fig. 11. Test to determine proliferation activity. The NK-92 cell line was used for the test. The test was carried out in a 96-well culture plate. The suspension contained NK-92 cells, the test antibody at the concentration indicated on the graph. All suspension components were prepared in RPMI-1640 medium containing fetal bovine serum and glutamine. After adding all components, the plates were incubated at 37°C, 5% CO ₂ . Next, Alamar Blue dye was added to the wells. After incubation, the fluorescence intensity in the wells was measured.

Fig. 12. Test to determine proliferation activity. For the test, we used isolated natural killer cells isolated from PBMCs of healthy donors using the negative selection method. The test was carried out in a 96-well culture plate. The suspension contained natural killer cells and the test antibody at the concentration indicated on the graph. All components of the suspension were prepared in a culture medium containing autologous human plasma. After adding all components, the plates were incubated at 37°C, 5% CO ₂ . Next, Alamar Blue dye was added to the wells. After incubation, the fluorescence intensity in the wells was measured.

Fig. 13. Test for determination of aPD1 spec. activity. For the test, we used the Jurkat NFAT-FLuc PD-1 cell line, created on the basis of the Jurkat cell line, stably expressing PD-1 on the surface and containing the gene encoding firefly luciferase under the control of the NFAT promoter; and the Raji PDL1 cell line, derived from the Raji cell line, stably expressing PDL1 on the surface. The test was carried out in a 96-well culture plate. The suspension in each well contained Jurkat NFAT-FLuc PD-1 cells, Raji PDL1 cells, aCD3/aTAA1 at a concentration of 1 ng/ml, and the test antibody at the concentration indicated on the graph. After adding all components, the plates were incubated at 37°C, 5% CO ₂ and then, using a luminescence assessment kit, the intensity of lucifera in the wells was measured.

Fig. 14. Analysis of the specific activity of anti-PDL1/IL15SA antibodies on a reporter cell line.

Fig. 15. Test to determine the effect of natural killer cells on ADCC. For the test, natural killer cells isolated from PBMCs of healthy donors using the negative selection method were used. The test was carried out 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 concentration indicated on the graph. All suspension components were prepared in a culture medium containing fetal bovine serum and glutamine. After adding all components, the plates were incubated at 37°C, 5% CO2. Next, the culture liquid was collected and the lactate dehydrogenase content was examined using an LDH determination kit.

Fig. 16. Results of testing the specific activity of immunocytokines in relation to enhancing the ADCC ability of rituximab against the TAA reporter line.

Fig. 17. Study of cytotoxicity for immunocytokine variants, including a heterodimeric protein complex based on IL-15 and IL-15Ra (IL15SA), on NK cells. The results indicate the absence of a significant cytotoxic effect of the candidates, that is, no more than a 10% decrease in the number of immunoreactive cells in comparison with the negative control AB (absence of any exogenous antibodies).

Fig. 18. Cytotoxicity study for IL15SA variants on B cells. The results indicate the absence of a significant cytotoxic effect of the candidates, that is, no more than a 10% decrease in the number of immunoreactive cells in comparison with the negative control AB (absence of any exogenous antibodies).

Fig. 19. Cytotoxicity study for IL15SA variants on CD3+ cells. The results indicate the absence of a significant cytotoxic effect of the candidates, that is, no more than a 10% decrease in the number of immunoreactive cells in comparison with the negative control AB (absence of any exogenous antibodies).

Fig. 20. Cytotoxicity study for IL15SA variants on CD4+ cells. The results indicate the absence of a significant cytotoxic effect of the candidates, that is, no more than a 10% decrease in the number of immunoreactive cells in comparison with the negative control AB (absence of any exogenous antibodies).

Fig. 21. Cytotoxicity study for IL15SA variants on CD8+ cells. The results indicate the absence of a significant cytotoxic effect of the candidates, that is, no more than a 10% decrease in the number of immunoreactive cells in comparison with the negative control AB (absence of any exogenous antibodies).

Fig. 22. Chromatogram. This chromogram demonstrates the high homogeneity (the content of aggregates in the solution was no more than 5%) of the immunocytokine, which includes a heterodimeric protein complex based on IL-15 and IL-15Ra (IL15 superagonist).

Fig. 23. Scheme of an experiment to determine the antitumor activity of immunocytokine preparations, including a heterodimeric protein complex based on IL-15 and IL-15Ra (BCD-225) in an in vivo mouse tumor model.

Fig. 24. Results of an experiment to determine the antitumor activity of variants of an immunocytokine drug, including a heterodimeric protein complex based on IL-15 and IL-15Ra (BCD-225) (graph) in an in vivo mouse tumor model.

Fig. 25. Results of an experiment to determine the antitumor activity of variants of an immunocytokine drug, including a heterodimeric protein complex based on IL-15 and IL-15Ra (BCD-225) (diagram) in an in vivo mouse tumor model.

Description of the invention

General definitions and general methods.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have meanings that are commonly understood by those skilled in the art.

In addition, unless the context otherwise requires, terms in the singular include plural terms, and plural terms include singular terms. In general, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry, and hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art. widely used in this field. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is typically carried out in the art, or as described herein.

Unless specifically stated otherwise, the expressions biologically active, and biological activity, and biological characteristics, in relation to a polypeptide of this invention, mean having the ability to bind to a biological molecule.

The term recombinant protein means a protein (polypeptide) that is expressed in a cell or cell line containing the nucleotide sequence(s) that encodes the protein, wherein the nucleotide sequence(s) are not naturally associated with the cell.

The term bispecific antibody means an antibody containing an antigen binding domain or antigen binding domains that are capable of specifically binding to two different epitopes on one biological molecule or capable of specifically binding to epitopes on two different biological molecules. A bispecific antibody is also referred to herein as having dual specificity or being a dual-specific antibody.

The term monoclonal antibody or mAb refers to an antibody that is 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, which are typically produced by fusing individual B lymphocytes from immunized animals with individual lymphocytic tumor cells. Hybridomas are a type of engineered cell and do not occur in nature.

The term Ka, as used herein, refers to the rate of association of a particular antibody-antigen interaction.

The term Kd, as used herein, refers to the rate of dissociation of a particular antibody-antigen interaction.

Binding affinity generally refers to the strength of the aggregate non-covalent interactions between a single binding site of a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise stated, binding affinity refers to the intrinsic (true) binding affinity, which reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can usually be represented by the dissociation constant (Kd). It is desirable that the Kd value is about 200, 150, 100, 60, 50, 40, 30, 20, 10, 8, 6, 4, 2, 1 nM or less. Affinity can be measured by conventional methods known in the art, including methods described herein. Various methods for measuring binding affinity are known in the art, any of these methods can be used for the purposes of the present invention.

The term peptide linker as used herein means any peptide capable of connecting domains of length depending on the domains it links, containing any amino acid sequence. Preferably, the peptide linker is greater than 5 amino acids in length and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.

The term in vitro refers to a biological entity, biological process, or biological reaction outside the body, simulated under artificial conditions. For example, in vitro cell growth should be understood as the growth of cells in an environment outside the body, such as a test tube, culture flask, or microplate.

In the present specification and in the following claims, unless the context otherwise requires, the words have, include and contain, or variations thereof such as has, having, includes, including, contains or containing, are to be understood to include the specified whole or group of wholes, but not to the exclusion of any other whole or group of wholes.

Detailed Description of the Invention

Immunocytokine.

In one aspect, the present invention relates to an immunocytokine for promoting the activation and/or proliferation of IL-15Rbeta/gamma positive cells, which includes a heterodimeric protein complex based on IL-15 and IL-15Ra, containing:

1) IL-15Ra, which is associated with:

a) the constant domain of the antibody light chain; or

b) constant domains of the antibody heavy chain, including the first (CH1) constant domain of the heavy chain and an Fc fragment monomer containing the second (CH2) and third (CH3) constant domains of the heavy chain;

2) IL-15, which is associated with:

a) constant domains of the antibody heavy chain, including the first (CH1) heavy chain constant domain and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; or

b) the constant domain of the light chain of the antibody, wherein the first constant domain of the heavy chain of the antibody and the constant domain of the light chain of the antibody as part of the heterodimer complex have or do not have a covalent association through a natural S-S bridge and, where, if IL-15 or IL-15Ra is associated with the antibody light chain constant domain, then another part of the heterodimeric protein complex selected from IL-15Ra or IL-15 is associated with the antibody heavy chain constant domains.

The format of the above immunocytokine is shown in FIG. 1 and 2.

The term immunocytokine refers to a molecule containing an antibody or fragments thereof, directly or indirectly linked by covalent bond to a cytokine or its derivatives. Said antibody and said cytokine may be linked using a linker peptide.

By immunocytokine according to the invention is meant an isolated immunocytokine.

The term isolated as used to describe the various immunocytokines herein means an immunocytokine that has been identified and isolated and/or recovered from the cell or cell culture in which it is expressed. Impurities (contaminants) from the natural environment are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes

- 11 043937 hormones and other protein or non-protein solutes. In preferred embodiments, the immunocytokine is purified (1) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spin glass sequencer (Edman sequencer), or (2) to homogeneity by SDSPAGE in non-reducing or reducing conditions using Coomassie brilliant blue or preferably silver staining. The isolated immunocytokine includes the immunocytokine in situ within the recombinant cells, since at least one component of the polypeptide's natural environment is missing. Typically, the isolated polypeptide is obtained through at least one purification step.

As used herein, a heterodimeric protein complex refers to a protein formed by a combination of IL-15 and IL-15Ra.

As used herein, IL-15, IL-15 peptide, or interleukin-15 may be any IL-15 (interleukin-15) or a mutant form thereof, such as human or non-human mammalian IL-15, or IL-15. 15 is not a mammal. Illustrative mammals other than humans include mammals such as pigs, rabbits, monkeys, chimpanzees, mice and the like; non-mammals include such as chickens and the like. Preferably, the mature human interleukin-15 molecule is found in the UniProtKB database, accession number P40933, 49-162aa. The term IL-15 variant refers to a variant of a molecule having increased or decreased affinity for its receptor or increased or decreased activity upon stimulation of T cells or NK cells as a result of one or more amino acid substitutions, additions or deletions.

IL-15Ra means interleukin 15 receptor α subunit and, according to the present invention, can be any IL-15Ra compound or functional fragment thereof, such as human IL-15Ra or non-human mammalian IL-15Ra or non-mammalian IL-15Ra . Illustrative mammals other than humans include mammals such as pigs, rabbits, monkeys, chimpanzees, mice and the like; non-mammals include such as chickens and the like. The preferred IL-15Ra molecule is found in the UniProt database, accession number Q13261 (https://www.uniprot.org/uniprot/Q13261).

A crystallizable fragment of an immunoglobulin (fragment crystallizable region, Fc region, Fc) is the terminal part of an immunoglobulin molecule that interacts with the Fc receptor on the cell surface and with some proteins of the complement system. This property allows antibodies to activate the immune system. The Fc region of IgG, IgA and IgD isotypes consists of two identical protein fragments, respectively, the second and third constant domains of two heavy chains; in the case of the IgM and IgE isotypes, Fc contains three heavy chain constant domains (CH2, CH3 and CH4 domains) in each polypeptide chain.

By Fc fragment monomer is meant the Fc region from the second and third constant domains of any one of the two heavy chains (for IgG, IgA and IgD isotypes); in the case of the IgM and IgE isotypes, the Fc monomer contains three constant domains of one of the two heavy chains (CH2, CH3 and CH4 domains).

In some embodiments, the immunocytokine includes a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex that are covalently associated through a natural S-S bridge.

In some embodiments, the immunocytokine includes 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 natural S-S bridge.

In some embodiments, the immunocytokine includes an antibody light chain constant domain selected from CK or CL.

In mammals, only two types of light chains are known, which are designated lambda (λ) and kappa (k). The light chain lambda constant domain is designated CL, and the light chain kappa constant domain is designated CK.

In some embodiments, the immunocytokine includes IL-15Ra with an amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP

SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASS (SEQ IDNO:9) or any known variant of IL-15Ra containing mutations with similar biological activity.

By IL-15Ra that contains mutations with similar biological activity to IL-15Ra is meant any IL-15Ra known in the art that contains mutations and has similar biological activity to wild-type IL-15Ra.

In some embodiments, the immunocytokine includes IL-15, which has an amino acid sequence that is represented by

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DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI

HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID

NO: 10) or any known variant of IL-15 containing mutations with similar biological activity.

By IL-15 that contains mutations with similar biological activity to IL-15 is meant any IL-15 known in the art that contains mutations and has similar biological activity to wild-type IL-15.

In some embodiments, the immunocytokine includes IL-15Ra, which is bound to the light chain constant domain of the antibody.

In some embodiments, the immunocytokine comprises IL-15Ra linked to an antibody light chain constant domain that has an amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPAL VHQRPAPPST VTTAGVTPQPESLSP SGKEPAAS SGAEGGGRTVAAP S VFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:1).

In some embodiments, the immunocytokine comprises IL-15Ra linked to an antibody light chain constant domain that has an amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPAL VHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEA (SEQ ID NO: 19).

The amino acid sequence, which is represented by SEQ ID NO: 19, differs from the amino acid sequence, which is represented by SEQ ID NO: 1, by one amino acid substitution of Cysteine (C) to Alanine (A) at position 216. This replacement makes it possible to obtain an immunocytokine that includes the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain as part of a heterodimeric complex, which do not have a covalent association through a natural S-S bridge.

In some embodiments, the immunocytokine includes IL-15, which is bound to the light chain constant domain of the antibody.

In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain that has an amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:3).

In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain that has an amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEA (SEQ ID NO:21).

The amino acid sequence, which is represented by SEQ ID NO: 21, differs from the amino acid sequence, which is represented by SEQ ID NO: 3, by one amino acid substitution of Cysteine (C) to Alanine (A) at position 233. This replacement makes it possible to obtain an immunocytokine that includes the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain as part of a heterodimeric complex, which do not have a covalent association through a natural S-S bridge.

In some embodiments, the immunocytokine includes a first (CH1) heavy chain constant domain and an Fc moiety monomer comprising a second (CH2) and third (CH3) heavy chain constant domain that are connected via a hinge.

In some embodiments, the immunocytokine includes IL-15 or IL-15Ra, which is associated with antibody heavy chain constant domains in the order CH1-hinge-CH2-CH3.

In some embodiments, the immunocytokine includes IL-15Ra, which is associated with the constant domains of the antibody heavy chain.

In some embodiments, the immunocytokine includes IL-15Ra linked to antibody heavy chain constant domains that have an amino acid sequence that is represented by

- 13 043937

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:4).

In some embodiments, the immunocytokine includes IL-15Ra linked to antibody heavy chain constant domains that have an amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGASTKGPSVFPL APS SKSTSGGT AALGCLVKD YFPEPVTVSWNSGALT SGVHTFP AVLQS SGL YSLS S VVT VPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSADKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTNQVS LTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:22).

The amino acid sequence, which is represented by SEQ ID NO: 22, differs from the amino acid sequence, which is represented by SEQ ID NO: 4, by one amino acid substitution of Cysteine (C) to Alanine (A) at position 212. This replacement makes it possible to obtain an immunocytokine that includes the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain as part of a heterodimeric complex, which do not have a covalent association through a natural S-S bridge.

In some embodiments, the immunocytokine includes IL-15, which is bound to the constant domains of the antibody heavy chain.

In some embodiments, the immunocytokine comprises IL-15 linked to antibody heavy chain constant domains that have an amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDT VENLIIL AND SL S SNGNVTES GCKECEELEEKNIKEFLQ SF VHIVQMFINT S AGGKPG GSAGGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:2).

In some embodiments, the immunocytokine comprises IL-15 linked to antibody heavy chain constant domains that have an amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSADKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:20).

The amino acid sequence, which is represented by SEQ ID NO: 20, differs from the amino acid sequence, which is represented by SEQ ID NO: 2, by one amino acid substitution of Cysteine (C) to Alanine (A) at position 229. This replacement makes it possible to obtain an immunocytokine that includes the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain as part of a heterodimeric complex, which do not have a covalent association through a natural S-S bridge.

In some embodiments, the immunocytokine includes

IL-15Ra bound to the light chain constant domain of an antibody that has the amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC (SEQ ID NO:1) or DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGRTVAAPSVFIF

- 14 043937

PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEA (SEQ ID NO: 19), a IL-15 linked to antibody heavy chain constant domains that have the amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:2) or DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSADKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:20).

In some embodiments, the immunocytokine includes

IL-15Ra bound to the light chain constant domain of an antibody that has the amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC (SEQ III NO:1), a IL-15 linked to antibody heavy chain constant domains that have the amino acid sequence represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ Ш NO:2).

In some embodiments, the immunocytokine includes

IL-15Ra bound to the light chain constant domain of an antibody that has the amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEA (SEQ ID NO: 19), a IL-15 linked to antibody heavy chain constant domains that have the amino acid sequence represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSADKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:20).

In some embodiments, the immunocytokine includes

IL-15Ra bound to antibody heavy chain constant domains that have the amino acid sequence represented by

- 15 043937

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:4) or

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSADKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:22), a IL-15 linked to the constant domain of an antibody light chain, which have the amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:3) or

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEA (SEQ ID NO:21). In some embodiments, the immunocytokine includes: IL-15Ra bound to antibody heavy chain constant domains that have an amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:4), and IL-15 linked to the light chain constant domain of an antibody, which have the amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:3).

In some embodiments, the immunocytokine includes

IL-15Ra bound to antibody heavy chain constant domains that have the amino acid sequence represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSADKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:22), and IL-15 bound to the light chain constant domain of an antibody, which have the amino acid sequence that is represented by

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DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI

HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG

GSAGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE

SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEA (SEQ ID

NO:21).

In some embodiments, the immunocytokine has mutations in the monomer of the Fc fragment, resulting in the absence of ADCC, CDC and/or ADCP properties of the specified immunocytokine.

Under mutations in the Fc fragment, modification(s) of the amino acid sequences of the antibodies described in this application are proposed. Antibody amino acid sequence variants are produced by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletion, insertion and substitution is performed to produce the final construct, provided that the final construct has the required characteristics.

An option for modifying the amino acid sequences of antibodies 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 the heading Preferred Substitutions.

Table. A Original balance Replacement examples Preferred Substitutions Ala (A) Vai; Leu.; He Vai Arg(R) Lys; Gin.; Asn Lys Asn(N) Gin.; His; Asp, Lys; Arg Gin Asp (D) Glu.; Asn. Glu Cys(C) Ser; Ala Ser Gln(Q) Asn.; Glu. Asn. Glu. (E) Asp; Gin. Asp Gly(G) Ala Ala His(H) Asn.; Gin.; Lys; Arg Arg He(I) Leu.; Vai; Met; Ala; Ph; Norleyschln Leu Leu. (L) Norleyschln; He; Vai; Met; Ala; Phe lie Lys (K) Arg; Gin.; Asn. Arg Met(M) Leu.; Ph; lie Leu Phe(F) Trp; Leu.; Vai; lie; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr(t) Vai; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Ph; Thr; Ser Phe Vai (V) lie; Leu; Met; Ph; Ala; Norleucine Leu

The term antibody effector function refers to the biological activities associated with the Fc region (native Fc region sequence or Fc region amino acid sequence variants) of an antibody and varies depending on the isotype of the antibody. Examples of antibody effector functions are: Clq binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor, BCR) and B cell activation.

Antibody-dependent cellular cytotoxicity or ADCC refers to a cell-mediated response in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on the target cell and then cause lysis or phagocytosis of the target cell. The primary cells for mediating ADCC, NK cells, express only FcyRIII, whereas monocytes express FcyRI, FcyRII, and FcyRIII. 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 the ADCC activity of a molecule of interest, in vitro ADCC assays such as

- 17 043937 assays described in US patent No. 5500362 or 5821337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as the model described in Clynes et al. PNAS (USA) 95: 652-656 (1998).

Human effector cells are leukocytes that express one or more FcRs and perform effector functions. Preferably the cells express at least FcyRIII and exert 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 can be isolated from their natural source, for example from blood or PBMC, as described herein.

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 (Clq) to a molecule (eg, antibody) complexed with its antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

In some embodiments, the immunocytokine includes an Fc moiety that is classified as IgG.

In some embodiments, the immunocytokine includes an Fc fragment that is selected from the group: human IgG1, IgG2, or IgG4.

In some embodiments, the immunocytokine includes the above two heterodimeric protein complexes based on IL-15 and IL-15Ra.

IL15 superagonist (otherwise known as BCD225 or IL15SA or (IL15SA)2-Fc) of the present invention means CK_IL15Ra_Hc_IL15CH1FcLALA and CKIL15_Hc_IL15RaCH1FcLALA.

The designations CK, IL15Ra, IL15, CH1FcLALA are explained in the text of the application.

CK_IL15Ra_Hc_IL15CH1FcLALA is the above immunocytokine comprising two heterodimeric protein complexes based on IL-15 and IL-15Ra, which include IL15Ra, which is associated with the antibody light chain constant domain CK and IL15, which is associated with the antibody heavy chain constant domains including the first (CH1 ) heavy chain constant domain and Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains, where Fc contains LALA mutations.

CK_IL15_Hc_IL15RaCH1FcLALA is the above immunocytokine comprising two heterodimeric protein complexes based on IL-15 and IL-15Ra, which include IL15, which is associated with the CK antibody light chain constant domain, and IL15Ra, which is associated with the antibody heavy chain constant domains, including the first ( CH1) heavy chain constant domain and Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains, where Fc contains LALA mutations.

The above immunocytokines can be made both in a format where the first constant domain of the heavy chain of the antibody and the constant domain of the light chain of the antibody as part of a heterodimeric complex have a covalent association through a natural S-S bridge, and in a format where the first constant domain of the heavy chain of the antibody and the constant domain of the light chain The antibody chains in the heterodimer complex do not have a covalent association through a natural S-S bridge; in a particular case, for this, both cessteins are replaced with alanine.

In experimental studies of the above immunocytokines, both in a format where the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain as part of a heterodimer complex have a covalent association through a natural S-S bridge, and in a format where the first constant domain of the antibody heavy chain and the constant domain of the light chain antibodies in the heterodimer complex do not have covalent association through a natural S-S bridge, no significant difference in the activity of the above immunocytokines was detected.

The format of the above immunocytokine is shown in FIG. 1 and 2.

In some embodiments, the immunocytokine is used as a therapeutic agent for the treatment of cancer or an autoimmune disease.

In some embodiments, the immunocytokine is used as a therapeutic agent for cancer therapy.

In one aspect, the present invention relates to an immunocytokine for promoting the activation and/or proliferation of IL-15Rbeta/gamma positive cells, which includes a heterodimeric protein complex based on IL-15 and IL-15Ra and an immunomodulatory antibody or antigen binding fragment thereof that specifically inhibits PD-1 pathway, where a heterodimeric protein complex based on IL-15 and IL-15Ra contains:

1) IL-15Ra, which is associated with:

a) the constant domain of the antibody light chain; or

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b) constant domains of the antibody heavy chain, including the first (CH1) constant domain of the heavy chain and an Fc fragment monomer containing the second (CH2) and third (CH3) constant domains of the heavy chain;

2) IL-15, which is associated with:

a) constant domains of the antibody heavy chain, including the first (CH1) heavy chain constant domain and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; or

b) the constant domain of the light chain of the antibody, wherein the first constant domain of the heavy chain of the antibody and the constant domain of the light chain of the antibody as part of the heterodimer complex have or do not have a covalent association through a natural S-S bridge and, where, if IL-15 or IL-15Ra is associated with the antibody light chain constant domain, then another part of the heterodimeric protein complex selected from IL-15Ra or IL-15 is associated with the antibody heavy chain constant domains.

The term antibody or immunoglobulin (Ig) as used herein includes whole antibodies and any antigen-binding fragment (ie, antigen-binding portion) or individual chains thereof. The term antibody refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. There are five known types of mammalian antibody heavy chains, which are designated by Greek letters: α, δ, ε, γ and μ. The type of heavy chain present determines the class of the antibody; these chains are found in antibodies of the IgA, IgD, IgE, IgG and IgM types, respectively. Different heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, and μ and ε consist of approximately 550 amino acids. Each heavy chain contains two regions, i.e. constant region and variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The heavy chains γ, α and δ contain a constant region, which consists of three constant domains CH1, CH2 and CH3 (arranged in a row) and a hinge region, which provides flexibility (Woof J., Burton D., Nat. Rev. Immunol. 4 , 2004, cc.89-99); The μ and ε heavy chains contain a constant region, which consists of four constant domains CH1, CH2, CH3 and CH4. In mammals, only two types of light chains are known, which are designated lambda (λ) and kappa (k). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The approximate length of the light chain is 211-217 amino acids.

Preferably the light chain is a kappa (k) light chain and the CL constant domain is preferably a C-kappa (k) chain.

The antibody of the invention may be of any class (eg, IgA, IgD, IgE, IgG and IgM, preferably IgG) or subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1).

The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed between regions that are more conserved called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (Clq) of the classical complement system.

The term antigen-binding portion of an antibody or antigen-binding fragment (or simply antibody portion or antibody fragment) as used herein refers to one or more antibody fragments that retain the ability to specifically bind 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 included in 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) F(ab') ₂ fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment, consisting of VL and VH domains in a single antibody arm, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH/VHH domain; and (vi) a dedicated complementarity determining region (CDR). In addition, the two regions of the Fv fragment, VL and VH, are encoded by different genes; they can be joined by recombinant methods using a synthetic linker, which makes it possible to obtain them in the form of a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single-chain

- 19 043937

Fv(scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci.

USA 85:5879-5883). It is intended that such single chain molecules are also included in the term antigen binding portion of an antibody. Such antibody fragments are prepared using conventional methods known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies.

Preferably, the CDR of the antigen binding region or the entire antigen binding region of the antibodies of the invention is of mouse, llama or human donor library origin, or is substantially human in origin with certain amino acid residues altered, for example, substituted with different amino acid residues, so as to optimize specific properties of the antibody, for example KD , koff, IC ₅₀ , EC ₅₀ , ED ₅₀ . Preferably, the framework regions of the antibody of the invention are of human origin or substantially human origin (at least 80, 85, 90, 95, 96, 97, 98 or 99% human origin).

In other embodiments, the antigen binding region of an antibody 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 human species.

The term variable refers to the fact that certain segments of variable domains vary widely in sequence among antibodies. Domain V mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the 110 amino acid variable domain region. In contrast, V regions are composed of invariant fragments called framework regions (FRs) of 15–30 amino acids separated by shorter regions of extreme variability called hypervariable regions or CDRs. Each variable domain of the native heavy and light chains contains four FRs, generally adopting a beta-sheet configuration, linked by three hypervariable regions that form binding loops and, in some cases, are part of a beta-sheet structure. The hypervariable regions on each chain are held together in close proximity by the FR and, with the hypervariable regions on the other chain, contribute to the formation of the antigen-binding site of antibodies. Constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term hypervariable region as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Typically, the hypervariable region contains amino acid residues from the complementarity determining region or CDR and/or such residues from the hypervariable loop.

In some cases, it may also be preferable to modify one or more amino acid residues of the CDR regions to increase binding affinity to the target epitope. This is known as affinity maturation and in some cases may be performed in conjunction with humanization, for example in situations where humanization of an antibody results in a decrease in binding specificity or affinity and it is not possible to sufficiently improve binding specificity or affinity using back mutations alone . Various affinity maturation methods are known in the art, for example, the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the in vitro stepwise affinity maturation method proposed by Wu et al., Proc Natl Acad Sci USA 95:6037 6042 (1998).

Framework regions (FR) are variable domain residues other than CDR residues. Typically, each variable domain has four FRs, defined as FR1, FR2, FR3 and FR4. If CDRs are defined according to Kabat, the light chain FR residues are located approximately in the region of residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4), and the heavy chain FR residues are located approximately in the region of residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3) and 103-113 (HCFR4) in the heavy chain. Where CDR regions contain amino acid residues from hypervariable loops, FR light chain residues are located approximately at 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 (HCFRI), 33-52 (HCFR2), 56-95 (HCFR3) and 102-113 (HCFR4) in the heavy chain residues. In some examples, when the CDR contains amino acids from both the Kabat CDR and amino acids from the hypervariable loop, the FRs are adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, heavy chain FR1 residues are at positions 1-25 and FR2 residues are at positions 36-49.

An antibody of the present invention that binds a target antigen is an antibody that binds the antigen with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent when targeting a protein or cell or tissue expressing the antigen and with little cross-reacts with other proteins to a degree. According to analytical methods: fluorescence-activated cell sorting (FACS), radioimmunoprecipitation assay (RIA) or ELISA, in such embodiments of the invention the degree of binding of the antibody to

- 20 043937 by a non-target protein (with a non-target protein) is less than 10% of the antibody binding to a specific target protein. With respect to the binding of an antibody to a target molecule, the term specific binding or expressions specifically binding to or specific to a particular polypeptide or epitope on a particular target polypeptide means binding that is markedly (measurably) different from a nonspecific interaction.

In some embodiments, the immunocytokine includes a first antibody heavy chain constant domain and an antibody light chain constant domain in a heterodimeric complex that are covalently associated through a natural S-S bridge.

In some embodiments, the immunocytokine includes 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 natural S-S bridge.

In some embodiments, the immunocytokine includes an antibody light chain constant domain selected from CK or CL.

In some embodiments, the immunocytokine includes IL-15Ra, which has an amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASS (SEQ IDNO:9) or any known IL-15Ra variant containing mutations with similar biological activity.

By IL-15Ra that contains mutations with similar biological activity to IL-15Ra is meant any IL-15Ra known in the art that contains mutations and has similar biological activity to wild-type IL-15Ra.

In some embodiments, the immunocytokine includes IL-15, which has an amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:10) or any known IL-15 variant containing mutations with similar biological activity.

By IL-15 that contains mutations with similar biological activity to IL-15 is meant any IL-15 known in the art that contains mutations and has similar biological activity to wild-type IL-15.

In some embodiments, the immunocytokine includes IL-15Ra, which is bound to the light chain constant domain of the antibody.

In some embodiments, the immunocytokine comprises IL-15Ra linked to an antibody light chain constant domain that has an amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC (SEQ ID NO:1).

In some embodiments, the immunocytokine comprises IL-15Ra linked to an antibody light chain constant domain that has an amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEA (SEQ ID NO: 19).

The amino acid sequence, which is represented by SEQ ID NO: 19, differs from the amino acid sequence, which is represented by SEQ ID NO: 1, by one amino acid substitution of Cysteine (C) to Alanine (A) at position 216. This replacement makes it possible to obtain an immunocytokine that includes the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain as part of a heterodimeric complex, which do not have a covalent association through a natural S-S bridge.

In some embodiments, the immunocytokine includes IL-15, which is bound to the light chain constant domain of the antibody.

In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain that has an amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:3).

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In some embodiments, the immunocytokine comprises IL-15 linked to an antibody light chain constant domain that has an amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEA (SEQ ID NO:21).

The amino acid sequence, which is represented by SEQ ID NO: 21, differs from the amino acid sequence, which is represented by SEQ ID NO: 3, by one amino acid substitution of Cysteine (C) to Alanine (A) at position 233. This replacement makes it possible to obtain an immunocytokine that includes the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain as part of a heterodimeric complex, which do not have a covalent association through a natural S-S bridge.

In some embodiments, the immunocytokine includes a first (CH1) heavy chain constant domain and an Fc moiety monomer comprising a second (CH2) and third (CH3) heavy chain constant domain that are connected via a hinge.

In some embodiments, the immunocytokine includes IL-15 or IL-15Ra, which is associated with antibody heavy chain constant domains in the order CH1-hinge-CH2-CH3.

In some embodiments, the immunocytokine includes IL-15Ra, which is associated with the constant domains of the antibody heavy chain.

In some embodiments, the immunocytokine includes IL-15Ra linked to antibody heavy chain constant domains that have an amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQV SLWCL VKGFYPSDIA VEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:6).

In some embodiments, the immunocytokine includes IL-15Ra linked to antibody heavy chain constant domains that have an amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSADKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQV SLWCL VKGFYPSDIA VEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:24).

The amino acid sequence, which is represented by SEQ ID NO: 24, differs from the amino acid sequence, which is represented by SEQ ID NO: 6, by one amino acid substitution of Cysteine (C) to Alanine (A) at position 212. This replacement makes it possible to obtain an immunocytokine that includes the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain as part of a heterodimeric complex, which do not have a covalent association through a natural S-S bridge.

In some embodiments, the immunocytokine includes IL-15, which is bound to the constant domains of the antibody heavy chain.

In some embodiments, the immunocytokine comprises IL-15 linked to antibody heavy chain constant domains that have an amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPCRDELTKNQVSLWCL VKGFYPSDIA VEW ESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:5).

In some embodiments, the immunocytokine comprises IL-15 linked to antibody heavy chain constant domains that have an amino acid sequence that is represented by

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DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSADKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ IDNO:23).

The amino acid sequence, which is represented by SEQ ID NO: 23, differs from the amino acid sequence, which is represented by SEQ ID NO: 5, by one amino acid substitution of Cysteine (C) to Alanine (A) at position 229. This replacement makes it possible to obtain an immunocytokine that includes the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain as part of a heterodimeric complex, which do not have a covalent association through a natural S-S bridge.

In some embodiments, the immunocytokine includes 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.

Specific binding can be quantified, for example, by measuring the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by a competitive reaction with another molecule similar to the target, for example, with an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term specific binding or expression specifically binds to or is specific to a particular polypeptide or epitope on a particular target polypeptide can be characterized by a molecule having a Kd to the 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 higher. In one embodiment, the term specific binding refers to binding in which a molecule binds to a specific polypeptide or epitope on a specific polypeptide without substantially binding to any other polypeptide or epitope on the polypeptide.

In some embodiments, the immunocytokine includes an immunomodulatory antibody that includes:

a) a light chain containing a light chain variable domain and a light chain constant domain;

b) a heavy chain containing a heavy chain variable domain and constant domains of the antibody heavy chain, including the first (CH1) constant domain of the heavy chain and an Fc fragment monomer containing the second (CH2) and third (CH3) constant domains of the heavy chain.

In some embodiments, the immunocytokine includes a light chain variable domain that includes LCDR 1, 2, and 3 (hypervariable regions 1, 2, and 3), which are respectively represented by amino acid sequences

GGNNIGSKNVH (SEQ ID NO: 14), RDSNRPS (SEQ ID NO: 15) and CQVWDSSTAV (SEQ ID NO: 16).

In some embodiments, the immunocytokine includes a light chain variable domain that includes an amino acid sequence that is represented by

QPVLTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYRDSNRPSGIPE RFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSTAVFGTGTKLTVLQ (SEQ ID NO: 18).

In some embodiments, the immunocytokine includes a heavy chain variable domain that includes HCDR 1, 2, and 3 (hypervariable regions 1, 2, and 3), which are respectively represented by amino acid sequences

FTFSSYWMY (SEQ Ш NO: И), AIDTGGGRTYYADSVKG (SEQ ID NO: 12) and CARDEGGGTGWGVLKDWPYGLDA (SEQ ID NO: 13).

In some embodiments, the immunocytokine includes a heavy chain variable domain that includes an amino acid sequence that is represented by

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQVPGKGLEWVSAIDTGGGR TYYADSVKGRFAISRVNAKNTMYLQMNSLRAEDTAVYYCARDEGGGTGWGVLKDWP YGLDAWGQGTLVTVSS (SEQ ID NO: 17).

In some embodiments, the immunocytokine includes:

1) light chain variable domain, which includes LCDR 1, 2 and 3 (hypervariable regions

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1, 2 and 3), which are respectively represented by amino acid sequences

GGNNIGSKNVH (SEQ ID NO: 14), RDSNRPS (SEQ ID NO: 15) and CQVWDSSTAV (SEQ ID NO: 16);

2) heavy chain variable domain, which includes HCDR 1, 2 and 3 (hypervariable regions 1, 2 and 3), which are respectively represented by amino acid sequences

FTFSSYWMY (SEQ ID NO: AND), AIDTGGGRTYYADSVKG (SEQ ID NO: 12) and CARDEGGGTGWGVLKDWPYGLDA (SEQ ID NO: 13).

In some embodiments, the immunocytokine includes:

1) light chain variable domain, which includes the amino acid sequence that is represented by

QPVLTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYRDSNRPSGIPE RFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSTAVFGTGTKLTVLQ (SEQ ID NO: 18);

2) heavy chain variable domain, which includes the amino acid sequence represented by

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQVPGKGLEWVSAIDTGGGR TYYADSVKGRFAISRVNAKNTMYLQMNSLRAEDTAVYYCARDEGGGTGWGVLKDWP YGLDAWGQGTLVTVSS (SEQ ID NO: 17).

In some embodiments, the immunocytokine includes an immunomodulatory antibody that includes a light chain comprising an amino acid sequence that is represented by

QPVLTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYRDSNRPSGIPE RFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSTAVFGTGTKLTVLQRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO 8).

In some embodiments, the immunocytokine includes an immunomodulatory antibody that includes a heavy chain comprising an amino acid sequence that is represented by

QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQVPGKGLEWVSAIDTGGGR TYYADSVKGRFAISRVNAKNTMYLQMNSLRAEDTAVYYCARDEGGGTGWGVLKDWP YGLDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSC AVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO 7).

In some embodiments, the immunocytokine includes an immunomodulatory antibody that includes:

a heavy chain containing the amino acid sequence represented by QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQVPGKGLEWVSAIDTGGGR TYYADSVKGRFAISRVNAKNTMYLQMNSLRAEDTAVYYCARDEGGGTGWGVLKDWP YGLDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KA KGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO Ό;

light chain containing the amino acid sequence represented by QPVLTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYRDSNRPSGIPE RFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSTAVFGTGTKLTVLQRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ Ш NO 8).

In some embodiments, the immunocytokine includes the immunomodulatory antibody prolgolimab (BCD100, WO 2018013017).

In some embodiments, the immunocytokine includes an immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway, which is an antibody that specifically binds PD-L1.

In some embodiments, the antibody that specifically binds to PD-L1 is BCD135, which is described in patent RU 2665790 (WO 2018194496).

In some embodiments, the immunocytokine includes:

a) a heterodimeric protein complex based on IL-15 and IL-15Roc, which contains:

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IL-15Ra bound to the light chain constant domain of an antibody that has the amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC (SEQ Ш NO:1), and IL-15 associated with antibody heavy chain constant domains that have the amino acid sequence represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:5);

b) an immunomodulatory antibody, which includes:

heavy chain containing the amino acid sequence represented by QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQVPGKGLEWVSAIDTGGGR TYYADSVKGRFAISRVNAKNTMYLQMNSLRAEDTAVYYCARDEGGGTGWGVLKDWP YGLDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS G ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO Ό;

light chain containing the amino acid sequence represented by QPVLTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYRDSNRPSGIPE RFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSTAVFGTGTKLTVLQRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ Ш NO 8).

In some embodiments, the immunocytokine includes:

a) a heterodimeric protein complex based on IL-15 and IL-15Ra, which contains:

IL-15Ra bound to the light chain constant domain of an antibody that has the amino acid sequence that is represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEA (SEQ ID NO: 19), and IL-15 associated with antibody heavy chain constant domains that have the amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSADKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:23);

b) an immunomodulatory antibody, which includes:

heavy chain containing the amino acid sequence represented by QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQVPGKGLEWVSAIDTGGGR TYYADSVKGRFAISRVNAKNTMYLQMNSLRAEDTAVYYCARDEGGGTGWGVLKDWP YGLDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS G ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO 7);

light chain containing the amino acid sequence represented by

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QPVLTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYRDSNRPSGIPE

RFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSTAVFGTGTKLTVLQRTVAAPSVFIF

PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ Ш NO 8).

In some embodiments, the immunocytokine includes:

a) IL-15Ra bound to antibody heavy chain constant domains, which have the amino acid sequence represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:6), and IL-15 bound to the constant domain of an antibody light chain, which have the amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:3);

b) an immunomodulatory antibody, which includes:

heavy chain containing the amino acid sequence represented by QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQVPGKGLEWVSAIDTGGGR TYYADSVKGRFAISRVNAKNTMYLQMNSLRAEDTAVYYCARDEGGGTGWGVLKDWP YGLDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS G ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREP QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO

Ό;

light chain containing the amino acid sequence represented by QPVLTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYRDSNRPSGIPE RFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSTAVFGTGTKLTVLQRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ Ш NO 8).

In some embodiments, the immunocytokine includes:

a) IL-15Ra bound to antibody heavy chain constant domains that have the amino acid sequence represented by

DTCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTP SLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGAEGGGASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSADKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:24), and IL-15 bound to the light chain constant domain of an antibody, which have the amino acid sequence that is represented by

DNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSAGGKPG GSAGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEA (SEQ ID NO:21);

b) an immunomodulatory antibody, which includes:

heavy chain containing the amino acid sequence, which is represented by

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QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQVPGKGLEWVSAIDTGGGR TYYADSVKGRFAISRVNAKNTMYLQMNSLRAEDTAVYYCARDEGGGTGWGVLKDWP YGLDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFP AVLQS SGL Y SLS S VVT VPS S SLGTQTYICNVNHKP SNTKVDKRVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTK NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO light chain containing the amino acid sequence represented by QPVLTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYRDSNRPSGIPE RFSGSNSGNTATLTISRA QAGDEADYYCQVWDSSTAVFGTGTKLTVLQRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO 8).

In some embodiments, the immunocytokine has mutations in the antibody constant domains causing heterodimerization of two different parts, one of which includes covalently or non-covalently associated IL-15Ra bound to the antibody constant domains and IL-15 bound to the antibody constant domain, and the other includes covalently associated or non-covalently associated light and heavy chains of an antibody.

In some embodiments, the immunocytokine includes: a first Fc monomer and a second Fc monomer, which are selected from the group: the first Fc monomer is an Fc modified to form a Knob and the second Fc monomer is an Fc modified to form a Hole, or where the second Fc monomer is is Fc modified to form Knob, and the first Fc monomer is Fc modified to form Hole.

knobs-into-holes is an approach that can get around the problem of mismatched byproducts. This approach aims to enhance the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the interface at the contact region. On one chain, large amino acids were replaced with amino acids with short side chains to create a cavity. Conversely, amino acids with larger side chains were introduced into the other CH3 domain, creating an overhang. By co-expressing these two heavy chains, high yields of heterodimeric formation (knob-valley) relative to homodimeric formation (valley-valley or knob-knob) were obtained (Ridgway J.B., Presta L.G., Carter P. and WO 1996/027011). The percentage of heterodimer can be further increased by remodeling the interfaces of the two CH3 domains using phage display technology and introducing a disulfide bridge to stabilize the heterodimers (Merchant A.M. et al., Nature Biotech 16, 1998, pp. 677-681; Atwell S., Ridgway J.B., Wells J.A., Carter P., J. Mol. Biol. 270, 1997, pp. 26-35).

In some embodiments, the immunocytokine includes a first Fc monomer that has the amino acid substitutions S354C/T366W and a second Fc monomer that has the amino acid substitutions Y349C/T366S/L368A/Y407V.

In some embodiments, the immunocytokine includes a first Fc monomer that has the amino acid substitutions Y349C/T366S/L368A/Y407 and a second Fc monomer that has the amino acid substitutions S354C/T366W.

In some embodiments, the immunocytokine includes an Fc moiety that is classified as IgG.

In some embodiments, an immunocytokine, wherein the Fc fragment isotype is selected from the group: human IgG1, IgG2, or IgG4.

In some embodiments, the immunocytokine includes mutations in the Fc fragment monomer resulting in the absence of ADCC, CDC and/or ADCP properties of the immunocytokine.

Bispecific IL15 superagonist or bispecific IL15 immunocytokine or immunocytokine anti-PD1/IL15SA (bispecific monoclonal antibody containing IL-15SA and a Fab fragment to PD-1) also means

Anti-PD-1 Fab-CK_IL15Ra_Hc IL 15 CHlFcknobLALA, Anti-PD-1 Fab-CK_IL15_Hc_IL15RaCHlFcknobLALA.

The immunocytokine anti-PD-Ll/IL15SA also means

Anti-PD-Ll Fab-CK_IL15Ra_Hc IL 15 CHlFcknobLALA, Anti-PD-Ll Fab- CK_IL15_Hc_IL15RaCHlFcknobLALA.

The designations Anti-PD-1 Fab, Anti-PD-Ll Fab, CK, IL15Ra, IL15, CHlFcknobLALA are explained in the text of the application.

Anti-PD-1 Fab-CK_IL15Ra_Hc IL 15CHlFcknobLALA is the above immunocytokine comprising the antigen binding Fab fragment of the anti-PD1 antibody, IL15Ra, which is associated with the CK antibody light chain constant domain and IL15, which is associated with the antibody heavy chain constant domains including the first (CH1 ) a heavy chain constant domain and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains, where the Fc contains mutations to form heterodimerization (for example, knob) and LALA.

Anti-PD-1 Fab- CK IL15 He IL15RaCHlFcknobLALA is the above immunocytokine, including the antigen-binding Fab fragment of the anti-PD1 antibody, IL15, which is associated with the constant domain of the light chain of the CK antibody and IL15Ra, which is associated with the constant domains of the heavy chain of the antibody, including the first (CH1 ) a heavy chain constant domain and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains, where the Fc contains mutations to form heterodimerization (eg knob) and LALA.

Anti-PD-Ll Fab-CK IL15Ra Hc IL15CHlFcknobLALA is the above-mentioned immunocytokine, including the antigen-binding Fab fragment of the antibody to PD-L1, IL15Ra, which is associated with the constant domain of the light chain of the antibody CK and IL15, which is associated with the constant domains of the heavy chain of the antibody, including the first (CH1) heavy chain constant domain and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains, where the Fc contains mutations to form heterodimerization (eg, knob) and LALA.

Anti-PD—LI Fab-CK IL15 He IL15RaCHlFcknobLALA is the above immunocytokine, which includes the antigen-binding Fab fragment of the antibody to PD-L1, IL15, which is associated with the constant domain of the light chain of the CK antibody and IL15Ra, which is associated with the constant domains of the heavy chain of the antibody, including the first (CH1) heavy chain constant domain and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains, where the Fc contains mutations to form heterodimerization (eg, knob) and LALA.

The above immunocytokines can be made both in a format where the first constant domain of the heavy chain of the antibody and the constant domain of the light chain of the antibody as part of a heterodimer complex have a covalent association through a natural S-S bridge, and in a format where the first constant domain of the heavy chain of the antibody and the constant domain of the light chain The antibody chains in the heterodimer complex do not have a covalent association through a natural S-S bridge; in a particular case, for this, both cessteins are replaced with alanine.

In experimental studies of the above immunocytokines, both in a format where the first constant domain of the antibody heavy chain and the constant domain of the antibody light chain as part of a heterodimer complex have a covalent association through a natural S-S bridge, and in a format where the first constant domain of the antibody heavy chain and the constant domain of the light chain antibodies in the heterodimer complex do not have covalent association through a natural S-S bridge, no significant difference in the activity of the above cytokines was detected.

By knob, as defined above, are meant mutations that produce a Knob-to-Hole structure between the first Fc variant and the second Fc variant in the CH3 domain.

The format of the above immunocytokine is shown in FIG. 3 and 4.

In some embodiments, the immunocytokine is used to treat cancer or an autoimmune disease.

In some embodiments, the immunocytokine is used to treat cancer.

Nucleic acid.

In one aspect, the present invention provides an isolated nucleic acid that encodes any of the above immunocytokines.

The terms nucleic acid, nucleic sequence or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence and nucleotide sequence, as used interchangeably herein, refer to a distinct sequence of nucleotides, modified or unmodified, defining a fragment or region of nucleic acid, whether or not containing unnatural nucleotides. and being either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of these DNAs.

It should also be mentioned here that this invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in its natural state. The sequences of the present invention have been isolated and/or purified, i.e. were taken directly or indirectly, for example by copying, and their environment was at least partially modified. Thus, it should also be understood here as isolated nucleic acids obtained by genetic recombination, for example by host cells, or obtained by chemical synthesis.

An isolated nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is typically associated in a natural source. The isolated nucleic acid molecule differs from the form or set in which it occurs naturally. Thus, the isolated nucleic acid molecule is different from the nucleic acid molecule that naturally exists in cells. However, the isolated nucleic acid molecule

- 28 043937 includes a nucleic acid molecule located in cells that normally express an immunocytokine, for example, if the nucleic acid molecule has a location on the chromosome that is different from its location in cells under natural conditions.

In some embodiments, the nucleic acid is DNA.

In one embodiment, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NO: 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 contain any combination of the above nucleotide sequences.

It will be apparent to one skilled in the art that a peptide with an amino acid sequence selected from SEQ ID NO: 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 can be encoded by a wide range of different DNA sequences, taking into account the degeneracy of the genetic code. It is well known to those skilled in the art to obtain such alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

Expression vector.

In one aspect, the present invention relates to an expression vector that contains the above nucleic acid.

The term vector as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e. a circular, double-stranded portion of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). In other embodiments, vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell, and thus replicate along with the host gene. Moreover, some 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).

The present invention relates to vectors containing the above nucleic acid molecules that encode any of the amino acid sequences of the above immunocytokine or constructive parts thereof as described herein.

In some embodiments, the immunocytokine of the invention is expressed by inserting DNA encoding part or all of the sequence of the first and second immunocytokine domains (e.g., a domain including IL15 or IL15Ra linked to antibody constant domain(s) and/or lung and antibody heavy chain) prepared as described above in expression vectors such that the genes are operably linked to necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmid, YAC, EBV-derived episomes and the like. DNA molecules can be ligated into a vector such that the transcription and translation control sequences in the vector perform the intended function of regulating DNA transcription and translation. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules encoding partial or entire sequences of the first and second binding domains (eg, heavy and light chain sequences, where the binding domain contains a heavy and light chain sequence) can be introduced into separate vectors. In one embodiment of the invention, any combination of the above DNA molecules is introduced into the same expression vector. DNA molecules can be introduced into the expression vector by standard methods (eg, by ligating complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if restriction sites are not present).

A suitable vector is one that encodes functionally complete human immunoglobulin CH or CL/CK sequences with an appropriate restriction site engineered so that any VH or VL or IL15 or IL15Ra sequence can be readily incorporated and expressed as described above. The NS and LC coding of genes in such vectors may contain intronic sequences, which leads to an overall increase in antibody protein products by stabilizing the corresponding mRNA. Intronic sequences are located in the environment of the splice donor and splice acceptor sites, which determine where RNA splicing will occur. The location of the intronic sequences can be either in the variable or constant regions of the antibody chains, or in both the variable and constant regions when multiple introns are used. Termination of polyadenylation and transcription can occur downstream of the native chromosome site of the encoded regions. The recombinant expression vector may also encode a signal peptide that facilitates production of an antibody chain by the host cell. An antibody chain gene or a chain gene comprising IL15 or IL15Ra can be cloned into a vector such that the signal peptide is linked to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a non-immunoglobulin protein signal peptide).

In addition to the antibody gene chain or the gene chain including IL15 or IL15Ra, recombinant expression vectors of the present invention may carry regulatory sequences that control gene expression in the host cell. Those skilled in the art will appreciate that the design of an expression vector, including the choice of regulatory sequences, may depend on factors such as the selection of the host cell for transformation, the level of expression of the desired protein, etc. Preferred regulatory sequences for a mammalian host expression cell include viral elements that provide high level expression of proteins in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR, cytomegalovirus (CMV) (eg, CMV promoter/enhancer), simian virus 40 (SV40) (eg, SV40 promoter/enhancer), adenovirus (eg, adenovirus large late promoter (AdMLP)), polyoma virus, as well as strong mammalian promoters such as the native immunoglobulin promoter or the actin promoter. For further description of viral regulatory elements and their sequences, see, for example, US patents 5168062, 4510245 and 4968615. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

In addition to the above genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate the replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, for example, US Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically a selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, to the host cell into which the vector is introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells for methotrexate selection/amplification), the neo gene (for G418 selection) and the glutamate synthetase gene.

The term expression control sequence as used herein refers to polynucleotide sequences that are necessary to influence the expression and processing of the coding sequences to which they are ligated. Expression control sequences include the corresponding transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (ie, the Kozak consensus sequence); sequences that increase protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. 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 beneficial, such as leader sequences and fusion sequences.

The expression control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes are, for example, a promoter, optionally an operator and a ribosome binding site. As is known, promoters, polyadenylation signals and enhancers are present in eukaryotic cells.

A nucleic acid is operably linked if it is in a functional relationship with another nucleotide sequence. For example, the DNA of a presequence or secretory leader sequence is operably linked to the DNA of 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 transcription of the sequence; the ribosome binding site is operably linked to the coding sequence if it is located in such a way that it can facilitate translation. Generally, operably linked means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, contiguous and in reading phase. However, enhancers do not have to be contiguous.

Host cells and method of obtaining them.

In one aspect, the present invention relates to a method of obtaining a host cell for producing the above immunocytokine, which includes transforming the cell with the above vector.

Nucleic acid molecules encoding the immunocytokines of the invention and vectors containing these nucleic acid molecules can be used to transfect a suitable mammal or cell thereof, a plant or cell thereof, or a bacterial or yeast host cell. Conversion can occur by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, transfection with a complex of nucleic acid and a positively charged polymer, transfection with a nucleic acid and calcium phosphate precipitate, polybrene-mediated transfection, protoplast fusion, transfection with liposome-encapsulated polynucleotides, and direct microinjection of DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods for transfecting cells are well known in the art. See, for example, US Pat. 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, including, 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.

In one aspect, the present invention relates to a host cell for producing the above immunocytokine, which contains the above nucleic acid.

In one aspect, the present invention relates to a method for producing a preparation containing the above immunocytokine, which consists of culturing the above host cells in a culture medium under conditions sufficient to obtain the specified immunocytokine, if necessary, followed by the isolation and purification of the resulting immunocytokine.

The term recombinant host cell (or simply host cell) as used herein means a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may include, for example, a vector in accordance with the present invention described above. The present invention also relates to host cells that include, for example, a nucleotide sequence encoding an immunocytokine or portions thereof of the invention. It should be understood that recombinant host cell and host cell mean not only the specific cell claimed, but also the progeny of such a cell. Since modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term host cell as used herein.

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, HEK293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, hamster kidney (HK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (eg Hep G2), A549 cells and a number of other cell lines. Cell lines are selected by determining which cell lines have high levels of expression and provide the desired characteristics of the protein produced. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When recombinant expression vectors encoding an immunocytokine of the invention, or a portion thereof, are introduced into mammalian host cells, the immunocytokines are produced by culturing the host cells for a time sufficient to express the immunocytokine, or a portion thereof, of the invention in the host cells, or, preferably, release the immunocytokine into the nutrient medium in which the host cells are grown. Immunocytokines can be isolated from culture media using standard protein purification methods. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Host bacterial cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

In addition, the level of production of the immunocytokine of this invention from a producing cell line can be enhanced using a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is common enough to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with patents EP 0216846,

- 31 043937

0256055,0323997 and 0338841.

It is likely that the immunocytokine or part thereof of the invention will differ in its glycosylation profile in different cell lines or host cells. However, the immunocytokine described herein, or a portion (domain) thereof containing the amino acid sequences set forth herein, is part of this invention, regardless of the glycosylation state of the binding molecules and, in general, regardless of the presence or absence of post-translational modifications.

Pharmaceutical composition.

In one aspect, the present invention relates to a pharmaceutical composition for promoting the activation and/or proliferation of IL-15Rbeta/gamma positive cells, which contains the above immunocytokine in a therapeutically effective amount, in combination with one or more pharmaceutically acceptable excipients.

Pharmaceutical composition means a composition comprising an immunocytokine according to the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, such as excipients, solvents, diluents, carriers, auxiliaries, dispensers, delivery vehicles, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, prolonged delivery regulators, the choice and ratio of which depends on the nature and method of administration and dosage. The pharmaceutical compositions of the present invention and methods for their manufacture will be readily apparent to those skilled in the art. The production of pharmaceutical compositions should preferably comply with GMP (Good Manufacturing Practices) requirements. The composition may include a buffer composition, tonic agents, stabilizers and solubilizers. Prolonged action of the composition can be achieved by using agents that slow down the absorption of the active pharmaceutical ingredient, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols, as well as mixtures thereof, oils and injectable organic esters.

A therapeutically effective amount is the amount of a therapeutic agent administered during treatment that will relieve, to a certain extent, one or more symptoms of the disease being treated.

Medicine (drug) - a substance or mixture of substances in the form of a pharmaceutical composition in the form of tablets, capsules, powders, lyophilisates, injections, infusions, ointments and other finished forms, intended to restore, correct or change physiological functions in humans and animals, and also for the treatment and prevention of diseases, diagnostics, anesthesia, contraception, cosmetology and other things. Any method of administering peptides, proteins or antibodies accepted in the art can be suitably used for the immunocytokine of this invention.

The term pharmaceutically acceptable means one or more compatible liquid or solid components that are suitable for administration to a mammal, preferably a human.

The term excipient or excipient is used herein to describe any component other than those previously described in this invention. These are substances of inorganic or organic origin, used in the production process of medicines to give them the necessary physical and chemical properties.

The term buffer, buffer composition, buffer agent refers to a solution capable of maintaining the pH value due to the interaction of the acidic and alkaline components included in its composition, which allows the immunocytokine preparation to exhibit resistance to changes in pH. In general, pH values of the pharmaceutical composition from 4.0 to 8.0 are preferred. As buffering agents, for example, acetate, phosphate, citrate, histidine, succinate, etc. can be used. buffer solutions, but not limited to them.

The terms tonic agent, osmolytic or osmotic agent as used herein refer to an excipient that can add osmotic pressure to a liquid antibody preparation. An isotonic drug is a drug that has an osmotic pressure equivalent to that of human blood. Isotonic drugs typically have an osmotic pressure of approximately 250 to 350 mOsm/kg. Polyols, mono- and disaccharides, amino acids, metal salts such as sodium chloride, etc., but not limited to, can be used as isotonic agents.

By stabilizer is meant an excipient or a mixture of two or more excipients that provide physical and/or chemical stability of the active agent. Amino acids can be used as stabilizers, for example, but not limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants, e.g. polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade name Poloxaner),

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Pluronic), sodium dodecyl sulfate (SDS), but not limited to; antioxidants, for example, but not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfuric acid salts, etc.; chelating agents, for example, but not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate and the like.

A pharmaceutical composition is stable if the active agent maintains its physical stability and/or chemical stability and/or biological activity through its stated shelf life at storage temperature, for example, 2-8°C. It is preferred that the active agent retains both physical and chemical stability as well as biological activity. The storage period is selected based on the results of stability studies during accelerated and natural storage.

The pharmaceutical composition of this invention may be manufactured, packaged or widely sold as a single unit dose or a plurality of unit dose units in a finished dosage form. As used herein, the term unit dose means a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of active ingredient is typically equal to the dosage of the active ingredient to be administered to the subject, or a convenient portion of such dosage, such as one-half or one-third of such dosage.

The pharmaceutical compositions of the present invention are generally suitable for parenteral administration in the form of sterile medicinal products intended for introduction into the human body without breaking the integrity of the skin or mucous membranes, bypassing the gastrointestinal tract by injection, infusion or implantation. For example, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous, intra-arterial, intrathecal, intraventricular, intraurethral, intracranial, intra-articular, transdermal injection or infusion; and renal dialysis infusion techniques. Intratumoral delivery, such as intratumoral injection, may also be applicable. Regional perfusion is also provided. Preferred embodiments of the invention include intravenous and subcutaneous routes. Any method of administering peptides or proteins accepted in the art can suitably be used for the immunocytokine of this invention.

Injectable drugs may be manufactured, packaged or sold in unit dosage form, such as, but not limited to, ampoules, vials, polymer containers, pre-filled syringes, auto-injection devices. Drugs for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous bases, pastes and the like.

Another embodiment of the invention is a medicament for parenteral administration, wherein the pharmaceutical composition is provided in dry form, i.e. powder or granules for dissolution in a suitable solvent (eg, sterile pyrogen-free water) before administration. Such a medicinal product can be obtained, for example, by lyophilization, i.e. a process known in the art as freeze drying, which involves freezing the preparation and then removing the solvent from the frozen contents.

The immunocytokine of this invention may also be administered intranasally or inhalation, alone, in the form of a mixture with a suitable pharmaceutically acceptable excipient from an inhaler, such as a pressurized aerosol container, pump, spray, nebulizer) or nebulizer, which may or may not use a suitable propellant, or in the form of nasal drops or spray.

The drug for parenteral administration may be immediate or modified release. Modified release drugs include delayed, sustained, pulsatile, controlled, targeted and programmable release.

In some embodiments, the pharmaceutical composition is used to treat cancer or an autoimmune disease.

In some embodiments, the pharmaceutical composition is used to treat an autoimmune disease.

The term autoimmune disease as used herein refers to a non-malignant disease or disorder arising from and directed against an individual's own (auto) antigens and/or tissues.

The definition of autoimmune disease includes, but is not limited to, rheumatoid arthritis, arthrosis, juvenile chronic arthritis, septic arthritis, Lyme arthrosis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, diabetes mellitus, thyroiditis , asthma, allergic diseases, psoriasis, atopic dermatitis, scleroderma, graft-versus-host disease, graft rejection, acute or chronic immune diseases associated with transplantation, sarcoidosis, Kawasaki disease, Graves' disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schönlein purpura, microscopic renal vasculitis, chronic active hepatitis, uvenita, septic shock, toxic shock syndrome, septic syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary Biliary cirrhosis, hemolytic anemia, adults (acute) respiratory distress syndrome, alopecia, focal alopecia, sernegative arthropathy, arthropathy, reitera, psoriatic arthropathy, arthropathy, atopic allergies, autoimmune bullet diseases, vulgaris bubbles, Lee, Lee, Lee Lee Bubility, Lee Hall bubbles, pemphigoid disease, linear IgA, autoimmune hemolytic anemia, Coombs positive hemolytic anemia, pernicious anemia, juvenile pernicious anemia, arthritis, primary sclerosing heppatitis A, cryptogenic autoimmune hepatitis, fibrosing lung diseases, cryptogenic fibrous alveolitis, post-inflammatory interstitial lung diseases, interstitial pneumonitis, chronic eosinophilic pneumonia chronic, post-infectious interstitial lung diseases, gouty arthritis, autoimmune heppatitis, autoimmune heppatitis type I (classical autoimmune heppatitis or lipoid), autoimmune heppatitis type II, osteoarthritis, primary sclerosing cholangitis, psoriasis type I, psoriasis type II, idiopathic leukopenia, autoimmune neutropenia, renal NOS disease [renal NOS-disease], glomerulonephritis, microscopic renal vasculitis, discoid lupus erythematosis, idiopathic or male NOS fertility, [autoimmunity to sperm], all subtypes of multiple sclerosis, sympathetic ophthalmia, secondary pulmonary hypertension in connective tissue diseases, Goodpasture's syndrome, pulmonary manifestation of nodular polyarthritis, acute rheumatic fever, rheumatoid spondylitis, ankylosing spondylitis, Still's disease, systemic sclerosis, Schoengren's syndrome, Takayasu's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroiditis, hyperthyroidism, Hoshimoto's disease, autoimmune atrophic hypothyroidism izm, primary mexidema , phacogenic uveitis, primary vasculitis, vitiligo, acute liver diseases, chronic liver diseases, allergies, asthma, mental illnesses (including depression and schizophrenia), diseases mediated by Th2 type and Th1 type, conjunctivitis, allergic contact dermatitis, allergic rhinitis, alpha deficiency 1-antitrypsin, amyotrophic lateral sclerosis, anemia, cystic fibrosis, diseases associated with cytokine therapy, demyelinating diseases, dermatitis, iridocyclitis/uveitis/optic neuritis, ischemic reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune premature ovarian failure and blepharitis.

In some embodiments, the pharmaceutical composition is used to treat cancer.

The terms cancer, cancer, or cancerous refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, peritoneal cancer, hepatic cell carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, cancer ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial and uterine carcinoma, salivary gland carcinoma, kidney cancer (renal cell carcinoma), prostate cancer (prostate cancer), cancer of the external female genitalia, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, melanoma and various types of head and neck cancer.

In some embodiments, the pharmaceutical composition is used to treat a cancer selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer without an identified primary source, 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 CRC (colorectal cancer with high microsatellite instability), leukemia (acute or myeloblastic), lymphoma, multiple myeloma, melanoma , breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory B-cell non-Hodgkin lymphoma , follicular lymphoma, marginal zone B-cell lymphoma, large B-cell diffuse lymphoma, squamous cell carcinoma of the head and neck, pancreatic cancer, ovarian cancer, acute myeloid leukemia and high-risk myelodysplastic syndrome.

The term antiproliferative action implies stopping or inhibiting the growth of proliferating cells, such as cancer cells.

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The term IC ₅₀ (50% inhibitory concentration) refers to the concentration of a drug at which a measurable activity or response, such as growth or proliferation of cells such as tumor cells, is inhibited by 50%. The IC ₅₀ value can be estimated using appropriate log dose response curves using special statistical curve processing programs.

The term GI50 (50% growth inhibition) refers to the drug concentration at which the proliferation of cells, such as tumor cells, is inhibited by 50%.

The term ED ₅₀ (EC ₅₀ ) (50% effective dose/concentration) refers to the drug concentrations at which 50% of the measured biological effect (may include cytotoxicity) is achieved.

Method of treatment/Use for treatment.

In one aspect, the present invention relates to a method of treating cancer, which includes administering to a subject in need of such treatment the above immunocytokine or the above pharmaceutical composition in a therapeutically effective amount.

Treat, cure, and therapy refer to a method of alleviating or eliminating a biological disorder and/or at least one of its accompanying symptoms. As used herein, the term ameliorate a disease, disorder or condition means reducing the severity and/or frequency of symptoms of the disease, disorder or condition. In addition, references to treatment contained herein include references to curative, palliative, and preventative therapies.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The above subject may be male or female of any age.

The term disease means any condition that can be improved by treatment according to the present invention. The definition of this term includes chronic and acute disorders or diseases.

In some embodiments of the treatment method, the cancer is selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer without an identified primary source, 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 CRC (colorectal cancer with high microsatellite instability), leukemia (acute or myeloblastic), lymphoma, multiple myeloma, melanoma, breast cancer , colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory B-cell non-Hodgkin lymphoma, follicular lymphoma, Marginal zone B-cell lymphoma, large cell diffuse lymphoma, squamous cell carcinoma of the head and neck, pancreatic cancer, ovarian cancer, acute myeloid leukemia and high-risk myelodysplastic syndrome.

In one aspect, the present invention relates to a method of activating the biological activity of a T cell population or NK cell population in a subject in need of such activation, which comprises administering to the subject an effective amount of the above immunocytokine or the above pharmaceutical composition.

In one aspect, the present invention relates to the use of the above immunocytokine or the above pharmaceutical composition for the treatment of a cancer in a subject in need of such treatment.

In some embodiments, the cancer is selected from the group of: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer without an identified primary source, 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 CRC (microsatellite instability-high colorectal cancer), leukemia (acute or myeloblastic), lymphoma, multiple myeloma, melanoma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory B-cell non-Hodgkin lymphoma, follicular lymphoma, B- marginal zone cell lymphoma, large cell diffuse lymphoma, squamous cell carcinoma of the head and neck, pancreatic cancer, ovarian cancer, acute myeloid leukemia and high-risk myelodysplastic syndrome.

In the case of a tumor (eg, a cancerous tumor), a therapeutically effective amount of the immunocytokine of the invention may reduce the number of cancer cells; reduce the initial size of the tumor; inhibit (ie, slow down to some extent and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (ie, slow down to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or alleviate, to some extent, one or more symptoms associated with the disorder. The immunocytokine of the invention can, to some extent, prevent the growth and/or kill existing cancer cells, it can cause a cytostatic and/or cytotoxic effect. In cancer therapy, in vivo efficacy can be determined, for example, by assessing survival, time to disease progression (TTP), tumor response rate (RR), duration of response, and/or quality of life.

The immunocytokines of this invention can be administered without additional therapeutic treatment, i.e. as an independent therapy. In addition, treatment with an immunocytokine according to this invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the immunocytokine may be co-administered or formulated with another drug/drug for the treatment of cancer.

As used herein, the terms co-administration, co-administered and in combination with refer to an immunocytokine of the invention with one or more other therapeutic agents and is intended to mean, refer to or include:

1) simultaneous administration of such a combination of an immunocytokine of this invention and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form from which said components are released substantially simultaneously to said patient;

2) the simultaneous administration of such a combination of an immunocytokine according to this invention and a therapeutic agent to a patient who needs treatment, when such components are formulated separately in different dosage forms, the administration of which occurs at substantially the same time to the specified patient, after which the specified components are released substantially simultaneously to the specified patient;

3) sequential administration of such a combination of an immunocytokine according to this invention and a therapeutic agent to a patient who needs treatment, when such components are formulated separately from each other in separate dosage forms that are taken in a sequential time specified by the patient with a significant time interval between each administration, after whereby said components are released at substantially different times to said patient; and

4) sequential administration of such a combination of an immunocytokine according to this invention and a therapeutic agent to a patient who needs treatment, when such components are formulated together in a single dosage form from which the release of said components occurs in a controlled manner, after which they are simultaneously, sequentially or jointly released in one and the same time and/or different times to the specified patient, where each part can be administered by one or different routes.

The immunocytokine of this invention may be combined with a therapeutic agent that is selected from the group consisting of a cytotoxic agent, a chemotherapeutic agent, an anti-hormonal agent, or other therapeutic antibody.

The term cytotoxic agent as used herein refers to a substance that inhibits or prevents the functioning of cells and/or causes cell destruction. The term is intended to include radioactive isotopes (e.g. At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu radioactive isotopes), chemotherapeutic agents and toxins such as low molecular weight toxins or enzymatically active toxins originating from bacteria, fungi, plants or animals, including fragments and/or variants thereof.

A chemotherapy agent is a chemical compound used to treat a malignant tumor. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (eg bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including synthetic analogue of topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (eg, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (eg calicheamicin, e.g. calicheamicin gamma II and calicheamicin omega II (see, for example, Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dinemycin, including dinemycin A; esperamycin; as well as the neocarcinostatin chromophore and related chromophores of chromoproteins - enediyne antibiotics, aclacinomysins, actinomycin, outramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo -5oxo-L-norleucine, doxorubicin (including ADRIAMICIN®, morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, doxorubicinHC1 in injectable liposomes (DOXOL®), liposomal doxorubicin TLC D-99 (MYOCET®), PEGylated liposomal doxorubicin (CAELYX ®) and deoxydoxorubicin ), epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancytabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; drugs that suppress adrenal function, such as aminoglutethimide, mitotane, trilostane; a folic acid compensator such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diazichon; elfornitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenomet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2-trichlorotriethylamine; trichothecenes (eg, T-2 toxin, verracurine A, roridin A and anguidine); urethane; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (ara-C); thiotepa; a taxoid, such as paclitaxel (TAXOL®), an engineered albumin-associated nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum-based agents such as cisplatin, oxaliplatin and carboplatin; Vinca alkaloids, which prevent the polymerization of tubulin from nascent microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®) and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (eg, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID® ) or risendronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside analogue of cytosine); antisense oligonucleotides, for example oligonucleotides that inhibit gene expression in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; topoisomerase 1 inhibitor (eg LURTOTECAN®); rmRH (eg ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitor (eg, celecoxib or etoricoxib), proteasome inhibitor (eg, PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (811577); orafenib, ABT510; a Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantron; 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, short for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, short for oxaliplatin treatment regimen (ELOXATINTM) in combination with 5-FU and leucovorin.

Also included in this definition are antihormonal agents that act by regulating or inhibiting the action of hormones on tumors, such as antiestrogens with a mixed agonist/antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, trioxifene, toremifene (FARESTON®); idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene 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 metabolism, and/or decrease ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and non-steroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide and other aromatase inhibitors, including vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, imidazole; luteinizing hormone releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin and

- 37 043937 tripterelin; sex steroids, including progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin and androgens/retinoids such as fluoxymesterone, all-trans retinoic acid and fenretinide; onapristone; antiprogesterones; estrogen receptor down-regulators (ERDs); antiandrogens such as flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as a combination of two or more of the above means.

Another therapeutic agent that may be used in combination with an immunocytokine of the present invention may be a therapeutic antibody selected from the group: anti-PD1 antibodies (e.g., prolgolimab, pembrolizumab, or nivolumab), anti-PD-L1 antibodies, anti-CTLA4 antibodies, anti-4-1BB, anti-OX40, anti-GITR, anti-CD20 (eg, rituximab), anti-HER2 (eg, trastuzumab or pertuzumab), anti-VEGF (eg, bevacizumab), or combinations thereof.

Another therapeutic agent that can be used in combination with the immunocytokine of this invention may be a therapeutically active antitumor compound that is selected from the group of innate or adaptive immune activators.

It is intended that the immunocytokine of this invention may be used in methods of treatment as described above, may be used in treatment as described above, and/or may be used in the manufacture of medicaments for treatment as described above.

Doses and routes of administration.

The immunocytokine of this invention will be administered in an amount effective to treat the condition in question, i.e. in doses and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the specific condition being treated, the age, sex, and weight of the patient, and whether administration of the immunocytokine is a stand-alone treatment or is administered in combination with one or more additional drugs or treatments. .

Drug regimens can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally decreased or increased depending on the acuity of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage.

Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for patients/subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specification for unit dosage forms of the present invention is generally dictated by and directly depends on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the technique of compounding such active compound for treating sensitivity in subjects.

Thus, those skilled in the art will appreciate from the disclosure provided herein that dosages and dosage regimens will be adjusted in accordance with methods well known in the therapeutic field. This means that the maximum tolerated dose can be easily determined and the effective amount to provide a detectable therapeutic effect for the patient can also be determined, as well as the timing requirements for each agent to achieve a detectable therapeutic effect for the patient. Thus, although certain dosages and dosage regimens are given as examples herein, these examples are not intended to limit in any way the dosages and dosage regimens that may be needed for a patient to practice the present invention.

It should be noted that dosage levels may vary depending on the type and severity of the condition to be alleviated and may include one or more doses. In addition, it should be understood that for any particular patient, specific dosage regimens must be adjusted over time according to individual need and at the discretion of the healthcare professional administering or supervising the administration of the compositions, and that the concentration ranges given herein are provided only by way of example and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen of the compositions of this invention can be based on various factors, including the type of disease, age, weight, gender, health conditions of the patient, severity of the condition, route of administration and the specific immunocytokine of the invention. Thus, the dosage regimen may vary widely, but can be determined regularly using standard methods. For example, dosages may be adjusted based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects such as toxic effects or laboratory values. Thus, the present invention covers individual dose escalation, which is determined by a qualified specialist. Determination of the required dosage and regimens are well known in the relevant art and will be apparent to one skilled in the art upon familiarization with the concepts disclosed herein.

Examples of suitable routes of administration are provided above.

It is expected that a suitable dose of the immunocytokine of this invention will range from 0.1-200 mg/kg, preferably 0.1-100 mg/kg, including about 0.5-50 mg/kg, for example about 1-20 mg/kg. The immunocytokine may be administered, for example, at a dose of at least 0.25 mg/kg, such as at least 0.5 mg/kg, including at least 1 mg/kg, such as at least 1.5 mg/kg , for example as well as 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 a maximum of 50 mg/kg, including up to a maximum of 30 mg/kg, for example, up to a maximum of 20 mg/kg, including up to a maximum of 15 mg/kg. Administration will be repeated usually at suitable intervals, for example once a week, once every two weeks, once every three weeks or once every four weeks, and for as long as is considered appropriate by the responsible physician, who may in some cases increase or decrease dose if necessary.

Examples

For a better understanding of the invention, the following examples are provided. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any form.

All publications, patents and patent applications referenced in this specification are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example in order to avoid ambiguity, those skilled in the art will appreciate from the teachings disclosed herein that certain changes and modifications may be made without departing from the spirit and scope of the appended embodiments. implementation of the invention.

Materials and general methods.

Recombinant DNA methods.

For DNA manipulation, standard methods were used 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 manufacturers' instructions.

Gene synthesis.

The required gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments ranging from 300 to 4000 bp in length, which are flanked by unique restriction sites, were assembled by annealing and ligation of oligonucleotides, including PCR amplification and subsequent cloning through the specified restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

Determination of DNA sequences.

DNA sequences were determined by Sanger sequencing.

Analysis of DNA and protein sequences and processing of sequence data.

Infomax's Vector NTI Advance suite, version 8.0, and SnapGene Viewer were used to generate, map, analyze, annotate, and illustrate sequences.

Example 1.

Production of recombinant proteins in suspension culture of mammalian cells.

To prepare recombinant IL15 superagonist proteins, schematically presented in FIG. 1-4, constructs containing fragments of the human IL15 ligand sequence (https://www.uniprot.org/uniprot/P40933) and IL15Ra (https://www.uniprot.org/uniprot/Q13261) were synthesized. The sequences of both genes were synthesized from oligonucleotides by PCR.

To generate IL15 superagonist variants as represented by CK_IL15Ra_Hc_IL15CH1FcLALA in FIG. 1 and CK_IL15_Hc_IL15RaCH1FcLALA in FIG. 2 (hereinafter referred to as IL15SA or BCD225), these genes were cloned into plasmids as an N-terminus fusion protein with the first constant kappa domain of the antibody light chain into the vector pEE-IL15Ra-CK (or pEE-IL15-CK) at restriction sites SalI/BsiWI (Fig. 5) and with the first constant domain of the heavy chain of the antibody into the vector pEEIL15-CH1-Fc-LALA (or pEE-IL15Ra-CH1-Fc-LALA) IgG1 no restriction sites SalI/NheI (Fig. 6 ) and both vectors are combined at the transfection stage.

To generate bispecific IL15 superagonist variants as represented by FabCK_IL15Ra_Hc_IL15CH1FcknobLALA (hereinafter referred to as IL15SA/PD1 or anti-PD1/IL15SA or IL15SA/PDL1 or anti-PDL1/IL15SA) in FIG. 4 and Fab-CK_IL15_Hc_IL15RaCH1FcknobLALA in FIG. 3, the IL15 ligand and receptor genes were cloned into N-terminus fusion plasmids with the first constant kappa domain of the antibody light chain into the vector pEE-IL15Ra-CK (or pEE-IL15-CK) at the SalI/BsiWI restriction sites (Fig. 5 ) and with the first constant domain of the antibody heavy chain into the pEEIL15-CH1-Fc-knob-LALA (or pEE-IL15Ra-CH1-Fc-knob-LALA) IgG1 vector at the SalI/NheI restriction sites

- 39 043937 (Fig. 9). In addition, to form the Fab portion of an asymmetric antibody, as shown in FIG. 3 and fig. 4, the genes for the variable domains of anti-PD1 (prolgolimab) (hereinafter as IL15SA/PD1 or antiPD1/IL15SA) or anti-PDL1 (BCD-135) (hereinafter as IL15SA/PDL1 or anti-PDL1/IL15SA) antibodies were cloned into plasmids in as an N-terminus fusion with the first constant domain of the kappa light chain of the antibody into the vector pEE-BCD100-02-VL-CK at the SalI/BsiWI restriction sites (Fig. 7) and with the first constant domain of the heavy chain of the antibody into the vector pEE-BCD100-02 -VH-CH1-Fc-hole-LALA IgG1 at SalI/NheI restriction sites (Fig. 8). As a result, the four described vectors were combined at the transfection stage to synthesize 4 separate polypeptides in one cell and produce a bispecific immunocytokine.

All of the above plasmids were produced in the required quantities in E. Coli cells and purified using the Maxiprep Qiagen kit.

Recombinant proteins containing IL15SA were transiently produced in cells of a Chinese hamster ovary cell line (Cno-Κι line) obtained 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 protein gene (Epstein-Barrvirus nuclear antigen 1) were used. Suspension cultivation was carried out in flasks on an orbital shaker, using serum-free media from Life Technologies Corporation and according to the manufacturer's instructions. For transient expression, cells were transfected at a concentration of 2x10 ⁶ /ml using linear polyethylenimine (PEI MAX, Polysciences). The DNA/PEI ratio was 1:3/1:10. 5-7 days after transfection, the culture medium was centrifuged at 2000 g for 20 min and filtered through a filter with a pore size of 0.22 μm. Target proteins were isolated from the culture liquid using affinity chromatography.

Recombinant proteins were isolated and purified from the culture fluid using a Protein A affinity chromatography column. The clarified culture fluid was passed through a 5 mL HiTrap rProtein A Sepharose FF column (GE Healthcare) equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with 5 column volumes of PBS to wash away nonspecifically binding components. Bound antigen was eluted using 0.1 M glycine buffer pH 3. The major protein peak of the elution was collected and adjusted to pH neutrality with 1 M Tris buffer (pH 8). All stages were carried out at a flow rate of 110 cm/h. Next, the protein was transferred to PBS (pH 7.4) by dialysis using SnakeSkin Dialysis Tubing technology, filtered (0.22 μm), transferred to tubes and stored at -70°C.

The purity of the resulting protein solution was assessed using SDS gel electrophoresis (example Fig. 10).

Example 2.

Kinetic analysis of the interaction of (IL15SA)2-Fc with the human IL2eY receptor.

Binding affinity constants for (IL15SA)2-Fc (hereinafter referred to as CK_IL15Ra_Hc_IL15CH1FcLALA in Fig. 1 and CK_IL15_Hc_IL15RaCH1FcLALA in Fig. 2) to human ^2βγ were obtained using an OctetRed 96 instrument according to ForteBio instructions. AR2G biosensors were prehydrated for an hour in mQ. After activation of the biosensors, (IL15SA)2-Fc preparations at a concentration of 25 μg/ml in acetate buffer pH4 were nonspecifically (according to NH ₂ groups) immobilized on the surface of the biosensor. Then the sensors were immersed in wells with solutions of Human CD122/IL-2RB Protein (Fc Tag) (1 and 0.5 μg/ml) at 30°C in PBS buffer containing 0.1% Tween-20 and 0.1% BSA . In this solution, the association of the IL15SA complex and ^2βγ occurred. After this, the sensors were immersed in a buffer solution for the subsequent dissociation step. The assay was performed at 30°C using PBS containing 0.1% Tween 20 and 0.1% BSA as running buffer.

The obtained sensorgrams are presented in table. 1 for CK_IL15Ra_Hc_IL15CH1FcLALA and for CK_IL15_Hc_IL15RaCH1FcLALA, which were analyzed base-subtracted using Octet Data Analysis (version 8.0) according to a standard procedure using a 1:1 interaction model. The resulting affinity constants are given in Table. 1. Both tested variants showed high affinity and specificity for human ^2βγ.

Table 1. Affinity constants of (IL15SA)2-Fc immunocytokine when interacting with human IL2eY-Fc

Loading Sample ID Cone. (nM) Response KD(M) kon(l/Ms) kdis{1/s) CK_IL15Ra_Hc_IL15CH1FcLA LA 19.4 0.3774 2.47E-10 3.69E+05 9.11E-05 CK_IL15Ra_Hc_IL15CH1FcLA L.A. 9. €9 0.3301 3.80E-10 3.02E+05 1.15E-04 CK_IL15_Hc_IL15 RaCH1FcLA L.A. 19.4 0.325 2.13E-10 3.58E+O5 7.63E-O5 CK_IL15_Hc_IL15 RaCH1FcLA LA 9.69 0.2832 2.12E-10 3.57E+O5 7.55E-O5

- 40 043937

Example 3.

Kinetic analysis of the interaction of bispecific IL15 superagonists with human II,2βγ receptor and other cellular receptor of immune cells.

The binding affinity constants of IL15SA bispecific immunocytokines (hereinafter will be called anti-PD1-Fab-CK_IL15Ra_Hc_IL15CH1FcknobLALA and anti-PD-Ll-FabCK_IL15Ra_Hc_IL15CH1FcknobLALA) with IL2py and human PD-1 and PD-L1 receptors were obtained using the OctetRed 96 device according to ForteBio instructions . AR2G biosensors were prehydrated for an hour in mQ. After activation of the biosensors, bispecific ones at a concentration of 25 μg/ml in acetate buffer pH4 were nonspecifically (by NH ₂ groups) immobilized on the surface of the biosensor. The sensors were then immersed in wells containing solutions of Human CD122/IL-2RB Protein (Fc Tag) or PD-1-Fc or PD-L1-Fc receptor (1 and 0.5 μg/ml) in PBS buffer containing 0.1% Tween-20 and 0.1% BSA for the association of the bispecific superagonist and the receptor of interest. After this, the sensors were immersed in a buffer solution for the subsequent dissociation step.

Instrumental data were analyzed base-subtracted using Octet Data Analysis (version 8.0) according to a standard procedure using a 1:1 interaction model.

The resulting affinity constants are given in Table. 2. Both tested variants showed high affinity and specificity for both human Π-2βγ and paired receptors and lack nonspecific activity for the other receptor.

Table 2. Affinity constants of bispecific IL15 superagonist antibodies when interacting with human IL2eY-Fc and the second receptor

Loading Sample ID PD1 IL2Rb PDL1 Response KD(M) Response KD(M) Responses KD(M) Anti-PD-1 Fab- CK_IL15Ra_Hc _IL15CHIFcknobLAL A 0.7606 3.713E-10 0.4504 1.25E-10 0 0 Anti-PD-Ll Fab - CK_IL15Ra_Hc _IL15CHIFcknobLAL A 0 0 0.3251 2.39E-11 0.3034 2.33E-11 Anti-PD-1 piolgolimab 0.9316 1.36E-10 0 0 0 0 Anti-PDL-1 ECD-135 0 0 0 0 0.3133 <1.OE-12

Example 4.

Cell test to determine the proliferation activity of IL15 superagonists on the NK92 line.

The NK-92 cell line was used for the test. The test was carried out in a 96-well culture plate. The suspension contained cells and NK-92, the test antibody, at the concentration indicated on the graph. All suspension components were prepared in RPMI-1640 medium containing fetal bovine serum and glutamine. After adding all components, the plates were incubated at 37°C, 5% CO2. Next, Alamar Blue dye was added to the wells. After incubation, the fluorescence intensity in the wells was measured. The drugs IL15SA (CK_IL15Ra_Hc_IL15CH1FcLALA), IL15SA/PD1 (Anti-PD-1 Fab-CK_IL15Ra_Hc_IL15CH1FcLALA) and HIL15SA/PDL1 (Anti-PDL-1 FabCK_IL15Ra_Hc _IL15CH1FcLALA) were studied comparatively.

The results of testing the specific activity of the above immunocytokines revealed high proliferative activity against the NK92 line. The calculated EC50 for all candidates turned out to be identical, and the activation level determined by the upper plateau also completely coincided (Fig. 11). It should be noted that the valency of the IL15SA part, and accordingly different avidity, did not have a significant effect on this activity.

Example 5.

Cellular test to determine the proliferation activity of IL15 superagonists on a natural killer cell line.

For the test, we used isolated natural killer cells isolated from PBMCs of healthy donors using the negative selection method. The test was carried out in a 96-well culture plate. The suspension contained natural killer cells and the test antibody at the concentration indicated on the graph. All components of the suspension were prepared in a culture medium containing autologous human plasma. After adding all components, the plates were incubated at 37°C, 5% CO2. Next, Alamar Blue dye was added to the wells. After incubation, the fluorescence intensity in the wells was measured. The effect of human drugs IL15SA (CK_IL15Ra_Hc_IL15CH1FcLALA), IL15SA/PD1 (Anti-PD-1 Fab-CK_IL15Ra_Hc _IL15CH1FcknobLALA) and interleukin-2 (IL2) (Ronleukin NPO Biotech) on the proliferative ability of natural killer cells was comparatively studied.

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The results of testing the specific activity of the above immunocytokines revealed high proliferative activity against natural killer cells (Fig. 12). The calculated EC ₅₀ for all candidates turned out to be identical within the error limits and slightly weaker than the IL2 cytokine. In addition, the level of activation determined by the upper plateau was also completely consistent. It should be noted that the valency of the IL15SA part and, accordingly, different avidity did not have a significant effect on this activity.

Example 6.

Cellular test to determine the anti-PD1 antagonistic activity of IL15 superagonists on a reporter line.

For the test, we used the Jurkat NFAT-FLuc PD-1 cell line, created on the basis of the Jurkat cell line, stably expressing PD-1 on the surface and containing the gene encoding firefly luciferase under the control of the NFAT promoter; and the Raji PDL1 cell line, derived from the Raji cell line, stably expressing PDL1 on the surface. The effect of the drugs IL15SA (CK_IL15Ra_Hc_IL15CH1FcLALA), anti-PD1/IL15SA (Anti-PD-1 Fab-CK_IL15Ra_Hc _IL15CH1FcknobLALA) and anti-PD1 antagonist (prolgolimab) on the activation ability in a reporter cell system was comparatively studied.

The test was carried out in a 96-well culture plate. The suspension in each well contained Jurkat NFAT-FLuc PD-1 cells, Raji PDL1 cells, aCD3/aTAA1 at a concentration of 1 ng/ml, and the test antibody at the concentration indicated on the graph. After adding all components, the plates were incubated at 37°C, 5% CO ₂ and then, using a luminescence assessment kit, the intensity of lucifera in the wells was measured.

The results of testing the specific activity of the above immunocytokines revealed an order of magnitude weaker EC ₅₀ value of specific activity for anti-PD1/IL15SA in comparison with the therapeutically effective monoclonal anti-PD1 antibody prolgolimab (Fig. 13). In addition, the level of maximum activity (upper plateau) is also reduced by 30%. The lower specific activity of anti-PD1/IL15SA than prolgolimab can only be explained by the monovalent Fab binding PD1 in the anti-PD1/IL15SA molecule than the classical antibody. This is also confirmed 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 value indicated for prolgolimab. It should be noted that the antagonistic activity of IL15SA appeared as background, that is, it was practically absent.

Example 7.

Cellular test to determine the anti-PDL1 antagonistic activity of IL15 superagonists on a reporter line.

For the test, we used the Jurkat NFAT-FLuc PD-1 cell line, created on the basis of the Jurkat cell line, stably expressing PD-1 on the surface and containing the gene encoding firefly luciferase under the control of the NFAT promoter; and the Raji PDL1 cell line, derived from the Raji cell line, stably expressing PDL1 on the surface. The effect of IL15SA (CK_IL15Ra_Hc_IL15CH1FcLALA), anti-PDL1/IL15SA (Anti-PDL1 Fab-CK_IL15Ra_Hc _IL15CH1FcknobLALA) and anti-PDL1 antagonist (atezolizumab) on activation ability in a reporter cell system was comparatively studied.

The test was carried out in a 96-well culture plate. The suspension in each well contained Jurkat NFAT-FLuc PD-1 cells, Raji PDL1 cells, aCD3/aTAAl at a concentration of 1 ng/ml, and the test antibody at the concentration indicated on the graph. After adding all components, the plates were incubated at 37°C, 5% CO ₂ and then, using a luminescence assessment kit, the intensity of lucifera in the wells was measured.

The results of testing the specific activity of the above immunocytokines revealed a 30x weaker EC ₅₀ value of specific activity for anti-PD-L1/IL15SA in comparison with the therapeutically effective monoclonal anti-PD-L1 antibody atezolizumab (Fig. 14). However, the level of maximum activity (upper plateau) was reduced by only 10%. The lower specific activity of anti-PD-L1/IL15SA than atezolizumab can only be explained by the monovalent Fab binding PD-L1 in the anti-PD-L1/IL15SA molecule than the classical antibody. However, the antagonistic activity of IL15SA was not significantly recorded.

Example 8.

Cellular test to determine the effect of IL15 superagonists in the presence of an antibody specific against tumor-associated antigen (TAA) on antibody-dependent cellular cytotoxicity (ADCC) of natural killer cells against a TAA reporter line.

For the test, natural killer cells isolated from PBMCs of healthy donors using the negative selection method were used. The test was carried out 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 concentration indicated on the graph. All suspension components were prepared in a culture medium containing fetal bovine serum and glutamine. After adding all components, the plates were incubated at 37°C, 5% CO2. Next, the culture liquid was collected and the lactate dehydrogenase content was examined using an LDH determination kit. The effect of drugs IL15SA (CK_IL15Ra_Hc_IL15CH1FcLALA), anti-PD1/IL15SA (Anti-PD-1 FabCK_IL15Ra_Hc _IL15CH1FcknobLALA) on the ADCC ability of rituximab in a reporter cell system was comparatively studied.

The results of testing the specific activity of the above immunocytokines revealed high activity in enhancing the ADCC ability of rituximab against the TAA reporter line (Fig. 15 and Fig. 16). The efficacy of rituximab-dependent ADCC, as measured by the percentage of lysed Raji cells overexpressing the CD20 receptor on their surface, increased by 30% in the presence of each candidate tested compared with the presence of rituximab alone. Apparently, this effect is explained by the proliferation of active natural killer cells under the influence of IL15SA and antiPD1/IL15SA in the test, according to the results given in examples 4 and 5. The calculated EC50 for all candidates turned out to be identical within the error limits. It should be noted that the valence of the IL15SA part, and accordingly different avidity, did not have a significant effect on this activity, which is also confirmed by examples 4 and 5.

Example 9.

Cellular test to determine the cytotoxicity of IL15 superagonists on PBMCs from whole blood of healthy donors.

PBMCs were isolated from whole blood of healthy donors by Ficoll density gradient centrifugation.

The test was carried out in a 96-well culture plate. The suspension in each well contained freshly isolated PBMCs and the antibody indicated on the graph at a concentration of 0.1 μg/ml. After mixing the PBMCs and antibodies, the plate was incubated for 16 hours at 37°C, 5% CO ₂ . Next, the proportion of CD56+, CD19+, CD3+, CD4+ and CD8+ subpopulations of PBMCs in the suspensions was assessed using direct staining of the suspensions with fluorescently labeled antibodies against the corresponding CDs and subsequent analysis of the cells on a flow cytometer. For CD56+, CD 19+, CD3+ cells, the graphs show the proportion of all cells in the analyzed suspension, for CD4+, CD8+ - the proportion of CD3+ cells. The cytotoxic effect of the drugs IL15SA (CK_IL15Ra_Hc_IL15CH1FcLALA), antiPD1/IL15SA (Anti-PD-1 Fab-CK_IL15Ra_Hc _IL15CH1FcknobLALA), anti-PD1 (prolgolimab), a mixture of antiPD1 (prolgolimab) + IL15SA, anti-CD20 GA101 antibody on PBMCs cells was comparatively studied. person.

The results presented in Fig. 17-21 indicate the absence of a significant cytotoxic effect of the candidates, that is, no more than a 10% decrease in the number of immunoreactive cells in comparison with the negative control AB (those in the absence of any exogenous antibodies). At the same time, the control antibody anti-CD20 naturally showed complete depletion of the CD20+ B cell population. Thus, all candidates considered do not have significant nonspecific cytotoxicity in vitro on human blood cells.

Example 10.

Determination of aggregation stability of IL15 superagonist under thermostress conditions.

The IL15 superagonist drug with a concentration of 9 mg/ml in PBS buffer was heated for 12 hours at a temperature of 50°C. Determination of aggregation after thermal stress was carried out by gel filtration high-performance chromatography. Chromatography was carried out on an HPLC system (Agilent) 1100 on a Tosoh TSK-Gel G3000SWXL column, 7.8 mmx30 cm, cat. No. 08541 with pre-column Tosoh TSKgel Guard SWXL, 6.0 mmx4.0 cm, particle diameter 7 µm, cat. No. 08543. Elution was carried out in isocratic mode with a mobile phase: 50 mM NaPb, 0.3 M NaCl, pH 7.0 at a flow rate of 0.5 ml/min. Detection was carried out at wavelengths of 214 and 280 nm. Antibody samples were diluted with PBS buffer, pH 7.5, to a concentration of ~1 mg/ml. The volume of the injected sample is 10 µl. The calibration mixture Gel filtration standard (from Bio-Rad), cat. No. 151-1901. In fig. Figure 22 shows a chromatogram in which the IL15 superagonist demonstrates high homogeneity (the content of aggregates in the solution was no more than 5%).

Example 11.

Evaluation of the antitumor activity of IL15 superagonist drugs in the syngeneic B16F10 melanoma model.

To evaluate the effectiveness, immunocompetent mice of the C57BL/6 line were used, which were inoculated with the B16F10 tumor line. Each animal in the group was injected subcutaneously into the right flank with 1.5 x 105 tumor cells. On day 7 after cell inoculation (day 1 of the experiment), the animals were divided into groups, as presented in Table. 3. Efficacy was assessed using a single dose of BCD-225, IL15SA/a-mPD-1, a bispecific monoclonal antibody containing IL-15SA and a Fab fragment to mouse PD-1, a derivative of the mouse antibody RMP1-14. The RMP1-14 antibody variable domain sequence was kindly provided by Hideo Yagita, PhD, Juntendo University School of Medicine. Genes and antibody preparations RMP1-14 variable domains were synthesized according to example 1. In addition, a reference drug ALT-803 was synthesized

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[Han KP et al., Cytokine. 2011 Dec;56(3):804-10.] (positive control) and placebo (negative control). The antitumor activity of the drugs in combination with the antibody to mouse PD-1 (a-mPD-1) was also assessed.

Table 3. Distribution of animals into groups in the experiment to determine the antitumor activity of IL15 superagonist drugs

Group Number of animals Dose BCD-325 (IL15SA) 9 0.2 mg/kg IL15ZA/a-mPD-1 9 0.2 mg/kg ALT-803 9 0.2 mg/kg anti-mPD-1 10 10 mg/kg BCD-225 (IL15SA) + antimPD-1 9 0.2 mg/kg + 10 mt/kg IL15SA/a-mPD-1 + antimPD-1 9 0.2 mg/kg + 10 mt/kg ALT-8 03 4- anti-mPD-1 9 0.2 mg/kg + 10 mt/kg Negative control 10 -

The drugs BCD-225 (IL15SA), IL15SA/a-mPD-1 and ALT-803 were administered intraperitoneally in a volume of 0.2 ml on days 1, 4, 8 and 11 of the experiment. The anti-mPD-1 drug was administered intraperitoneally in a volume of 0.2 ml on days 2, 5, 9 and 12 of the experiment. The negative control group was injected with sodium acetate buffer on days 1, 2, 4, 5, 8, 9, 11 and 12 of the experiment in a volume of 0.2 ml (Fig. 23).

During the experiment, the body weight of the mice and the linear dimensions of the tumor were measured. To calculate the tumor volume, we used the formula

V=LxWxWxn/6.

As a criterion for the effectiveness of the study drug, we used the tumor growth inhibition index (TGR), which is calculated taking into account the median value of the tumor volume in the negative control group (V _c ) and the drug group (V _t ) according to the formula

Z L K TRO (%) = * 100

The results of the experiment are shown in Fig. 24 and 25. Data are presented for the 11th day of the experiment due to the death of animals from the tumor load in almost all experimental groups and the impossibility of reliably assessing the antitumor activity of the drugs on the 15th day.

According to the study results, the drug BCD-225 and the reference drug ALT-803 showed comparable antitumor activity: TPO in the BCD-225 group was 52%, in the ALT803 group - 53%. There was a trend towards an increase in the TPO value in the IL15SA/anti-mPD-1 bispecific antibody group compared to the TPO values in the BCD-225 and ALT-803 groups. TPO in the IL15SA/anti-mPD-1 group was 58% on the 11th day of the experiment.

TPO indicators in the groups of the combination of drugs BCD-225 + anti-mPD-1 and ALT-803 + anti-mPD-1 were equal to 72% on the 11th day of the experiment, in the group of the combination IL15SA/a-mPD-1H anti-mPD-1 - 69%. The obtained TPO values indicate a more pronounced antitumor activity of the combination of the studied drugs with anti-mPD-1 compared to the activity of these drugs in monotherapy.

Example 12.

Evaluation and analysis of comparative mortality of mice with IL15 superagonists and ALT803 in the syngeneic B16F10 melanoma model.

For a comparative assessment of the mortality of mice as a result of the use of IL15 superagonist drugs, the model and series of animals described in example 11 were used. Survival was assessed by counting animals that died or were euthanized in an agonizing state.

The results are presented in table. 4. As follows from the data obtained, the mortality rates in the IL15SA/a-mPD-1 and IL15SA/a-mPD-1 + anti-mPD-1 drug groups are on average lower in terms of the number of mice and survival time than for IL15SA (BCD225) , IL15SA + anti-mPD-1, ALT-803, ALT-803 + anti-mPD-1. The obtained mortality values indicate that the bispecific immunocytokine IL15SA/a-mPD-1 is likely less toxic than the monospecific bivalent IL15SA and ALT-803. The presence or absence of anti-PD1 antibody did not have a statistically significant effect on mortality. In addition, analysis of the organs of the deceased mice from the IL15SA and ALT-803 series revealed the presence of hydropic dystrophy in the liver with necrosis of hepatocytes, and compression of the sinusoids. Lymphohistiocytic infiltrates in the perinchyma and accumulation of lymphocytes in the sinusoids. These findings are consistent with previously reported findings in the literature that IL15 superagonists are capable of causing liver injury in mice.

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Claims (66)

Table 4. Distribution of animals by groups in the experiment to determine mortality from drugs IL15 superagonists and ALT803 superagonists and their combinations with anti-PD 1 antibody Group number Treatment Number of dead animals Day of experiment Total number 1) the light chain variable domain includes the amino acid sequence represented by SEQ ID NO: 18; 1) the light chain variable domain includes LCDR 1, 2 and 3 (hypervariable regions 1, 2 and 3), which are respectively represented by the amino acid sequences SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; 1) IL-15Ra, which is associated with: a) the constant domain of the antibody light chain; or b) constant domains of the antibody heavy chain, including the first (CH1) constant domain of the heavy chain and an Fc fragment monomer containing the second (CH2) and third (CH3) constant domains of the heavy chain, 1) IL-15Ra, which is associated with: a) the constant domain of the antibody light chain; or b) constant domains of the antibody heavy chain, including the first (CH1) constant domain of the heavy chain and an Fc fragment monomer containing the second (CH2) and third (CH3) constant domains of the heavy chain, 1. An immunocytokine for stimulating the activation and/or proliferation of IL-15Rbeta/gamma-positive cells, including a heterodimeric protein complex based on IL-15 and IL-15Ra, containing: 1 IL15SA (BCD-225) 2 2 10 14 4 2) the heavy chain variable domain includes the amino acid sequence represented by SEQ ID NO: 17. 2) the heavy chain variable domain includes HCDR 1, 2 and 3 (hypervariable regions 1, 2 and 3), which are respectively represented by the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. 2) IL-15, which is associated with: a) constant domains of the antibody heavy chain, including the first (CH1) heavy chain constant domain and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; or b) the constant domain of the light chain of the antibody, wherein the first constant domain of the heavy chain of the antibody and the constant domain of the light chain of the antibody as part of the heterodimer complex have or do not have a covalent association through a natural S-S bridge and, where, if IL-15 or IL-15Ra is associated with the antibody light chain constant domain, then another part of the heterodimeric protein complex selected from IL-15Ra or IL-15 is associated with the antibody heavy chain constant domains. 2. The immunocytokine of claim 1, wherein the antibody light chain constant domain is selected from CK (kappa light chain constant domain) or CL (lambda light chain constant domain). 2) IL-15, which is associated with: a) constant domains of the antibody heavy chain, including the first (CH1) heavy chain constant domain and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; or b) the constant domain of the light chain of the antibody, wherein the first constant domain of the heavy chain of the antibody and the constant domain of the light chain of the antibody as part of the heterodimer complex have or do not have a covalent association through a natural S-S bridge, and where, if IL-15 or IL-15Ra is associated with the antibody light chain constant domain, then another part of the heterodimeric protein complex selected from IL-15Ra or IL-15 is associated with the antibody heavy chain constant domains. 2 IL15SA/a-mPD-1 2 15 2 3. The immunocytokine according to claim 1, wherein IL-15Ra has the amino acid sequence represented by SEQ ID NO: 9, or any known variant of IL-15Ra containing mutations with similar biological activity. 3 ALT-303 2 7 2 4. The immunocytokine according to claim 1, wherein IL-15 has the amino acid sequence represented by SEQ ID NO: 10, or any known variant of IL-15 containing mutations with similar biological activity. 4 anti-mPD-1 3 14 3 5. The immunocytokine of claim 1, wherein IL-15Ra is linked to the light chain constant domain of the antibody. 5 IL15SA (BCD-225) + anti-mPD-1 1 14 1 6. The immunocytokine according to claim 5, wherein the IL-15Ra bound to the antibody light chain constant domain has the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19. 6 TLlbSA/a-mPD-1 + anti-mPD-1 1 1 eleven 14 2 7. The immunocytokine of claim 1, wherein IL-15 is linked to the light chain constant domain of the antibody. 7 ALT-3 03 4- antimPD-1 3 7 3 Θ Placebo 1 14 1 CLAIM 8. The immunocytokine of claim 7, wherein the IL-15 linked to the antibody light chain constant domain has the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 21. 9. An immunocytokine according to claim 1, wherein the first (CH1) heavy chain constant domain and the Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains are connected through a hinge. 10. The immunocytokine of claim 1, wherein IL-15 or IL-15Ra is linked to antibody heavy chain constant domains in the order CH1-hinge-CH2-CH3. 11. The immunocytokine of claim 10, wherein IL-15Ra is linked to antibody heavy chain constant domains. 12. The immunocytokine of claim 11, wherein the IL-15Ra bound to the antibody heavy chain constant domains has the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 22. 13. The immunocytokine of claim 10, wherein IL-15 is linked to the constant domains of the antibody heavy chain. 14. The immunocytokine of claim 13, wherein the IL-15 linked to the antibody heavy chain constant domains has the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 20. 15. Immunocytokine according to claim 1, where IL-15Ra bound to the antibody light chain constant domain has the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19, and IL-15 bound to the antibody heavy chain constant domains has the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 20. 16. Immunocytokine according to claim 1, where IL-15Ra bound to the antibody heavy chain constant domains has an amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 22, and IL-15 bound to the antibody light chain constant domain has the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 21. 17. An immunocytokine according to claim 1, having mutations in the monomer of the Fc fragment, leading to the absence of ADCC, CDC and/or ADCP properties of the specified immunocytokine. 18. Immunocytokine according to claim 1, where the Fc fragment refers to IgG. 19. The immunocytokine according to claim 18, where the isotype of the Fc fragment is selected from the group: human IgG1, IgG2 or IgG4. 20. An immunocytokine according to any of the above claims 1-19, including the above two heterodimeric protein complexes based on IL-15 and IL-15Ra. 21. An immunocytokine for stimulating the activation and/or proliferation of IL-15Rbeta/gamma-positive cells, comprising a heterodimeric protein complex based on IL-15 and IL-15Ra and an immunomodulatory antibody or an antigen-binding fragment thereof, which specifically inhibits the PD-1 pathway, where the heterodimeric protein complex based on IL-15 and IL-15Ra contains: 22. The immunocytokine of claim 21, wherein the antibody light chain constant domain is selected from CK (kappa light chain constant domain) or CL (lambda light chain constant domain). 23. The immunocytokine according to claim 21, wherein IL-15Ra has the amino acid sequence represented by SEQ ID NO: 9, or any known variant of IL-15Ra containing mutations with similar biological activity. 24. The immunocytokine according to claim 21, wherein IL-15 has the amino acid sequence represented by SEQ ID NO: 10, or any known variant of IL-15 containing mutations with similar biological activity. 25. The immunocytokine of claim 21, wherein IL-15Ra is linked to the light chain constant domain of the antibody. 26. The immunocytokine of claim 25, wherein the IL-15Ra bound to the antibody light chain constant domain has the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19. 27. The immunocytokine of claim 21, wherein the IL-15 is linked to the light chain constant domain of the antibody. 28. The immunocytokine of claim 27, wherein the IL-15 linked to the antibody light chain constant domain has the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 21. 29. The immunocytokine according to claim 21, wherein the first (CH1) heavy chain constant domain and the Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains are connected through a hinge. 30. The immunocytokine of claim 21, wherein IL-15 or IL-15Ra is linked to antibody heavy chain constant domains in the order CH1-hinge-CH2-CH3. 31. The immunocytokine according to claim 30, wherein IL-15Ra is linked to constant domains of the antibody heavy chain. 32. The immunocytokine of claim 31, wherein the IL-15Ra bound to the antibody heavy chain constant domains has the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 24. 33. The immunocytokine of claim 30, wherein the IL-15 is linked to the constant domains of the antibody heavy chain. 34. The immunocytokine of claim 33, wherein the IL-15 linked to the antibody heavy chain constant domains has the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 23. 35. The immunocytokine of claim 21, 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. 36. The immunocytokine according to claim 35, where the immunomodulatory antibody includes: a) a light chain containing a light chain variable domain and a light chain constant domain; b) a heavy chain containing a heavy chain variable domain and constant domains of the antibody heavy chain, including the first (CH1) constant domain of the heavy chain and an Fc fragment monomer containing the second (CH2) and third (CH3) constant domains of the heavy chain. 37. The immunocytokine of claim 36, wherein the light chain variable domain includes LCDR 1, 2 and 3 (hypervariable regions 1, 2 and 3), which are respectively represented by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO : 16. 38. The immunocytokine of claim 37, wherein the light chain variable domain comprises the amino acid sequence represented by SEQ ID NO: 18. 39. The immunocytokine according to claim 36, wherein the heavy chain variable domain includes HCDR 1, 2 and 3 (hypervariable regions 1, 2 and 3), which are respectively represented by the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO : 13. 40. The immunocytokine of claim 39, wherein the heavy chain variable domain comprises the amino acid sequence represented by SEQ ID NO: 17. 41. Immunocytokine according to claim 36, where: 42. Immunocytokine according to claim 41, where: 43. The immunocytokine of claim 36, wherein the immunomodulatory antibody comprises a light chain comprising the amino acid sequence represented by SEQ ID NO: 8. 44. The immunocytokine of claim 36, wherein the immunomodulatory antibody comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 7. 45. The immunocytokine according to claim 36, where the immunomodulatory antibody includes a heavy chain containing the amino acid sequence represented by SEQ ID NO: 7; a light chain containing the amino acid sequence represented by SEQ ID NO: 8. - 45 043937 46. The immunocytokine of claim 21, wherein the immunomodulatory antibody or antigen-binding fragment thereof that specifically inhibits the PD-1 pathway is an antibody that specifically binds to PD-L1. - 46 043937 - 47 043937 b) the immunomodulatory antibody includes a heavy chain containing the amino acid sequence represented by SEQ ID NO: 7; a light chain containing the amino acid sequence represented by SEQ ID NO: 8. 47. Immunocytokine according to claim 21, where: a) a heterodimeric protein complex based on IL-15 and IL-15Ra contains IL-15Ra bound to the antibody light chain constant domain has the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19, and IL-15 bound to the antibody heavy chain constant domains has an amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 23; b) the immunomodulatory antibody includes a heavy chain containing the amino acid sequence represented by SEQ ID NO: 7; a light chain containing the amino acid sequence represented by SEQ ID NO: 8. 48. Immunocytokine according to claim 21, where: a) IL-15Ra bound to the antibody heavy chain constant domains has an amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 24, and IL-15 bound to the antibody light chain constant domain has the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 21; 49. An immunocytokine according to any one of claims 21 or 36, having mutations in the constant domains of the antibody causing heterodimerization of two different parts, one of which includes covalently or non-covalently associated IL-15Ra associated with the constant domains of the antibody, and IL-15 associated with the constant domain of the antibody, and the other includes the covalently or non-covalently associated light and heavy chains of the antibody. 50. The immunocytokine according to any one of claims 21 or 36, wherein the first Fc monomer and the second Fc monomer are selected from the group: the first Fc monomer is an Fc modified to form a Knob and the second Fc monomer is an Fc modified to form a Hole, or when the second Fc monomer is an Fc modified to form a Knob and the first Fc monomer is an Fc modified to form a Hole. 51. Immunocytokine according to claim 50, where: a) the first Fc monomer has amino acid substitutions S354C/T366W, and the second Fc monomer has amino acid substitutions Y349C/T366S/L368A/Y407V; or b) the first Fc monomer has amino acid substitutions Y349C/T366S/L368A/Y407, and the second Fc monomer has amino acid substitutions S354C/T366W. 52. Immunocytokine according to any one of claims 21 or 36, where the Fc fragment refers to IgG. 53. The immunocytokine according to claim 52, where the isotype of the Fc fragment is selected from the group: human IgG1, IgG2 or IgG4. 54. An immunocytokine according to any one of claims 21 or 36, having mutations in the monomer of the Fc fragment, leading to the absence of ADCC, CDC and/or ADCP properties of the specified immunocytokine. 55. Immunocytokine according to any one of claims 21-54 for the treatment of cancer or autoimmune disease. 56. An isolated nucleic acid that encodes an immunocytokine according to any one of claims 1 to 54. 57. The nucleic acid according to claim 56, wherein the nucleic acid is DNA. 58. An expression vector containing a nucleic acid according to any one of claims 56, 57. 59. A method for obtaining a host cell for producing an immunocytokine according to any one of claims 1 to 54, including transforming the cell with a vector according to claim 58. 60. A host cell for producing an immunocytokine according to any one of claims 1 to 54, containing a nucleic acid according to any of claims 56, 57. 61. A method for producing a drug containing an immunocytokine according to any one of claims 1 to 54, consisting in cultivating the host cell according to claim 60 in a culture medium under conditions sufficient to obtain said immunocytokine, if necessary, followed by isolation and purification of the resulting immunocytokine. 62. Pharmaceutical composition for stimulating the activation and/or proliferation of IL15Rbeta/gamma-positive cells, containing the immunocytokine according to any one of claims 1 to 54 in a therapeutically effective amount, in combination with one or more pharmaceutically acceptable excipients. 63. Pharmaceutical composition according to claim 62 for the treatment of cancer or autoimmune disease. 64. The pharmaceutical composition according to claim 63, where the oncological disease is selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer without an identified primary source, 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 CRC (colorectal cancer with high microsatellite instability), leukemia (acute or myeloblastic), lymphoma, multiple myeloma, melanoma , breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory B-cell non-Hodgkin lymphoma , follicular lymphoma, marginal zone B-cell lymphoma, large B-cell diffuse lymphoma, squamous cell carcinoma of the head and neck, pancreatic cancer, ovarian cancer, acute myeloid leukemia and high-risk myelodysplastic syndrome. 65. A method of treating cancer, comprising administering an immunocytokine according to any one of claims 1 to 54 or a pharmaceutical composition according to claim 62 to a subject in need of such treatment in a therapeutically effective amount. 66. The treatment method according to claim 65, where the oncological disease is selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer without an identified primary source, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer ), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), cancer -