Modified IL-2 molecules and their uses

Abstract

The present invention discloses a modified IL-2 molecule, in which the modification involves replacing a region in the IL-2 molecule that mediates binding of the IL-2 molecule to IL2Rα with a region in the IL-15 molecule that mediates binding of the IL-15 molecule to IL15Rα, wherein the region in the IL-15 molecule that mediates binding of the IL-15 molecule to IL15Rα comprises nine or more amino acid residues. Furthermore, the present invention discloses a protein heterodimer comprising the modified IL-2 molecule and IL15Rα (sushi domain) or a mutant thereof. The modified IL-2 molecule and / or the protein heterodimer comprising it of the present invention significantly reduces affinity for IL2Rα, thereby significantly reducing side effects in clinical treatment and enhancing the potential for drug development.

Description

[Technical Field]

[0001] This invention belongs to the biopharmaceutical field and specifically relates to modified IL-2 molecules and their uses. [Background technology]

[0002] Publicly available data from the World Health Organization (WHO) in February 2017 revealed the following: More than 14 million new cancer cases occur worldwide each year, and this number is projected to increase to over 21 million by 2030. 8.8 million people die from cancer each year, with cancer deaths accounting for approximately one-sixth of all deaths worldwide. Cancer prevention and treatment are urgent global challenges that must be addressed in the medical field.

[0003] At present, conventional methods used in cancer treatment include surgery, chemotherapy, hormone therapy, and radiotherapy (see, for example, Stockdale, 1998, *Principles of Cancer Patient Management*, *Scientific American: Medicine*, Vol. 3, edited by Rubenstein and Federman, Chapter 12, Section IV). However, surgery may not be possible depending on the patient's health condition or the advanced stage of the disease, and even if surgery is performed, it is often not possible to completely eliminate cancer cells from the patient's body. Radiotherapy is only effective if the tumor tissue is more sensitive to radiation than normal tissue, and high-dose radiotherapy often causes severe side effects. Hormone therapy is rarely used as a single drug and, while it can be effective, is usually used to prevent or delay cancer recurrence after most cancer cells have been eliminated by other treatments. Furthermore, the biggest challenge with chemotherapy is the possibility that patients may develop resistance to anticancer drugs in a short period of time.

[0004] Furthermore, immunomodulatory factors (e.g., interleukins) can also be applied to animal models and cancer patients, exhibiting antitumor effects. While they can avoid the shortcomings of the conventional therapies mentioned above, their use is severely limited by systemic toxicity and side effects associated with applied immunomodulatory factors. Therefore, there is a need to develop effective treatments that can reduce the toxicity and side effects of immunomodulatory factors. [Overview of the project]

[0005] In light of the limitations of the prior art, one aspect of the present invention provides a modified IL-2 molecule. The modification includes substituting a region in the IL-2 molecule that mediates the binding of the IL-2 molecule to IL2Rα with a region in the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα. The region in the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα contains nine or more amino acid residues.

[0006] In some embodiments, the region in the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα is a continuous amino acid sequence.

[0007] In some embodiments, the region in the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα is not a continuous amino acid sequence.

[0008] In some embodiments, the region in the IL-15 molecule that mediates the binding of the IL-15 molecule to IL15Rα includes one or more non-contiguous amino acid positions.

[0009] In some embodiments, the region in the IL-2 molecule that mediates the binding of the IL-2 molecule to IL2Rα is a continuous amino acid sequence.

[0010] In some embodiments, the region in the IL-2 molecule that mediates the binding of the IL-2 molecule to IL2Rα is not a continuous amino acid sequence.

[0011] In some embodiments, the region in the IL-2 molecule that mediates the binding of the IL-2 molecule to IL2Rα includes one or more non-contiguous amino acid positions.

[0012] In some embodiments, the modification can reduce the affinity of the modified IL-2 molecule for IL2Rα.

[0013] In some embodiments, the modification can eliminate the affinity of the modified IL-2 molecule for IL2Rα.

[0014] In some embodiments, the affinity is measured by biolayer interferometry (BLI).

[0015] In some embodiments, the region mediating the binding of the IL-2 molecule to IL2Rα includes loop M or a variant thereof. Loop M includes the sequence indicated by SEQ ID NO. 1.

[0016] In some embodiments, the region mediating the binding of the IL-2 molecule to IL2Rα includes loop N or a variant thereof. Loop N includes the sequence indicated by SEQ ID NO. 5.

[0017] In some embodiments, the region mediating the binding of the IL-2 molecule to IL2Rα includes helix B or a variant thereof. Helix B includes the sequence indicated by SEQ ID NO. 2.

[0018] In some embodiments, the region mediating the binding of the IL-2 molecule to IL2Rα includes a loop M or a variant thereof and a helix B or a variant thereof. The loop M includes the sequence indicated by SEQ ID NO. 1. The helix B includes the sequence indicated by SEQ ID NO. 2.

[0019] In some embodiments, the region mediating the binding of the IL-2 molecule to IL2Rα includes helix B or its variant and loop N or its variant. The helix B includes the sequence represented by SEQ ID NO. 2. The loop N includes the sequence represented by SEQ ID NO. 5.

[0020] In some embodiments, the region mediating the binding of the IL-2 molecule to IL2Rα includes loop M or its variant and loop N or its variant. The loop M includes the sequence represented by SEQ ID NO. 1. The loop N includes the sequence represented by SEQ ID NO. 5.

[0021] In some embodiments, the region mediating the binding of the IL-2 molecule to IL2Rα includes, from the N-terminus to the C-terminus, loop M or its variant, helix B or its variant, and loop N or its variant in sequence. The loop M includes the sequence represented by SEQ ID NO. 1. The helix B includes the sequence represented by SEQ ID NO. 2. The loop N includes the sequence represented by SEQ ID NO. 5.

[0022] In some embodiments, the region mediating the binding of the IL-15 molecule to IL15Rα includes loop m or its variant. The loop m includes the sequence represented by SEQ ID NO. 8.

[0023] In some embodiments, the region mediating the binding of the IL-15 molecule to IL15Rα includes loop n or its variant. The loop n includes the sequence represented by SEQ ID NO. 12.

[0024] In some embodiments, the region that mediates the binding of the IL-15 molecule to IL15Rα includes helix b or a variant thereof. The helix b includes the sequence represented by SEQ ID NO. 9.

[0025] In some embodiments, the region that mediates the binding of the IL-15 molecule to IL15Rα includes loop m or a variant thereof and helix b or a variant thereof. The loop m includes the sequence represented by SEQ ID NO. 8. The helix b includes the sequence represented by SEQ ID NO. 9.

[0026] In some embodiments, the region that mediates the binding of the IL-15 molecule to IL15Rα includes helix b or a variant thereof and loop n or a variant thereof. The helix b includes the sequence represented by SEQ ID NO. 9. The loop n includes the sequence represented by SEQ ID NO. 12.

[0027] In some embodiments, the region that mediates the binding of the IL-15 molecule to IL15Rα includes loop m or a variant thereof and loop n or a variant thereof. The loop m includes the sequence represented by SEQ ID NO. 8. The loop n includes the sequence represented by SEQ ID NO. 12.

[0028] In some embodiments, the region that mediates the binding of the IL-15 molecule to IL15Rα includes, from the N-terminus to the C-terminus, loop m or a variant thereof, helix b or a variant thereof, and loop n or a variant thereof. The loop m includes the sequence represented by SEQ ID NO. 8, the helix b includes the sequence represented by SEQ ID NO. 9, and the loop n includes the sequence represented by SEQ ID NO. 12.

[0029] In some embodiments, the modification involves substituting a loop M or a variant thereof in the region that mediates the binding of an IL-2 molecule to IL2Rα with a loop m or a variant thereof in the region that mediates the binding of an IL-15 molecule to IL15Rα.

[0030] In some embodiments, the modification involves substituting helix B or a variant thereof in the region mediating the binding of an IL-2 molecule to IL2Rα with helix b or a variant thereof in the region mediating the binding of an IL-15 molecule to IL15Rα.

[0031] In some embodiments, the modification involves substituting loop N or a variant thereof in the region mediating the binding of an IL-2 molecule to IL2Rα with loop n or a variant thereof in the region mediating the binding of an IL-15 molecule to IL15Rα.

[0032] In some embodiments, the modification includes substituting loop M or a variant thereof in the region mediating the binding of IL-2 molecules to IL2Rα with loop m or a variant thereof in the region mediating the binding of IL-15 molecules to IL15Rα, and substituting helix B or a variant thereof in the region mediating the binding of IL-2 molecules to IL2Rα with helix b or a variant thereof in the region mediating the binding of IL-15 molecules to IL15Rα.

[0033] In some embodiments, the modification includes substituting helix B or a variant thereof in the region mediating the binding of IL-2 molecules to IL2Rα with helix b or a variant thereof in the region mediating the binding of IL-15 molecules to IL15Rα, and substituting loop N or a variant thereof in the region mediating the binding of IL-2 molecules to IL2Rα with loop n or a variant thereof in the region mediating the binding of IL-15 molecules to IL15Rα.

[0034] In some embodiments, the modification includes substituting loop M or a variant thereof in the region mediating the binding of an IL-2 molecule to IL2Rα with loop m or a variant thereof in the region mediating the binding of an IL-15 molecule to IL15Rα, and substituting loop N or a variant thereof in the region mediating the binding of an IL-2 molecule to IL2Rα with loop n or a variant thereof in the region mediating the binding of an IL-15 molecule to IL15Rα.

[0035] In some embodiments, the modification includes substituting loop M or a variant thereof in the region mediating the binding of IL-2 molecules to IL2Rα with loop m or a variant thereof in the region mediating the binding of IL-15 molecules to IL15Rα; substituting helix B or a variant thereof with helix b or a variant thereof in the region mediating the binding of IL-15 molecules to IL15Rα; and substituting loop N or a variant thereof in the region mediating the binding of IL-2 molecules to IL2Rα with loop n or a variant thereof in the region mediating the binding of IL-15 molecules to IL15Rα.

[0036] In some embodiments, the modification allows the modified IL-2 molecule to bind to IL15Rα or the sushi domain of IL15Rα.

[0037] In some embodiments, the modification involves substituting loop X or a variant thereof in the IL-2 molecule with loop x or a variant thereof in the IL-15 molecule. Loop x includes the sequence indicated by SEQ ID NO. 10. Loop X includes the sequence indicated by SEQ ID NO. 3.

[0038] In some embodiments, the modification includes substituting helix C or a variant thereof in the IL-2 molecule with helix c or a variant thereof in the IL-15 molecule. Helix c comprises the sequence indicated by SEQ ID NO. 11. Helix C comprises the sequence indicated by SEQ ID NO. 4.

[0039] In some embodiments, the substitution can improve the stability of the modified IL-2 molecule compared to a modified IL-2 molecule that does not contain the substitution.

[0040] In some embodiments, the modification involves making an amino acid substitution in the helix D region of the IL-2 molecule. The hydrophobic core is stabilized by the substituted amino acid being able to form an aromatic ring interaction with phenylalanine (F) at position 43 in helix b. Here, the amino acid position is determined by the EU index of the KABAT number, based on the amino acid sequence of the natural IL-15 molecule. Helix D contains the sequence indicated by SEQ ID NO. 6.

[0041] In some embodiments, the amino acid sequence of the native IL-15 molecule is represented by SEQ ID NO. 13.

[0042] In some embodiments, the amino acid substitution includes W121F, where the amino acid position is determined by the EU index of the KABAT number, based on the amino acid sequence of the native IL-2 molecule.

[0043] In some embodiments, the amino acid sequence of the native IL-2 molecule is represented by SEQ ID NO. 7.

[0044] In some embodiments, the modified native IL-2 molecule contains the sequence indicated by SEQ ID NO. 14.

[0045] In another aspect of the present invention, the present invention provides a protein heterodimer comprising the modified IL-2 molecule described in the present invention and IL15Rα or a variant thereof. Alternatively, the protein heterodimer comprises the modified IL-2 molecule described in the present invention and the sushi domain of the IL15Rα receptor or a variant thereof.

[0046] In some embodiments, the IL15Rα includes the sequence indicated by SEQ ID NO. 16.

[0047] In some embodiments, the sushi domain includes a sequence indicated by SEQ ID NO. 15 or SEQ ID NO. 20.

[0048] In some embodiments, the protein heterodimer has a higher affinity for IL2Rβ than for the IL-2 molecule, without binding to the IL2Rα receptor. The IL-2 molecule includes the native IL-2 molecule and its functional variants.

[0049] In some embodiments, the functional variant of the native IL-2 molecule includes the modified IL-2 molecule.

[0050] In some embodiments, the IL15Rα variant or the sushi domain variant includes an Fc fragment.

[0051] In some embodiments, the Fc fragment is located at the C-terminus of the IL15Rα or the sushi domain.

[0052] In some embodiments, the Fc includes one of human IgG1Fc, human IgG4Fc, and mouse IgG2aa.1Fc.

[0053] In some embodiments, Fc includes the sequence shown in SEQ ID NO. 18, SEQ ID NO. 24, or SEQ ID NO. 25.

[0054] In some embodiments, the protein heterodimer includes the sequence shown in SEQ ID NO. 14 and the sequence shown in SEQ ID NO. 19, SEQ ID NO. 21, or SEQ ID NO. 22.

[0055] In some embodiments, the modified IL-2 molecule and the protein heterodimer described in the present invention can reduce the proliferative activity of Treg cells compared to the unmodified IL-2 molecule.

[0056] In another aspect of the present invention, nucleic acid molecules encoding the modified IL-2 molecule described in the present invention are provided.

[0057] In another aspect of the present invention, a) a modified IL-2 molecule as described in the present invention, and b) a combination of nucleic acid molecules encoding IL15Rα or a variant thereof, or the sushi domain or a variant thereof, as described in the present invention.

[0058] In another aspect of the present invention, a vector comprising a nucleic acid molecule or a combination of nucleic acid molecules described in the present invention is provided.

[0059] In some embodiments, the nucleic acid molecule encoding the modified IL-2 molecule described in the present invention and the nucleic acid molecule encoding IL15Rα or its variant, or the sushi domain or its variant, described in the present invention are located in the same vector.

[0060] In some embodiments, the vector comprises a first vector and a second vector. The first vector comprises a nucleic acid molecule encoding the modified IL-2 molecule described in the present invention. The second vector comprises a nucleic acid molecule encoding IL15Rα or a variant thereof, or the sushi domain or a variant thereof, as described in the present invention.

[0061] In another aspect of the present invention, cells are provided that contain a modified IL-2 molecule as described in the present invention, a protein heterodimer as described in the present invention, a nucleic acid molecule as described in the present invention, a combination of nucleic acid molecules as described in the present invention, or a vector as described in the present invention.

[0062] In another aspect of the present invention, a drug composition is provided comprising a modified IL-2 molecule as described in the present invention, a protein heterodimer as described in the present invention, a nucleic acid molecule as described in the present invention, a combination of nucleic acid molecules as described in the present invention, a vector as described in the present invention, or cells as described in the present invention.

[0063] In another aspect of the present invention, the modified IL-2 molecule described in the present invention, the protein heterodimer described in the present invention, the nucleic acid molecule described in the present invention, the combination of nucleic acid molecules described in the present invention, the vector described in the present invention, and the use of the cell described in the present invention in drug production, where the drug is used for the prevention and / or treatment of tumors.

[0064] In some embodiments, the tumors include anal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, gastric cancer, head and neck squamous cell carcinoma, liver cancer, solid tumors, Merkel cell carcinoma, mesothelioma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, cutaneous squamous cell carcinoma, small cell lung cancer, thymic cancer, thyroid cancer, colon cancer, and / or melanoma.

[0065] Clinically, under low-dose conditions, IL-2 preferentially binds to high-affinity receptors with α chains on the surface of Treg cells, resulting in immunosuppression and failure to achieve therapeutic efficacy. High-dose IL-2 neutralizes the immunosuppression caused by Treg activation by activating a large number of effector T cells, but simultaneously causes greater toxicity, side effects, and apoptosis (activation-induced cell apoptosis). In this invention, by substituting the polypeptide sequence that binds to the receptor α chain in IL-2 with the polypeptide sequence that binds to the receptor α chain in IL-15, binding to the α receptor is reduced or eliminated, thereby weakening the function of IL-2 that promotes the proliferation activity of regulatory T cells. At the same time, by reducing binding to endothelial cell α receptors, the toxicity and side effects caused by IL-2 therapy are reduced or eliminated. Due to these advantages, the modified IL-2 molecule provided in this invention is expected to have broad clinical application potential. [Brief explanation of the drawing]

[0066] [Figure 1] Figure 1 shows the results of SEC-HPLC identification of the IL-2 / 15 chimera 1 and the heterodimer purified product of the sushi-Fc protein in Example 1 of the present invention. Figure 1a is complex 1, Figure 1b is complex 2, and Figure 1c is complex 3. [Figure 2] Figure 2 shows the results of SEC-HPLC identification of the IL-2 / 15 chimera 2 and the purified heterodimer of sushi-hIgG4Fc protein in Example 1 of the present invention. [Figure 3] Figure 3 shows the results of the proliferation effect of complex 1 protein on CTLL-2 (T cells) in Example 4 of the present invention. [Figure 4] Figure 4 shows the results of a predictive toxicology experiment in Example 6 of the present invention. [Figure 5] Figure 5 shows the antitumor effect of the complex against a typical cancer in Example 7 of the present invention. [Figure 6]Figure 6 is a schematic diagram of the four-helix structure of IL-2 and IL-15, where the four α-helices of IL-2 are designated as helix A / B / C / D, and the four α-helices of IL-15 are designated as helix a / b / c / d. [Figure 7] Figure 7 is a schematic diagram comparing the structural superpositions of the complexes formed by IL-15 and IL-2 with their respective receptors. In the central IL-2 or IL-15 region, gray represents IL-2 and black represents IL-15. [Modes for carrying out the invention]

[0067] In this invention, the term "IL-2" usually refers to natural interleukin-2 or its functional variants. Natural interleukin-2 is a cytokine mainly produced by T cells (it can also be produced by B cells, NK cells, and mononuclear macrophages) and is a globular glycoprotein that plays an important role in maintaining the normal function of T lymphocytes and NK cells. It was discovered in 1976 and was then called T cell growth factor (TCGF). In humans, IL-2 is encoded by a gene on chromosome 4. IL-2 is a polypeptide consisting of 133 amino acid residues, with a molecular weight of approximately 15 kD, and has three cysteine ​​residues located at positions 58, 105, and 125, respectively. Post-translational modifications include Thr glycosylation at position 3, a disulfide bond formed by cysteine ​​residues at positions 58 and 105, and a higher structure consisting mainly of four α-helices and multiple linkage sequences (loops) that are essential for its function (Bazan et al., Science 257, 410-413 (1992)). For example, in the present invention (as shown in Figure 6), the four α-helices from the N-terminus to the C-terminus of IL-2 are designated as helix A, helix B, helix C, and helix D, respectively. The linkage sequence (loop) between helices A and B is designated as loop M. The linkage sequence (loop) between helices B and C is designated as loop X. The linkage sequence (loop) between helices C and D is designated as loop N. For example, helix B may include the amino acid sequence indicated by SEQ ID NO. 2, helix C may include the amino acid sequence indicated by SEQ ID NO. 4, helix D may include the amino acid sequence indicated by SEQ ID NO. 6, loop M may include the amino acid sequence indicated by SEQ ID NO. 1, loop X may include the amino acid sequence indicated by SEQ ID NO. 3, and loop N may include the amino acid sequence indicated by SEQ ID NO. 5.

[0068] IL-2 mediates its action by binding to the IL-2 receptor (IL2R). The native IL-2 receptor consists of three subunits: the α (CD25), β (CD122), and γ (CD132) receptor subunits (shown in Figure 7). Here, the α receptor is mainly expressed on the surface of regulatory T cells (Treg) and some endothelial cells, while the β and γ receptor subunits are highly expressed on effector T cells (Teff) and NK cells. The affinity of IL-2 differs depending on the complex configuration of the different receptor subunits. The affinity of IL-2 is highest for the complex consisting of the α, β, and γ receptor subunits, and moderate (approximately 100-fold decrease) for the complex consisting of the β and γ receptor subunits. IL-2 can combine with two forms of receptor subunits, and after binding, both can transmit signals (Minami et al., Annu Rev Immunol 11, 245-268 (1993)).

[0069] In this invention, the term "IL-15" usually refers to natural interleukin-15 or its functional variants. Interleukin-15 (IL-15) is a type of cytokine produced by many cells, including activated mononuclear macrophages, epithelial cells, or fibroblasts. The mature peptide of natural human interleukin-15 contains 114 amino acids, is approximately 12-14 kD, consists of four α-helices and multiple binding sequences (loops), and contains four cysteine ​​residues. Here, Cys35 and Cys85, and Cys42 and Cys88 are bound, and the two pairs of intramolecular disulfide bonds formed play an important role in maintaining the three-dimensional structure and biological activity of IL-15 (Lowe DC, et al J. Mol. Biol. 406:160-175, 2011). For example, in the present invention (as shown in Figure 6), the four α-helices from the N-terminus to the C-terminus of IL-15 are denoted as helix a, helix b, helix c, and helix d, respectively. The linking sequence (loop) between helices a and b is denoted as loop m, the linking sequence (loop) between helices b and c is denoted as loop x, and the linking sequence (loop) between helices cd is denoted as loop n. For example, helix b may contain the amino acid sequence shown in SEQ ID NO. 9, helix c may contain the amino acid sequence shown in SEQ ID NO. 11, loop m may contain the amino acid sequence shown in SEQ ID NO. 8, loop x may contain the amino acid sequence shown in SEQ ID NO. 10, and loop n may contain the amino acid sequence shown in SEQ ID NO. 12.

[0070] IL-15 acts in the normal immune response of an organism, such as promoting the development and proliferation of T cells, B cells, and natural killer (NK) cells. While IL-15 and IL-2 share the same IL2 / 15β and γ chain receptors, they differ in their α chain receptors (as shown in Figure 7). Therefore, IL-2 can activate Treg cells and apoptosis (AICD). IL-15, however, lacks Treg activation and AICD functions (Waldmann TA et al, Nature Reviews Immunol 6:595-601, 2006), and because it consists of different receptor subunits, IL-15 has a more pronounced effect on NK cell proliferation and the maintenance of memory T cells. Accordingly, this invention utilizes the properties of IL-2 and IL-15 by substituting a partial sequence in IL-2 with a partial sequence in IL-15, thereby blocking binding to the IL2Rα receptor while maintaining binding to the IL2 / 15Rβ and γ receptors.

[0071] In the present invention, the term "region mediating the binding of the IL-15 molecule to IL15Rα" usually refers to all or part of a sequence in the IL-15 molecule (including the natural IL-15 molecule or its functional variant) that allows the IL-15 molecule to bind to its receptor α subunit. The region mediating the binding of the IL-15 molecule to IL15Rα may be a continuous amino acid sequence in the polypeptide sequence of the IL-15 molecule, or a non-contiguous amino acid sequence, for example, a combination of multiple non-adjacent amino acid sites.

[0072] In the present invention, the term "region mediating the binding of the IL-2 molecule to IL2Rα" usually refers to all or part of a sequence in an IL-2 molecule (including the natural IL-2 molecule or its functional variant) that allows the IL-2 molecule to bind to its receptor α subunit. The region mediating the binding of the IL-2 molecule to IL2Rα may be a continuous amino acid sequence in the polypeptide sequence of the IL-2 molecule, or a non-contiguous amino acid sequence, for example, a combination of multiple non-adjacent amino acid sites.

[0073] In this invention, the term "biolayer interferometry (BLI)" is also known as biolayer interferometry and refers to an experimental method commonly used in dynamic and quantitative measurement experiments between biomolecules. For example, by utilizing it to detect interactions between biomolecules in real time, it is widely used to measure the dynamic constants of proteins, nucleic acids, and other biomolecules. For instance, it can provide dynamic information including the binding rate constant (Ka), dissociation rate constant (Kd), and affinity constant (KD). For example, the principle is that a biomolecule A can bind to a biosensor end (for example, made of an optical fiber) to create a biolayer. The method includes the following steps: when molecule A at the end of the sensor binds to molecule B to be detected, the molecular weight of the sensor end changes, which in turn changes the thickness of the biolayer; after light passes through the biolayer of the sensor, interference waves are formed by transmission and reflection, and the change in the thickness of the biolayer causes a relative displacement in the interference waves; the interference waves before and after the binding of the biomolecule are detected by a spectrometer and shown by the real-time displacement (nm) of the formed interference spectrum; and finally, the molecule to be detected is analyzed based on the change in the spectrum before and after the molecule binds.

[0074] In this invention, the terms "natural" or "naturally occurring" usually refer to substances that exist in nature (e.g., compound molecules such as proteins and nucleic acids) that have not been artificially altered in structure after separation and identification. Examples include "natural IL-15 molecule" and "natural IL-2 molecule."

[0075] In the present invention, the term "mutant" usually refers to obtaining a polypeptide sequence by modifying a natural polypeptide sequence with amino acids (e.g., by substituting a group), or by inserting, substituting, and / or deleting one or more amino acids (e.g., deleting a partially truncated amino acid sequence). The mutant may retain all or part of the function of the original natural polypeptide sequence, may not retain the function of the original natural polypeptide sequence, or may have improved function compared to the original natural polypeptide sequence (e.g., improved affinity to the corresponding ligand or receptor). Here, a mutant that retains all or part of the function of the original natural polypeptide sequence, or a mutant that has improved function compared to the original natural polypeptide sequence, may be called a "functional mutant." For example, the functional mutant may have better biological activity (or function) than the original sequence. For example, the retention does not necessarily have to be complete retention. For example, the functional mutant can basically retain the function of the original sequence. For example, it retains at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the function of the original sequence. For example, it has 105%, 110%, 115%, 125%, 140%, 160%, 180%, 200%, 230%, or 260% of the function of the original sequence. For example, the amino acid sequence of the functional variant may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% the same as the original amino acid sequence.

[0076] In this invention, the term "C-terminus" usually refers to one of the two ends of a polypeptide chain. The amino acid residue at this terminus has a free α-carboxyl group (-COOH).

[0077] In this invention, the term "N-terminus" usually refers to one of the two ends of a polypeptide chain. The amino acid residue at this terminus has a free α-amino group (-NH2).

[0078] In the present invention, the term "containing, in order from the N-terminus to the C-terminus, ..." usually refers to the positional order of each included sequence fragment or element. Here, each sequence fragment or element does not necessarily have to be directly joined.

[0079] In the present invention, the term “drug composition” usually refers to a composition suitable for administration to a patient, for example, a human patient. For example, a drug composition described in the present invention may include a nucleic acid molecule described in the present invention, a vector described in the present invention and / or a cell described in the present invention, and an optional pharmaceutically acceptable adjuvant. The drug composition may also include a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable components of the composition are non-toxic to the recipient at the drug dosage and concentration used. The drug compositions of the present invention include, but are not limited to, liquid, cryogenic, and lyophilized compositions.

[0080] In the present invention, the term "vector" generally refers to a nucleic acid delivery means that inserts a polynucleotide encoding a protein therein and enables the expression of that protein. A vector enables the expression of its DNA elements within a host cell by transmuting, transmitting, or transfecting the host cell. For example, vectors include plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), and bacteriophages (e.g., λ bacteriophages or M13 bacteriophages and viral vectors). A vector may contain multiple elements that control expression, including a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene. A vector may also contain a replication origin. A vector may further contain components that assist in entry into cells, including, but not limited to, virions, liposomes, or protein capsids.

[0081] In the present invention, the term "prevention" generally refers to the prophylactic administration of a composition to a healthy patient so as to prevent the onset of the diseases and symptoms described in the present invention. The term "prevention" also refers to the prophylactic administration of such a composition to a patient in the pre-stage of a treatable allergic disease. The term "prevention" does not require 100% elimination of the possibility of occurrence. More specifically, "prevention" means that, given the existence of the drug composition or method, the likelihood of occurrence has been reduced.

[0082] In the present invention, the term "treatment" usually refers to administering or supplying a therapeutic agent to a patient, or administering or supplying a therapeutic agent to tissue or cell lines isolated from a patient. The purpose is to treat, cure, alleviate, mitigate, modify, reverse, improve, enhance, or influence the disease, symptoms of the disease, or tendencies toward the disease of a patient who suffers from the disease and exhibits symptoms or tendencies toward the disease. This may include improvement of the condition, elimination of lesions, or improvement of the prognosis.

[0083] In this invention, the terms “tumor” and “cancer” generally refer to cells in which proliferation control has been lost, at least partially, during normal growth and / or development. For example, common tumor or cancer cells typically have lost contact inhibition and can be invasive and / or metastatic.

[0084] In some embodiments of the present invention, bioinformatics and protein engineering techniques are used to replace amino acids primarily involved in binding to IL2Rα in the IL-2 molecule with corresponding sequences of IL-15. Specifically, loops M (SEQ ID NO. 1), B (SEQ ID NO. 2), and N (SEQ ID NO. 5) between helix A and B, and helix C and D, in the IL-2 molecule are replaced with corresponding sequences of IL-15 (i.e., loop m (SEQ ID NO. 8), helix b (SEQ ID NO. 9), and loop n (SEQ ID NO. 12)). This blocks the binding of IL-2 to IL2Rα. Furthermore, to improve the stability of the IL-2 / 15 chimera (modified IL-2 molecule) structure, loops X (SEQ ID NO. 3) and C (SEQ ID NO. 4) between helix B and C of IL-2 are replaced with loop x (SEQ ID NO. 12) of IL-15. 10) The objective is to substitute helix c (SEQ ID NO. 11) and further mutate the tryptophan (W) at position 115 in helix D to phenylalanine (F), thereby forming an aromatic ring interaction with the phenylalanine (F) at position 55 in the introduced IL-15 helix b, while simultaneously stabilizing the hydrophobic core. This stabilizes the 4-helix bundle structure, ultimately enabling the newly formed protein molecule IL-2 / 15 chimera to not bind to the IL2Rα receptor but to retain binding to the β-chain and γ-chain receptors.

[0085] Clinically, under low-dose IL-2 conditions, it preferentially binds to high-affinity receptors with α chains on the surface of Treg cells, resulting in immunosuppression and failure to achieve therapeutic efficacy. High-dose IL-2 neutralizes the immunosuppression caused by Treg activation by activating a large number of effector T cells, but at the same time, it causes more toxicity, side effects, and apoptosis (activation-induced cell apoptosis). The technical solution employed in this application is advantageous in weakening the function of IL-2, which promotes the proliferation activity of regulatory T cells, by reducing or eliminating binding to α receptors. At the same time, it reduces binding to endothelial cell α receptors, thereby reducing or eliminating the toxicity and side effects caused by IL-2 therapy. Because IL-15 lacks Treg activation and AICD functions (Waldmann TA et al, Nature Reviews Immunol 6:595-601, 2006), and because it consists of different receptor subunits, IL-15 shows clearer effects in NK cell proliferation and memory T cell maintenance. Therefore, the present invention utilizes the properties of IL-2 and IL-15 to block binding to the IL2Rα receptor while maintaining binding to the IL2 / 15Rβ and γ receptors.

[0086] In some embodiments, the IL-15 protein sequence introduced into IL-2 can bind to IL15Rα, resulting in co-expression of the IL15Rα receptor (sushi domain) and the IL-2 / 15 chimera, forming a heterodimer complex. This stabilizes the IL-2 / 15 chimera while simultaneously improving its affinity for the IL2 / 15Rβ receptor, leading to better biological activity. Furthermore, Fc is fused and expressed at the C-terminus of the sushi domain (e.g., SEQ ID NO.15 or SEQ ID NO.20), enabling prolonged action. Fc may be human IgG1Fc, human IgG4Fc, or mouse IgG2aa.1Fc.

[0087] The present invention will be further described below in conjunction with examples. These examples are illustrative and do not limit the scope of protection of the present invention.

[0088] In the following examples, experimental procedures where specific conditions are not described shall follow general conditions, such as those described in Sambrook et al.'s Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer. Unless otherwise specified, all reagents used are commercially available or publicly available.

[0089] Example 1: Production of IL-2 / 15 chimeras from mammalian cells 1.1 Synthesis and Construction of Expression Plasmids We commissioned Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize target gene fragments for four proteins: IL-2 / 15 Chimera 1 (SEQ ID NO. 14), sushi-hIgG1Fc (SEQ ID NO. 19), sushi-hIgG4Fc (SEQ ID NO. 21), and sushi-mIgG2aa.1Fc (SEQ ID NO. 22). These fragments were cloned between the EcoRI and HindIII sites of a pZD vector to obtain four expression plasmids. The plasmid numbers are PM619, PM432, PM657, and PM599, respectively. Based on plasmid PM619, we performed a point mutation according to the method described in "Molecular Cloning," mutating phenylalanine (F) at position 115 (corresponding to position 121 of the natural IL-2 molecular amino acid sequence) to tryptophan (W), thereby obtaining plasmid IL-2 / 15 Chimera 2 (SEQ ID NO. 23). The plasmid number is PM824. The synthesized or constructed plasmids were transmuted to DH10B, sequenced, and stored.

[0090] 1.2 Plasmid Extraction and Preparation of HEK293 Cells 1.2.1 Plasmid Extraction Following the procedures described in the "Qiagen Mini-prep Kit" and the "Qiagen Endofree Maxi-prep Kit," the two IL-2 / 15 chimeras and three sushi-Fc plasmids mentioned above were prepared.

[0091] 1.2.2 Preparation of HEK293 cells Density is 1 - 1.2 × 10 6 Newly passaged HEK293 cells (National Research Council, Canada) with a concentration of / mL were used for transient expression.

[0092] 1.3 Transient HEK293 expression 1.3.1 Reagent Preparation A) For G418 solution, 250 mg of Geneticin TM The sample was weighed, 4.5 ml of ultrapure water was added and dissolved, the volume was adjusted to 5 ml with ultrapure water, filtered through a 0.22 μm filtration membrane, and stored at -20°C. B) For the PEI solution, 50 mg of PEI was weighed out, added to 45 ml of ultrapure water and dissolved, the pH was adjusted to 7.0 with 1 M NaOH, the volume was reduced to 50 ml with ultrapure water, filtered through a 0.22 μm filtration membrane, and stored at -20°C. C) Regarding the culture medium, 1L of FreeStyle TM 293 Expression Medium, 10ml Pluronicd TM F-68 and 500 μL of G418 were added. D) The plasmid was pre-prepared in a 2 mL deendotoxinated centrifuge tube. E) Depending on the volume required for transfection, newly passaged 1-1.2 × 10 6 A cell suspension was prepared at a concentration of cells / mL.

[0093] 1.3.2 Preparation of the transfection reagent-plasmid complex Solution A: Plasmid 1 μg / mL + Opti-MEM TM 33.3 μL / mL Solution B: PEI 2 μg / mL + Opti-MEM TM 33.3 μL / mL 1 mL of solution B was added to 1 mL of solution A, mixed uniformly, incubated for 10 minutes, and then the cell suspension was added.

[0094] IL-2 / 15 chimeric 1 and three kinds of sushi-Fc were mixed at a mass ratio of 1:1 (total amount 1 μg / mL) and transfected. IL-2 / 15 chimeric 2 and sushi-hIgG4Fc were mixed at a mass ratio of 1:1 (total amount 1 μg / mL) and transfected.

[0095] 1.3.3 Exchange of liquid After culturing at 115 rpm, 36.8 °C, and 5% CO2 for 4 hours, centrifuged at 800 g for 5 minutes, and replaced with FreeStyle TM 293 Expression Medium without the addition of Pluronicd TM F-68 and G418.

[0096] 1.3.4 Expression and collection of culture After culturing at 115 rpm, 36.8 °C, and 5% CO2 for 5 days, centrifuged at 8500 rpm for 15 minutes, and the cell supernatant was collected and purified.

[0097] 1.4 Purification preparation IL-2 / 15 chimera 1 was co-expressed with sushi-hIgG1Fc, sushi-hIgG4Fc, and sushi-mIgG2aa.1Fc, respectively. During expression, heterodimer complexes were formed, designated as complex 1, complex 2, and complex 3. All three complexes possessed an Fc tag. Affinity purification was performed using Mabselect sure (Protein A, GE Healthcare). The purification method can be found in the purification packing manual. Further purification was performed via gel filtration chromatography (Superdex 200 pg, GE Healthcare) to obtain a highly pure protein, from which polymers were removed. Purity was analyzed using high-efficiency liquid-phase chromatography molecular sieve column (SEC-HPLC), and the results are shown in Figure 1(a-c). The SEC main peak of the purified protein was a single peak, indicating that IL-2 / 15 chimera 1 formed complexes with each of the three Sushi-Fc types. The purity was high, and polymers and fragments accounted for less than 5% overall. Liquid-phase chromatography-mass spectrometry revealed that the purified protein contained two components: Sushi-Fc and IL-2 / 15. This demonstrated successful protein expression and purification. IL-2 / 15 chimera 2 was also co-expressed with sushi-hIgG4Fc, forming a complex and being purified (shown in Figure 2).

[0098] Example 2: Production of IL-2 / 15 chimeras from E. coli 2.1 Synthesis and Construction of Expression Plasmids We commissioned Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the IL-2 / 15 chimera 1 gene fragment (SEQ ID NO. 26), cloned it between the NdeI and XhoI sites of the pET41a vector, and obtained an expression plasmid. The number is 1187.

[0099] 2.2 Expression and Purification of IL-2 / 15 Chimeras in Escherichia coli Plasmid extraction was performed according to the procedure described in "Molecular Cloning," and transgenic expression of strain BL21(DE3) was carried out. IL-2 / 15 chimera 1 was expressed according to conventional methods for prokaryotic protein expression, and then purified using traditional denaturation and regeneration techniques and chromatography (see, for example, Yunier Rodriguez-Alvarez et al, Preparative Biochemistry and Biotechnology, 47:9, 889-900) to obtain a relatively pure chimeric protein.

[0100] Example 3: Measurement of the binding ability of IL-2 / 15 chimeras to IL2Rα and IL2 / 15Rβ The affinity between the target protein and the receptor was measured using biolayer interferometry (BLI). This was done in reference to the method described in Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):p.270-8. The receptor proteins IL-15Rα-his, IL2Rα-his, and IL2 / 15Rβ-Fc / Fc used in the experiment were all manufactured by our company. The IL-2 derivatives and IL-2 complexes without IL2Rα binding were manufactured according to the description in published patent application CN111018961A. The IL-15(N72D) / sushi-hIgG1Fc complex was manufactured according to the description in the literature (K.-p. Han et al. / Cytokine 56 (2011) 804-810). The buffer solution consisted of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.1% BSA, and 0.05% tween20. Receptor proteins were pre-immobilized onto their respective sensors, and then subjected to baseline, loading, baseline, association, and dissociation steps using established methods (see Estep, P, etc., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p.270-8). Data acquisition and analysis were performed using Fortebio Octet RED96 software, Data acquisition 11.0 and Data analysis 11.0, respectively. As shown in Table 1 (lower values ​​indicate stronger affinity), IL-2 / 15 chimera 1 and IL-2 / 15 chimera 1-sushi-Fc (complex 1, complex 2, or complex 3) did not bind to the IL2Rα receptor compared to IL-2.Compared to IL-15, the affinity of IL-2 / 15 chimera 1 to the IL-15Rα receptor is less than 10 times lower, and complexes 1, 2, or 3 do not bind to IL-15Rα. In comparison with the affinity to IL-2 / 15Rβ, IL-2 / 15 chimera 1 is clearly weaker than IL-15 and slightly weaker than IL-2. The affinity of complexes 1, 2, or 3 is stronger than IL-2 or IL-15, but weaker than the IL-15(N72D) / sushi-hIgG1Fc complex. [Table 1]

[0101] Example 4: Experiment to promote T cell proliferation The proliferation experiment of CTLL-2 (T cells) is a commonly used experiment to measure interleukin-stimulated immune cell activity at the cellular level. In this example, the biological activity of IL-2 / 15 chimera 1, prepared in Example 1, was measured by the proliferation experiment of CTLL-2 cells. CTLL-2 cells were cultured using a culture medium containing FBS and Rat-T-Stim (purchased from ATCC, product number ATCC(registered trademark) TIB-214). TM The CTLL-2 cells, quantified at 21,000 cells / well, were inoculated into a 96-well culture plate. Simultaneously, the positive control (IL-2) and the target sample complex 1 protein were serially diluted and added to the plate. The initial working concentration was set to 0.0078125 nM, with 3 wells prepared for each dilution concentration. A culture medium control well (100 μL cells + 100 μL culture medium) was also prepared. The plates were incubated at 37°C and 5% CO2 for 72 hours. Subsequently, 20 μl of CellTiter96® AQueous One Solution Reagent (purchased from Promega, catalog number Cat# G3581)) was added to each well, and the plates were incubated at 37°C and 5% CO2 for 2-4 hours. The absorbance (A) at a wavelength of 490 nm was measured using a microplate reader, and the EC50 value was calculated. The results are shown in Table 2 and Figure 3. Complex 1 protein clearly promotes the proliferation activity of CTLL-2 (T cells), and this activity is higher than that of the positive control IL-2. [Table 2]

[0102] Example 5: Experiment to promote Mo7e cell proliferation Mo7e cell lines (expressing IL-2 / 15Rβ and γ receptors) were cultured in complete medium (RPMI1640 + 10% FBS + GM-CSF (10 ng / mL)) at 37°C and 5% CO2. Cells in the logarithmic growth phase were obtained. The cells were washed twice with PBS to wash away cytokines, and then resuspended in cytokine-free medium. Cell density was adjusted in cytokine-free medium and inoculated into 96-well cell culture plates. 90 μL was inoculated into each well. The total number of cells was 5000. For later use, the cells in the 96-well plates were cultured at 37°C and 5% CO2. A 10-fold drug solution was prepared, resulting in nine concentrations and 3.16-fold dilutions with a final working concentration of 0.020116 μM. Subsequently, 10 μL of each serial dilution sample was transferred to the corresponding experimental wells of the 96-well cell plate, creating three wells for each drug concentration. Cells in a 96-well plate containing the drug were cultured continuously at 37°C and 5% CO2 for 72 hours. Subsequently, CTG (Celltiter Glo assay kit, Promega) analysis was performed. The same volume of CTG solution was added to each well. The cell plates were left at room temperature for 20 minutes to stabilize the luminescence signal. Luminescence values ​​were read and data were collected. The data were analyzed using GraphPad Prism 7.0 software, and dose-response curves were obtained by fitting the data using nonlinear S-curve regression, and EC50 values ​​were calculated.

[0103] Cell viability (%) = (Lum drug-treated sample - Lum medium control) / (Lum solvent control - Lum medium control) × 100%.

[0104] In this experiment, neither hIgG1 nor hIgG4 promoted cell proliferation when used as controls. The results showed that the activity of IL-2 / 15 chimera 1 alone was between that of IL-15 and IL-2, approximately 10 times lower than that of IL-15. The IL-2 / 15 chimera 1 complex with Sushi showed weaker activity than the IL-15(N72D) / sushi-hIgG1Fc complex. Previously, IL-2 / 15Rβγ receptor agonists have been clinically shown to cause drug toxicity due to excessively high activity; therefore, the IL-2 / 15 chimera 1 complex with Sushi appears to be safer. [Table 3]

[0105] Example 6: Predictive toxicological testing of cynomolgus monkeys In the experiment, the animals were divided into two groups: one receiving complex 2 and the other receiving the IL-15(N72D) / sushi-hIgG1Fc complex. Each group contained one male and one female animal, and they were administered 1 mg / kg intravenously once a week for a total of two doses. The animals' clinical symptoms were observed daily, their body weight was measured, and drug tolerance was analyzed.

[0106] Experiments showed that females in the complex 2 group exhibited small amounts of yellowish loose stools on days 2-4, but both males and females maintained relatively stable body weight over 14 days (as shown in Figure 4), indicating tolerance to a 1 mg / kg dose. On the other hand, females and males in the IL-15(N72D) / sushi-hIgG1Fc complex group showed lethargy, decreased spontaneous activity, small to moderate amounts of yellowish loose stools, and orbital depression on days 3-4. They died on day 4. Thus, tolerance to a 1 mg / kg dose was not demonstrated in these groups.

[0107] Example 7: Pharmacokinetic evaluation of IL-2 / 15 chimeric complex in a C57BL / 6 mouse tumor model obtained by subcutaneous transplantation of mouse cutaneous melanoma B16F10. 5.0×10 6A B16F10 cell suspension at a concentration of cells / mL was pipetted to ensure uniformity and placed on ice. The right hind limb of a mouse was dehaired and disinfected with an iodine cotton ball. Using a 1 mL syringe, 0.1 mL of the cell suspension was applied to the right hind limb of the mouse in 5 × 10⁻¹⁴⁻¹ 5 The cells were administered by subcutaneous injection at a single point. On the day of tumor cell inoculation, the mice were randomly divided into three groups of five based on their body weight. The day the groups were divided was designated Experiment Day 0, and the drug was administered from the following day (Day 1). Details of the administration and groups are shown in Table 4. [Table 4]

[0108] After inoculation with tumors, the tumor growth, motor function, food intake, body weight, eyes, coat, and other abnormal behaviors of the mice were regularly checked to provide humane endpoints. After administration to each group, the tumor volume of the mice was measured twice a week. A bidirectional measurement method was used to measure the tumor volume, with the long and short diameters of the tumor measured using calipers. Let the long diameter be a and the short diameter be b, and the formula T = 0.5 × a × b 2 The volume of the tumor was then calculated.

[0109] Relative Tumor Suppression Rate (TGI) (%): TGI = 1 - T / C (%). T / C% is the relative tumor growth rate, i.e., the percentage of the relative tumor volume or tumor weight of the treatment group compared to the control group at a given time point. T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively. The formula is as follows: T / C % = TRTV / CRTV × 100% (TRTV: mean RTV of the treatment group; CRTV: mean RTV of the solvent control group; RTV = V t -V0 (where V0 is the tumor volume of the animal in question when divided into groups, and Vt is the tumor volume of the animal in question after treatment).

[0110] The results are shown in Figure 5. In this experiment, the efficacy of the reagent administered alone was investigated in a C57BL / 6 female mouse model inoculated subcutaneously with mouse melanoma B16F10 cells. Here, the animals in the G1 control group had an average tumor volume of 2942 ± 545.3 mm² on Day 13.3 In group G2, complex 2 was administered at 2 mg / kg BIW until Day 13, and the TGI% in this group was 95%, showing a very significant difference compared to the control group (p<0.01). In group G3, complex 3 was administered at 2 mg / kg BIW until Day 13, and the TGI% in this group was 92%, showing a very significant difference compared to the control group (p<0.01). The data showed that in a C57BL / 6 mouse model inoculated with mouse melanoma tumor cells B16F10, both complex 2 and complex 3 reagents showed strong efficacy after 13 days of administration, with a statistically significant difference compared to the control group (p<0.01). Complex 2 and complex 3 showed no difference and both had clear tumor suppressor effects. [Sequence Listing Free Text]

[0111] SEQ ID NO: 14: Amino acid sequence of IL-2 / IL-15 chimera 1 Sequence ID 19: Amino acid sequence of Sushi-hlgG1Fc SEQ ID NO: 21: Amino acid sequence of Sushi-hIgG4Fc Sequence ID 22: Amino acid sequence of Sushi-mlgG2aa.1Fc SEQ ID NO: 23: Amino acid sequence of IL-2 / 15 chimera 2 SEQ ID NO: 26: Amino acid sequence of synthesized IL-2 / 15 chimera 1

Claims

1. A protein heterodimer comprising a modified IL-2 molecule indicated by SEQ ID NO. 14 and the Sushi domain of IL15R indicated by SEQ ID NO. 19, SEQ ID NO. 21, or SEQ ID NO.

22.

2. A drug characterized by comprising the protein heterodimer described in claim 1.

3. The drug according to claim 2, characterized in that it is used for the prevention and / or treatment of tumors.

4. The drug according to claim 3, characterized in that the tumor includes anal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, gastric cancer, head and neck squamous cell carcinoma, liver cancer, solid tumors, Merkel cell carcinoma, mesothelioma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, cutaneous squamous cell carcinoma, small cell lung cancer, thymic cancer, thyroid cancer, colon cancer, and / or melanoma.

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