A fusion molecule of CTLA4 and IL-15

Abstract

Provided herein is a soluble fusion protein complex comprising domains from IL-15, the IL-15 receptor and the αCTLA4 antibody for preventing, reducing the incidence of and / or treating cancer or an autoimmune disease or disorder in a subject. Provided herein is a method comprising administering to a subject a pharmaceutical composition comprising the soluble fusion protein complex.

Description

[Technical Field]

[0001] Cross-reference of related applications This patent application claims benefits under 35 U.S. SC § 119(e) of U.S. Provisional Patent Application No. 63 / 302,044, filed on 22 January 2022. The entire contents of U.S. Patent No. 63 / 302,044 are incorporated herein by reference.

[0002] Sequence listing This disclosure includes references to amino acid sequences and nucleic acid sequences submitted concurrently with this specification as a sequence listing ST.26 XML file named "000112wopoa_SequenceListing.XML" (file size 29.8 kilobytes (KB), created January 10, 2023). The above sequence listing is incorporated herein by reference in its entirety in accordance with 37 C. FR §1.52(e)(5).

[0003] This disclosure relates to multimeric soluble fusion protein complexes and their use in the treatment of diseases such as cancer and autoimmune diseases. [Background technology]

[0004] Cytotoxic T lymphocyte antigen 4 (CTLA4), a member of the immunoglobulin superfamily, is a molecule expressed by activated T cells. CTLA4 is analogous to the T cell costimulatory molecule CD28, and both molecules bind to B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells (APCs). However, CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals.

[0005] Interleukin-15 (IL-15) is CD8 + / CD4 + By stimulating T cells and natural killer (NK) cells, both innate and adaptive immune responses can be promoted, but T regulatory (T regIt does not show efficacy in activating cells or in inducing activation-associated death of effector T cells and NK cells. Therefore, IL-15 is a promising molecule for antitumor immunotherapy. Bessard & al. (2009) Mol Cancer Ther. 8(9):2736-45.

[0006] While IL-15 has great potential for therapeutic use, the main limitations in the clinical development of recombinant human IL-15 (rhIL-15) are its low production yield and short serum half-life in standard mammalian cell expression systems. Ward & al. (2009) Protein Expr Purif. 68(1):42-48. [Overview of the project] [Means for solving the problem]

[0007] A soluble fusion protein complex is disclosed herein, comprising a first domain containing an IL-15 peptide or a variant thereof, a second domain containing a fusion polypeptide comprising an IL-15RαSushi peptide (IL15RαSu) and an αCTLA4 antibody heavy chain, and a third domain containing an αCTLA4 antibody light chain. The IL-15 peptide or variant binds to the IL15RαSu peptide to form the soluble fusion protein complex. The soluble fusion protein complex and its pharmaceutical composition modulate the immune response. Methods for preventing or treating cancer by enhancing the immune response and methods for preventing or treating autoimmune diseases or disorders by reducing the immune response are also disclosed herein.

[0008] In some embodiments, the soluble fusion protein complex comprises (a) a first domain having an IL-15 peptide having sequence identity with SEQ ID NO: 17 (e.g., at least about 85% sequence identity); (b) a second domain having a fusion polypeptide comprising an IL15RαSu peptide and an αCTLA4 antibody heavy chain having sequence identity with SEQ ID NO: 10 (e.g., at least about 85% sequence identity); and (c) a third domain having an αCTLA4 antibody light chain having sequence identity with SEQ ID NO: 3 (e.g., at least about 85% sequence identity). In some embodiments, the IL15RαSu peptide has sequence identity with SEQ ID NO: 8 (e.g., at least about 85% sequence identity). The first domain may contain an IL-15 variant (IL-15N72D) including the N72D mutation. Furthermore, the IL-15 peptide in the first domain may or may not contain a leader sequence.

[0009] In some embodiments, the soluble fusion protein complex may include a complementation-determining region on the αCTLA4 antibody heavy chain of the second domain (e.g., one or more of SEQ ID NOs. 11-13) and / or on the αCTLA4 antibody light chain of the third domain (e.g., one or more of SEQ ID NOs. 14-16). The fusion polypeptide of the second domain may further include a linker between the IL15RαSu peptide and the αCTLA4 antibody heavy chain. The fusion polypeptide may have sequence identity with SEQ ID NOs. 4 or SEQ ID NOs. 5 (e.g., at least about 85% sequence identity).

[0010] In further embodiments, the soluble fusion protein complex may be formulated into a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. The pharmaceutical composition may be formatted for various routes of administration, including parenteral injection. The pharmaceutical composition may be formulated for subcutaneous, intravenous, intramuscular, intravesicular, intratumoral, or intraperitoneal injection.

[0011] A method for preventing or treating cancer is also described, comprising administering a soluble fusion protein complex described herein to a subject in need, comprising a first domain having an IL-15 peptide or a variant thereof, a second domain having a fusion polypeptide comprising an IL15RαSu peptide and an αCTLA4 antibody heavy chain, and a third domain having an αCTLA4 antibody light chain. The soluble fusion protein complex can be formulated into a pharmaceutical composition suitable for administration to a subject for the prevention or treatment of cancer.

[0012] Methods for preventing or treating autoimmune diseases or disorders are also described, comprising administering a soluble fusion protein complex described herein to a subject in need, comprising a first domain having an IL-15 peptide or a variant thereof, a second domain having a fusion polypeptide comprising an IL15RαSu peptide and an αCTLA4 antibody heavy chain, and a third domain having an αCTLA4 antibody light chain. The soluble fusion protein complex can be formulated into a pharmaceutical composition suitable for administration to a subject for preventing or treating autoimmune diseases or disorders.

[0013] Various purposes, features, embodiments, and advantages will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings. [Brief explanation of the drawing]

[0014] [Figure 1] The keys for interpreting Figures 2 and 3, along with two exemplary molecules illustrated according to these keys, are shown. [Figure 2] Three "TxM" configurations are shown. Configuration A (also called TxM-A, N-844-1) has an antibody domain conjugated to the IL15RαSu peptide. Configuration B (also called TxM-B, N-844-2) has an antibody domain conjugated to the IL-15 peptide. Configuration C (also called TxM-C, N-844-3) has an antibody domain conjugated to the CH3 half of the Fc domain. [Figure 3]Shows two CTLA4-targeted IL-15 molecules that are not assembled in the "TxM" configuration. As used herein, "IL-15 / αCTLA-4" also refers to N-844-4, and "αCTLA-4 / IL-15" also refers to N-844-5. [Figure 4] Graphical representation of differential scanning fluorimetry Tm analysis of various molecules shown in FIGS. 2 and 3 (Nogapentin alpha interferon (NAI or also known as ALT-803) (N-803); α-CTLA4-TxM-A (N-844-1); α-CTLA4-TxM-B (N-844-2); α-CTLA4-TxM-C (N-844-3); IL-15-α-CTLA4 (N-844-4); α-CTLA4-IL-15 (N-844-5)). [Figure 5] Graphical representation of mean tumor growth in mice inoculated with B16.F10 melanoma tumor cells in both flanks. Treatment was administered directly to the tumors on one flank ("injected tumor"). Tumor growth was monitored in both the injected tumor and the non-injected tumor on the other flank ("distal tumor"). [Figure 6A] Graphical representation of tumor growth in individual mice aggregated in FIG. 5. FIG. 6A shows tumors grown in individual mice treated with αOX40 antibody + αCTLA4-7. FIG. 6B shows tumors grown in individual mice treated with αOX40 antibody + α-CTLA4-TxM-A (N-844-2). FIG. 6C shows tumors grown in individual mice treated with αOX40 antibody + IL-15-α-CTLA4 (N-844-4). FIG. 6E shows tumors grown in individual mice treated with αOX40 antibody + αCTLA4-7 + NAI (N-803). [Figure 6B] Graphical representation of tumor growth in individual mice aggregated in FIG. 5. FIG. 6B shows tumors grown in individual mice treated with αOX40 antibody + α-CTLA4-TxM-A (N-844-2). [Figure 6C] Graphical representation of tumor growth in individual mice aggregated in FIG. 5. FIG. 6C shows tumors grown in individual mice treated with αOX40 antibody + IL-15-α-CTLA4 (N-844-4). [Figure 6D] Figure 5 shows a graph of tumor growth in individual mice. Figure 6A shows tumors grown in individual mice treated with αOX40 antibody + αCTLA4-7. Figure 6B shows tumors grown in individual mice treated with αOX40 antibody + α-CTLA4-TxM-A (N-844-2). Figure 6C shows tumors grown in individual mice treated with αOX40 antibody + IL-15-α-CTLA4 (N-844-4). Figure 6E shows tumors grown in individual mice treated with αOX40 antibody + αCTLA4-7 + NAI (N-803). [Figure 6E] Figure 5 shows a graph of tumor growth in individual mice. Figure 6E shows tumors grown in individual mice treated with αOX40 antibody + αCTLA4-7 + NAI (N-803). [Figures 7A-7B] This graph shows the efficacy of treatment in injected tumors and distal tumors on day 11, based on area under the curve (AUC) calculation. Figures 7A and 7B show the efficacy of treatment in injected tumors and distal tumors, respectively. Figure 7C shows the tolerability of treatment up to day 18. [Figure 7C] This graph shows the efficacy of treatment in injected and distal tumors on day 11, based on area under the curve (AUC) calculation. Figure 7C shows the tolerability of treatment up to day 18. [Modes for carrying out the invention]

[0015] Definitions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out the tests of the present invention, but preferred materials and methods are described herein. The following terms are used in the description and claims of the present invention. It should also be understood that the terms used herein are intended solely to describe and not to limit specific embodiments.

[0016] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “element” means one or more elements. Thus, the enumeration of “cells” includes, for example, multiple cells of the same type. Furthermore, to the extent that the terms “containing,” “inclusion,” “having,” “possessing,” or variations thereof are used in any part of the detailed description and / or claims, such terms are intended to be as comprehensive as the term “containing.”

[0017] As used herein and in the appended claims, “or” is used in its general sense, including “and / or,” unless the context clearly indicates otherwise.

[0018] As used herein, "approximately" when referring to measurable values ​​such as quantity or duration means that the value is rounded to the last significant digit. For example, the designation "approximately 2.5" means reading a range of values ​​from 2.45 (which is rounded up to 2.5) to 2.54 (which is rounded down to 2.5).

[0019] The term "improve" means to reduce, suppress, weaken, decrease, stop, or stabilize the onset or progression of a disease.

[0020] The terms “antibody” or “immunoglobulin” encompass both polyclonal and monoclonal antibodies. Preferred antibodies are monoclonal antibodies that react with an antigen. The term “antibody” also encompasses mixtures of two or more antibodies that react with an antigen (e.g., a cocktail of different types of monoclonal antibodies that react with the antigen). Furthermore, “antibody” encompasses whole antibodies, their biologically functional fragments, single-chain antibodies, and genetically modified antibodies, such as chimeric antibodies containing portions from two or more species, bifunctional antibodies, antibody conjugates, humanized antibodies, and fully human antibodies. Similarly, biologically functional antibody fragments that can be used are peptide fragments derived from an antibody sufficient to bind to an antigen. As used herein, “antibody” means the whole antibody as well as any antibody fragment (e.g., F(ab')2, Fab', Fab, Fv) that can bind to the desired epitope, antigen, or antigenic fragment. To “bind” to a molecule means to have physicochemical affinity for that molecule.

[0021] As used herein, the term "cancer" means a disease characterized by unregulated cell proliferation or replication, as is known in the art. "Cancer" includes colorectal cancer and AIDS-related cancers such as leukemia, e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia, Kaposi's sarcoma; breast cancer; bone cancers such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantinoma, and chordoma; brain tumors such as meningioma, glioblastoma, low-grade astrocytoma, oligodendroma, pituitary tumor, Schwann cell tumor, and metastatic brain tumor; head and neck cancers including various lymphomas such as mantle cell lymphoma, non-Hodgkin lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer; retinal cancers such as retinoblastoma; esophageal cancer; gastric cancer; and multiple cancers. Myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung cancer), pancreatic cancer, sarcoma, Wilms' tumor, cervical cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma, multidrug-resistant cancer; and proliferative disorders such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet / dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration, and other proliferative disorders and conditions such as restenosis and polycystic kidney disease, as well as other cancers or proliferative disorders that can respond to the modification of their environment, either alone or in combination with other therapies.

[0022] The term "disease" means any condition or disorder that impairs or interferes with the normal function of a cell, tissue, or organ. Examples of diseases that can be treated with the compositions disclosed herein include neoplasms, autoimmune diseases, viral infections, and senescent and age-related diseases.

[0023] The “effective dose” and “therapeutic effective dose” of a formulation or formulation component carry a sufficient amount of the formulation or component, either alone or in combination, to provide the desired effect. For example, the “effective dose” is the amount of soluble fusion protein complex needed, either alone or in combination, to improve the symptoms of the disease compared to an untreated patient. The effective dose of the soluble fusion protein complex used to carry out the present invention for the therapeutic treatment of a disease varies depending on the mode of administration, the age, weight, and overall health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate dose and administration regimen. Such a dose is referred to as the “effective” dose.

[0024] "High affinity" natural killer (haNK) cells are NK cells that have been engineered to incorporate a high-affinity receptor, i.e., a CD16 allele that binds to an administered antibody. T-haNK are haNK natural killer cells that have been further engineered to contain an antigen-targeting scFv.

[0025] In relation to two or more nucleic acid or polypeptide sequences, “identical” or “identical” percentage refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence across a comparison window. For the purposes of this disclosure, the degree of amino acid or nucleic acid sequence identity is determined using the BLAST algorithm, which is incorporated herein by reference by Altschul&al. (199) J. Mol. Biol. 215:403-10. The BLAST algorithm is publicly available through software provided by the National Center for Biotechnology Information (web address www.ncbi.nlm.nih.gov). This algorithm identifies high-scoring sequence pairs (HSPS) by identifying short words of length W in a query sequence that, when aligned with words of the same length in a database sequence, match or satisfy a threshold score T of some positive value, where T is called the neighboring word score threshold (Altschul&al., above). The first neighboring word hits act as a seed to initiate the search for longer HSPs containing them. Word hits are then extended in both directions along each sequence as long as they can increase the cumulative alignment score. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for a pair of matching residuals; always > 0) and N (penalty score for mismatch residuals; always < 0). For amino acid sequences, the cumulative score is calculated using a score matrix. The extension of word hits in each direction stops when the cumulative alignment score falls by an amount X from its maximum achieved value, and the cumulative score becomes 0 or less due to the accumulation of one or more negatively scoring residue alignments or reaches the end of either sequence. Default parameters of the BLAST program can be used to determine the identity percentage of amino acid sequences or nucleic acid sequences. For amino acid sequence analysis, the BLAST defaults are as follows: word length (W), 3; expected value (E), 10; and BLOSUM62 score matrix.For nucleic acid sequence analysis, the default settings for the BLASTN program are word length (W), 11; expected value (E), 10; M=5; N=-4; and comparison of both strands. The TBLASTN program (which queries nucleotide sequence databases using protein sequences) uses word length (W) 3, expected value (E) 10, and a BLOSUM 62 score matrix as default (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915, incorporated herein by reference).

[0026] In addition to calculating the sequence identity percentage, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-87, incorporated herein by reference). The minimum sum probability (P(N)) provides an indicator of the probability that the match between two nucleotide or amino acid sequences occurs by chance. For example, if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than approximately 0.01, the nucleic acid is considered similar to the reference sequence.

[0027] As used herein, the term “immune effector cell” refers to a cell involved in promoting an immune response, such as an immune effector response. Examples of immune effector cells include T cells, such as alpha / beta (α / β) T cells and gamma / delta (γ / δ) T cells, B cells, natural killer (NK) cells, natural killer T (NK-T) cells, mast cells, and bone marrow-derived phagocytic cells. “Immune effector function or immune effector response” refers, as used herein, to a function or response of immune effector cells that enhances or promotes an immune attack on target cells. For example, immune effector function or response refers to the properties of T or NK cells that promote the death or inhibition of growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector function or response.

[0028] As used herein, in relation to the administration of a treatment to a subject, the term “combined” refers to the use of two or more treatments for a therapeutic benefit. In relation to administration, the term “combined” may also refer to the prophylactic use of a treatment to a subject when used in conjunction with at least one additional treatment. The use of the term “combined” does not restrict the order in which the treatments (e.g., the first and second treatments) are administered to the subject. The treatment may be administered before (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before) or simultaneously with or following (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second treatment to a subject who has had, has, or is susceptible to cancer. The treatments are administered to the subject within a series of time intervals so that the treatments can act together. In certain embodiments, the treatment is administered to the subject within a series of time intervals to provide a greater benefit than if administered by other means. Any additional treatments may be administered in any order with other additional treatments.

[0029] Examples of "parenteral" administration of immunogenic compositions include subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques.

[0030] "Patient," "individual," or "subject" are used interchangeably herein and refer to a mammalian subject being treated, interchangeably to a mammal, preferably a human or a non-human primate, but also to domestic mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs) and agricultural mammals (e.g., horses, cattle, pigs, sheep). In certain embodiments, the subject may be a human being (e.g., an adult male, an adult female, adolescent male, an adolescent female, a boy, a girl) under the care of a physician or other healthcare professional. In certain embodiments, the subject may not be under the care of a physician or other healthcare professional.

[0031] A "pharmaceutically acceptable" ingredient / carrier is suitable for use in humans and / or animals without excessive adverse side effects (toxicity, irritation, allergic reactions, etc.) that are commensurate with a reasonable benefit-risk ratio.

[0032] "Preventing," "preventing," and "prevention" refer to the administration of a drug or composition to a clinically asymptomatic individual who is susceptible or prone to a particular adverse condition, disorder, or disease, and thus relate to the prevention of the development of symptoms and / or their underlying causes.

[0033] "Reduction" means a negative change in the condition, disability, or disease compared to a "healthy" or "appropriate" control subject who does not have the condition, disability, or disease. The negative change may be at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%.

[0034] "Specifically binding" means that a molecule, protein, peptide, antibody, or antibody fragment recognizes and binds to the polypeptide of the present invention, but does not substantially recognize and bind to other molecules in a sample that may contain the polypeptide of the present invention, such as a biological sample.

[0035] As used herein, “to treat,” “to treat,” and “to treat” mean administering a drug or formulation to a clinically symptomatic individual suffering from an adverse condition, disorder, or disease in order to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying causes, and / or promote improvement or repair of damage. Treatment may include the administration of a fusion molecule of CTLA4 and IL-15 or an analog thereof. It will be understood that treating a disorder or condition does not require the complete elimination of the disorder, condition, or symptoms associated with it, although this is not elimination.

[0036] Treatment for patients with neoplasms may include any of the following: adjuvant therapy (also called adjuvant or supportive therapy) to destroy any remaining tumor cells that may exist after the known tumor has been removed by the first treatment (e.g., surgery) and thereby prevent possible cancer recurrence; neoadjuvant therapy performed before surgical intervention to shrink the cancer; induction therapy, typically for acute leukemia, to induce remission; consolidation therapy (also called intensification therapy) given to maintain remission once remission has been achieved; maintenance therapy, performed at low doses or infrequently, to help prolong remission; first-line treatment (also called standard treatment); second-line (or third, fourth, etc.) therapy (also called salvage therapy) if the disease has not responded to or recurred after first-line treatment; and palliative care (also called supportive care) to manage symptoms without expecting a significant reduction in cancer.

[0037] A “tumor” refers to a mass of transformed cells characterized by neoplastic, uncontrolled cell proliferation and at least partially angiogenic vascular structures. Abnormal neoplastic cell growth is rapid and continues even after the stimulus that initiated the new growth has ceased. The term “tumor” is used more broadly to include tumor parenchymal cells and the supporting stroma containing neovascularization that infiltrates the tumor parenchymal cell mass. Tumors are generally malignant tumors, i.e., cancers with the ability to metastasize (i.e., metastatic tumors), but tumors can be non-malignant (i.e., non-metastatic tumors). Tumors are a prominent feature of cancer, and their natural course is that of a fatal neoplastic disease.

[0038] A particular methodology of the present invention includes a step of comparing values, levels, features, characteristics, properties, etc., with a “preferred control,” which is interchangeably referred to herein as “appropriate control.” A “preferred control” or “appropriate control” is a control or standard well known to those skilled in the art and useful for comparison purposes. In one embodiment, a “preferred control” or “appropriate control” is a value, level, feature, characteristic, property, etc., determined before carrying out the therapeutic and / or drug administration methodology described herein. For example, transcription rate, mRNA level, translation rate, protein level, biological activity, cell characteristics or properties, genotype, phenotype, etc., can be determined before introducing the therapeutic and / or drug of the present invention to the target. In another embodiment, a “preferred control” or “appropriate control” is a value, level, feature, property, etc., determined in a cell or organism exhibiting normal or healthy (e.g., non-disease) traits, e.g., a control or normal cell or organism. In yet another embodiment, a “preferred control” or “appropriate control” is a predetermined value, level, feature, characteristic, property, etc.

[0039] Genes: All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein are applicable. Where a gene or gene product from a particular species is disclosed, it is understood that this disclosure is intended to be illustrative only and should not be construed as limiting unless indicated by the context in which it appears. Thus, for example, with respect to genes or gene products disclosed herein, homologous and / or orthologous genes and gene products from other species are intended to be included.

[0040] The GenBank and NCBI submissions, indicated by accession numbers cited herein, are incorporated herein by reference.

[0041] Scope: Throughout this disclosure, various aspects of the invention can be presented in scope form. It should be understood that scope descriptions are merely for convenience and brevity and should not be interpreted as inflexible limitations on the scope of the invention. Therefore, scope descriptions should be considered to specifically disclose all possible sub-scopes and the individual numbers within those scopes. For example, a scope description such as 1-6 should be considered to specifically disclose sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within those scopes, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

[0042] Any composition or method provided herein may be combined with one or more other compositions and methods provided herein.

[0043] Interleukin-15. Human IL-15 (huIL-15) has high binding affinity (equilibrium dissociation constant (K)). D ) about 10 -11M) is a member of a small α-helix bundle family of cytokines that associate with the huIL-15 receptor α chain (huIL-15Rα). The resulting complex is trans-presented on the human IL-2 / 15 receptor β / common γ chain (huIL-15RβγC) complex, which is presented on the surface of T cells and NK cells. This cytokine / receptor interaction leads to the expansion and activation of effector T cells and NK cells, which play a crucial role in the eradication of virus-infected and malignant cells. Normally, huIL-15 and huIL-15Rα are co-produced in dendritic cells, form a complex intracellularly, are then secreted, and presented on the cell surface as heterodimer molecules. Therefore, the characteristics of the huIL-15 and huIL-15Rα interaction suggest that their interchain binding domains can function as human-derived immunostimulatory scaffolds for creating soluble dimer molecules capable of target-specific binding.

[0044] In certain embodiments of the soluble fusion protein complex of the present invention, the IL-15 peptide may be an IL-15 variant having a different amino acid sequence from the native IL-15 peptide. huIL-15 and its variants are referred to using the native amino acids, their positions in the mature sequence, and the variant amino acids. For example, huIL15N72D represents human IL-15 with a substitution of N to D at position 72. In one embodiment, the IL-15 variant functions as an IL-15 agonist, as demonstrated, for example, by increased binding activity to the IL-15RβγC receptor compared to the native IL-15 peptide. Alternatively, the IL-15 variant functions as an IL-15 antagonist, as demonstrated, for example, by reduced binding activity to the IL-15RβγC receptor compared to the native IL-15 sequence.

[0045] IL-15:IL-15Rα complex. The formation of an IL-15:IL-15Rα complex, in which both the IL-15 peptide and the IL-15 receptor α chain are co-expressed in the same cell, can stimulate immune effector cells carrying the IL-2βγC receptor via a trans-presentation mechanism. Furthermore, when the IL-15 peptide is bound to IL-15Rα, the affinity of the IL-15 peptide for IL-2Rβ increases by approximately 150-fold compared to free IL-15. The IL-15 peptide superagonist mutant (IL-15N72D) has increased IL-2Rβ binding ability (4-5 times higher than natural IL-15). By utilizing the strong interaction between IL-15N72D and soluble IL-15Rα, an IL-15 superagonist complex was constructed with IL-15N72D bound to IL-15RαSu. For example, a superagonist complex may be an IL-15 derivative bound to an IL-15Rα / IgG1 Fc fusion protein, also known as nogapentin alpha-invacixept (NAI). NAI may be N-803, ALT-803, or IL-15 N72D It is also known in the literature as IL-15RαSu / IgG1. U.S. Patent No. 9,328,159 describing NAI is incorporated herein by reference in its entirety. Clinical trials including N-803 are described in NCT04385849, which is incorporated herein by reference in its entirety.

[0046] Fusion protein complex. This disclosure provides a soluble fusion protein complex. The soluble fusion complex described herein comprises a first domain (for example, an interleukin-15 (IL-15) peptide which may be an IL-15 variant (also referred to herein as an IL-15 mutant, but also within the general range of "IL-15 peptide")), a second domain containing a fusion polypeptide, and a third domain. The IL-15 peptide in the first domain preferably contains an amino acid sequence different from that of the natural (or wild-type) IL-15 protein. The IL-15 peptide preferably binds to the IL-15Rα peptide and functions as an IL-15 agonist or antagonist. Preferably, an IL-15 peptide having agonist activity in the first domain may have superagonist activity. The IL-15 peptide can function as an IL-15 agonist or antagonist independently of association with IL-15Rα. IL-15 agonists are exemplified by biological activity equivalent to or increased compared to wild-type IL-15. IL-15 antagonists are exemplified by decreased biological activity or the ability to inhibit IL-15-mediated responses compared to wild-type IL-15. In some cases, the IL-15 peptide in the first domain binds with increased or decreased activity to the IL-15RβγC receptor. In some cases, the sequence of the IL-15 peptide in the first domain has at least one amino acid change, e.g., substitution or deletion, compared to the natural (or wild-type) human IL-15 peptide, and such change results in IL-15 agonist or antagonist activity. Preferably, the amino acid substitution / deletion is located in the region of the IL-15 peptide that interacts with IL-15Rβ and / or γC. More preferably, the amino acid substitution / deletion does not affect the ability to bind to the IL-15Rα polypeptide or produce the IL-15 peptide. Suitable amino acid substitutions / deletions for generating the IL-15 peptide in the first domain can be identified by rational or random mutagenesis and functional assays or other empirical methods, such as those provided herein, based on comparison with known structures of the putative or known IL-15 structure, molecules homologous to the IL-15 peptide (e.g., IL-2).Furthermore, preferred amino acid substitutions may be conservative or non-conservative changes and insertions of additional amino acids. Preferably, the IL-15 peptides of the present invention contain one or more amino acid substitutions / deletions at positions 6, 8, 10, 61, 65, 72, 92, 101, 104, 105, 108, 109, 111, or 112 of the mature human IL-15 sequence, in particular, D8N ("D8" refers to the position of the amino acid and residue in the natural mature human IL-15 sequence, and "N" refers to the substituted amino acid residue at that position in the IL-15 mutant), I6S, D8A, D61A, N65A, N72R, V104P, or Q108A substitutions result in an IL-15 peptide having antagonist activity, and the N72D substitution results in an IL-15 peptide having agonist activity. Alternative IL-15 peptides include those disclosed in U.S. Patent No. 8,163,879 and U.S. Patent No. 9,255,141, both of which are incorporated in their entirety by reference.

[0047] In some embodiments, the first domain may comprise a first biologically active polypeptide covalently bound to an interleukin-15 (IL-15) peptide or a functional fragment thereof, and the second domain may comprise a second biologically active polypeptide covalently bound to a soluble interleukin-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof (e.g., IL15RαSu peptide), wherein the IL-15 peptide of the first domain binds to the soluble IL-15Rα peptide or its functional fragment of the second domain to form a soluble fusion protein complex. The fusion protein complex may further comprise one or more immunoglobulin Fc peptides or functional fragments thereof linked to one or both of the first and second domains. Additionally or alternatively, Fc peptides linked to the first and / or second domains may interact to form a fusion protein complex. Such a complex may be stabilized by disulfide bond formation between immunoglobulin Fc peptides. The soluble fusion protein complex may comprise a first domain containing an IL-15 peptide (e.g., native or wild-type IL-15, an IL-15 variant, or a functional fragment thereof) and a second domain containing a soluble IL-15Rα polypeptide or a functional fragment thereof, wherein one or both of the IL-15 peptide and the IL-15Rα polypeptide further comprise an immunoglobulin Fc domain or a functional fragment thereof.

[0048] Additionally or alternatively, one or both of the first and second domains may further contain an antibody or a functional fragment thereof. For example, one of the domains may contain a soluble αCTLA4 single-chain antibody variable fragment (scFv) or αPD-L1 scFv or a functional derivative thereof. As a non-limiting example, a suitable αCTLA4 antibody is disclosed in International Publication No. 2021 / 250594, which is incorporated herein by reference in its entirety. In another example, the first or second domain may contain αCTLA4 scFv or a disease antigen-specific antibody or a functional derivative thereof. An example of scFv is disclosed in U.S. Patent No. 11,105,188, which is incorporated herein by reference in its entirety. Soluble fusion protein complexes containing a covalently bound IL-15 peptide and an IL-15Rα domain have several important applications. For example, a soluble fusion protein complex containing αCTLA4 scFv on a first or second domain can be used to deliver the IL-15:IL-15Rα complex to specific cells, such as NK cells. Alternatively, as will be discussed later, the NK cells may include high-affinity NK cells engineered to express the fusion protein. Thus, protein fusion or conjugate complexes provide a means for selectively damaging or killing cells containing ligands. Examples of cells or tissues that can be damaged or killed by protein fusion or conjugate complexes include tumors and virus-infected or bacterial-infected cells expressing one or more ligands. Cells or tissues that are susceptible to damage or death can be readily assayed by the methods disclosed herein.

[0049] Additionally or alternatively, the soluble fusion protein complex may further include a third domain which may contain another antibody or a functional fragment thereof. For example, the third domain may contain, for example, an antibody light chain (e.g., an antibody fragment that does not contain the Fc region). In certain examples, the third domain may contain an αCTLA4 antibody light chain or a peptide having sequence identity (e.g., at least about 85% sequence identity) with the αCTLA4 antibody light chain.

[0050] The IL-15 peptide and IL-15Rα peptide of the present invention correspond appropriately in amino acid sequences to naturally occurring IL-15 and IL-15Rα molecules, such as those of humans, mice, other rodents, or other mammals. The sequences of these polypeptides and coding nucleic acids, including human interleukin-15 (IL-15) mRNA-GenBank:U14407.1 (the whole thereof incorporated herein by reference), mouse interleukin-15 (IL-15) mRNA-GenBank:U14332.1 (the whole thereof incorporated herein by reference), human interleukin-15 receptor alpha chain precursor (IL-15Rα) mRNA-GenBank:U31628.1 (the whole thereof incorporated herein by reference), and mouse interleukin-15 receptor, alpha chain-GenBank:BC095982.1 (the whole thereof incorporated herein by reference), are publicly known in the literature.

[0051] In some situations, it may be useful to make the protein fusion or conjugate complex of the present invention polyvalent, for example, by increasing the valency of a single-chain antibody. In particular, the interaction between the IL-15 peptide and the IL-15Rα peptide of the fusion protein complex provides a means for generating a polyvalent complex. Furthermore, polyvalent fusion proteins can be prepared by covalently or non-covalently bonding one protein to four proteins (the same or different) by, for example, using standard biotin-streptavidin labeling techniques or by conjugation to a suitable solid support such as latex beads. Chemically crosslinked proteins (e.g., crosslinked to dendrimers) are also suitable polyvalent species. For example, proteins can be modified by including modifyable tag sequences such as biotinylated BirA tags or sequences encoding amino acid residues having chemically reactive side chains such as Cys or His. Such amino acid tags or chemically reactive amino acids can be located at various positions in the fusion protein, preferably distal to the active site of a biologically active polypeptide or effector molecule. For example, the C-terminus of a soluble fusion protein complex can be covalently bonded to a tag or other fusion protein containing such reactive amino acids. Preferred side chains may be included to chemically link two or more fusion proteins to a suitable dendrimer or other nanoparticle to obtain a polyvalent molecule. Dendrimers are synthetic chemical polymers that may have one of several different functional groups on their surface (D. Tomalia, Aldrichimica Acta, 26:91:101 (1993), the whole of which is incorporated herein by reference). Exemplary dendrimers for use according to the present invention include, for example, E9 starburst polyamine dendrimers and E9 combust polyamine dendrimers that can link cysteine ​​residues. Exemplary nanoparticles include liposomes, core-shell particles, or PLGA-based particles.

[0052] Additionally or alternatively, one or both of the first and second domains of the soluble fusion protein complex may include an immunoglobulin domain. Alternatively, a protein-binding domain-IL-15 fusion protein may be further ligated to the immunoglobulin domain. The immunoglobulin domain includes a region that enables interaction with other immunoglobulin domains to form a multi-chain protein such as the one provided above. For example, IgG1 C H 2-C H The immunoglobulin heavy chain regions of 3 can stably interact to create an Fc region. Preferred immunoglobulin domains containing an Fc domain also include regions having effector functions, including Fc receptor or complement protein binding activity, and / or regions having glycosylation sites. In some embodiments, the immunoglobulin domain of the fusion protein complex contains mutations that decrease or enhance Fc receptor or complement binding activity, or glycosylation or dimerization, thereby affecting the biological activity of the resulting protein. For example, using an immunoglobulin domain containing a mutation that reduces binding to the Fc receptor, it is possible to generate the fusion protein complex of the present invention with lower binding activity to Fc receptor-carrying cells, which may be advantageous for reagents designed to recognize or detect specific antigens.

[0053] A soluble fusion protein complex comprising a first domain containing an IL-15 peptide, a second domain containing a fusion polypeptide comprising an IL15RαSu peptide and an αCTLA4 antibody heavy chain, and a third domain containing an αCTLA4 antibody light chain is also described herein.

[0054] The IL-15 peptide may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 17, and this does not include the leader sequence. The IL-15 peptide may include SEQ ID NO: 17. An exemplary nucleotide sequence encoding SEQ ID NO: 17 is given herein as SEQ ID NO: 18. Alternatively, the IL-15 peptide may further include a leader sequence, and this may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 1, and this includes the leader sequence. The IL-15 peptide may include SEQ ID NO: 1. An exemplary nucleotide sequence encoding SEQ ID NO: 1 is given herein as SEQ ID NO: 2.

[0055] The IL15RαSu peptide may have at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% sequence identity with SEQ ID NO: 8. The IL15RαSu peptide may contain SEQ ID NO: 8. The αCTLA4 antibody heavy chain may have at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% sequence identity with SEQ ID NO: 10. The αCTLA4 antibody heavy chain may contain SEQ ID NO: 10. The αCTLA4 antibody light chain may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 3. The αCTLA4 antibody light chain may contain SEQ ID NO: 3.

[0056] In particular, the soluble fusion protein complex may comprise a first domain having an IL-15 peptide with at least approximately 85% sequence identity with SEQ ID NO: 17, a second domain having an IL15RαSu peptide and an αCTLA4 antibody heavy chain with at least approximately 85% sequence identity with SEQ ID NO: 10, and a third domain having an αCTLA4 antibody light chain with at least 85% sequence identity with SEQ ID NO: 3. The IL15RαSu peptide and αCTLA4 antibody heavy chain may be part of the fusion polypeptide. The IL-15 peptide of the first domain may bind to the IL15RαSu peptide of the second domain to form a soluble fusion protein complex. The IL15RαSu peptide may have at least 85% sequence identity with SEQ ID NO: 8. Additionally or alternatively, the heavy and light chains of the αCTLA4 antibody may include a complementation-determining region (CDR). The CDR region of the αCTLA4 antibody heavy chain may have at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% sequence identity with one or more of SEQ ID NOs: 11-13. Alternatively, the CDR region of the αCTLA4 antibody heavy chain may contain each of SEQ ID NOs: 11-13. The CDR region of the αCTLA4 antibody light chain may have at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% sequence identity with one or more of SEQ ID NOs: 14-16. Alternatively, the CDR region of the αCTLA4 antibody light chain may contain each of SEQ ID NOs: 14-16. In particular, the soluble fusion protein complex may contain an αCTLA4 antibody heavy chain having at least approximately 90% sequence identity with SEQ ID NO: 10, and may further contain an αCTLA4 antibody light chain having approximately 90% sequence identity with SEQ ID NOs: 11-13 and SEQ ID NO: 3, and SEQ ID NOs: 14-16. Alternatively, the soluble fusion protein complex may contain an αCTLA4 antibody heavy chain having at least approximately 95% sequence identity with SEQ ID NO: 10, and may further contain an αCTLA4 antibody light chain having approximately 95% sequence identity with SEQ ID NOs: 11-13 and SEQ ID NO: 3, and SEQ ID NOs: 14-16.Instead, the soluble fusion protein complex contains the αCTLA4 antibody heavy chain with SEQ ID NO: 10 and the αCTLA4 antibody light chain with SEQ ID NO: 3.

[0057] The IL-15 peptide in the soluble fusion protein complex may or may not contain a leader sequence. For example, the soluble fusion protein complex may contain an IL-15 peptide lacking a leader sequence, and the IL-15 peptide may contain SEQ ID NO: 17. Alternatively, the soluble fusion protein complex may contain an IL-15 peptide containing a leader domain, and the IL-15 peptide may contain SEQ ID NO: 1.

[0058] An αCTLA4 antibody (e.g., an αCTLA4 antibody heavy chain) may be directly conjugated to an IL-15 peptide. Alternatively, the αCTLA4 antibody may be conjugated to an IL-15 receptor (e.g., an IL15RαSu peptide), and the IL-15 peptide may be associated with an anti-CTLA4 / IL15RαSu chimera. Additionally or alternatively, the IL-15 peptide or IL15RαSu peptide may be conjugated to the Fab terminus of an αCTLA4 antibody (e.g., an αCTLA4 antibody heavy chain), and in other embodiments, the IL-15 peptide or IL15RαSu peptide may be conjugated to the Fc terminus of an αCTLA4 antibody heavy chain. The αCTLA4 antibody may be a complete antibody or only a Fab fragment, but in other embodiments, αCTLA4 also includes an Fc domain. In certain embodiments, the αCTLA4 molecule is an IgA, IgE, IgG, or IgM antibody.

[0059] Additionally or alternatively, a soluble fusion protein complex may further contain peptide linkers between one or more domains of the complex. For example, if the fusion polypeptide of the second domain of a soluble fusion protein complex contains the IL15RαSu peptide and the αCTLA4 antibody heavy chain, the IL15RαSu peptide and the αCTLA4 antibody heavy chain may be directly conjugated via a peptide linker. The peptide linker may be between the amino terminus of the IL15RαSu peptide and the carboxyl terminus of the αCTLA4 antibody heavy chain. In particular, the fusion polypeptide may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 5. Alternatively, the fusion polypeptide may contain SEQ ID NO: 5.

[0060] Alternatively, the peptide linker may be located between the carboxyl terminus of the IL15RαSu peptide and the amino terminus of the αCTLA4 antibody heavy chain in the second domain. In particular, the fusion polypeptide may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 4. Alternatively, the fusion polypeptide may contain SEQ ID NO: 4.

[0061] Amino Acid Mutations: There are several methods by which researchers have mutated the amino acid sequence of the IgG Fc region to modulate FcγR-based effector function. Some examples include point mutations, design algorithms, yeast display, and asymmetric engineering. The results from each of these different techniques have led to numerous mutations that alter the Fc-FcγR interaction and the resulting effector function. For example, Fc-optimized immunoglobulin molecules based on amino acid substitutions have been found to enhance ADCC. These include the following amino acid substitutions that increase FcγRIIIa binding: F243L / R292P / Y300L / V305I / P396L;S239D / I332E;S298A / E333A / K334A. Another mutation with increased FcγRIIIa binding and decreased FcγRIIb binding is 239D / I332E / A330L. Other mutations that increased FcγRIIIa binding include L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the opposite heavy chain (Xinhua & al. (2017) Protein & Cell 9(1):63-73, the whole is incorporated herein by reference). Combining several identified mutations (e.g., S298A / E333A / K334A) enhanced ADCC compared to IgG1. A series of Fc variants with optimized Fcγ receptor affinity, optimized using computational design algorithms and high-throughput screening, identified S239D / I332E and S239D / I332E / A330L as two variants with enhanced ADCC activity. The crystal structure of the Fc fragment containing the mutation S239D / A330L / I332E was elucidated, and modeling studies suggested that additional hydrogen bonding, hydrophobic contact, and / or electrostatic interactions lead to enhanced binding to FcγRIIIa. Addition of G236A to the S239D / I332E mutation resulted in up to a 70-fold improvement in binding to FcγRIIa, a 13-fold improvement in the FcγRIIa / FcγRIIb binding ratio (activation / inhibition ratio), and enhanced phagocytosis of antibody-coated target cells by macrophages.The mutant F243L / R292P / Y300L / V305I / P396L showed a more than 100-fold increase in ADCC activity (Stavenhagen & al. (2007) Cancer Res. 67:8882-90, the entire report is incorporated herein by reference). Antibody variants with asymmetrically manipulated Fc domains, created by introducing different amino acid changes into each Fc domain, demonstrated that L234Y / L235Q / G236W / S239M / H268D / D270E / S298A alters one Fc domain, while D270E / K326D / A330M / K334E alters the other Fc domain, increasing affinity for FcγRIIIa F158 by more than 2000-fold and affinity for FcγRIIIa V158 by more than 1000-fold (Mimoto&al. (2013) MAbs.5:229-36, the entire report is incorporated herein by reference).

[0062] Glycotechnology: IgG contains a conserved glycosylation site at amino acid N297 in the CH2 domain. The core structure of the glycan consists of N-acetylglucosamine (GlcNAc) and mannose, and further modifications may include bisecting GlcNAc, fucose, galactose, and sialic acid. One of the first reports linking glycotechnology with enhanced Fc effector function demonstrated that when IgG1 antibodies produced in Chinese hamster ovary (CHO) cell lines expressing β(1,4)-N-acetylglucosaminyltransferase III express bisecting GlcNAc, ADCC activity is enhanced compared to IgG1 (Umana & al. (1999) Nat Biotechnol. 17:176-80, the whole is incorporated herein by reference). Furthermore, fucose-deficient IgG1 showed up to a 50-fold increase in FcγRIIIa binding and enhanced ADCC compared to IgG1 (Shields & al. (2002) J Biol Chem. 277:26733-40, the whole is incorporated herein by reference). Subsequently, it was demonstrated that fucose-deficient antibodies have improved ADCC function compared to antibodies containing bisecting GlcNAc (Shinkawa & al. (2003) J Biol Chem. 278:3466-73, the whole is incorporated herein by reference). Amino acid N162 in FcγRIIIa contains glycan, and the absence of fucose allows for greater carbohydrate-carbohydrate interaction with Fc, which increases the overall binding strength.

[0063] Antibody-dependent cell-mediated cytotoxicity (ADCC): ADCC is a process in which an antibody coats target cells and recruits effector cells to induce target cell death via a non-phagocytic mechanism. Antibodies can bind to their specific antigens on the surface of target cells via their antigen-binding fragment (Fab) portion and interact with effector cells via their fragment crystallizable region (Fc) portion, thereby acting as a crosslink that connects effectors to the target. Several classes of human antibodies, including IgG, IgA, and IgE, can mediate ADCC, but IgG1 is the most widely used subclass for cancer treatment antibodies (Zahavi & al. (2018) Antibody Therapeutics 1(1):7-12, the whole of which is incorporated herein by reference). For effector cells to perform ADCC, they must express an antibody-binding Fc receptor (FcR). Known classes of FcR include FcγR, which binds to IgG, FcαR, which binds to IgA, and FcεR, which binds to IgE. Fcγ R is crucial for tumor cell clearance by myeloid cells and consists of activated Fcγ RI (CD64), Fcγ RIIA (CD32A), Fcγ RIIIA (CD16A), and inhibitory Fcγ RIIB (CD32B) receptors. When Fcγ R binds to an antibody, it triggers receptor crosslinking and downstream signaling. They activate Fcγ R signaling through their immunoreceptor tyrosine system activating motifs, while activating inhibitory Fcγ R signaling through their immunoreceptor tyrosine system inhibitory motifs. Many effector cells also express other receptor types, such as inhibitory killer inhibitory receptors (e.g., KIR) and activated NKG2D receptors on natural killer (NK) cells. A delicate balance between activating and inhibitory pathway signaling ultimately determines the effector cell response. Myeloid cells that can act as ADCC effectors include NK cells, monocytes, macrophages, neutrophils, eosinophils, and dendritic cells. When these effector cells are activated, they mediate target cell death through three key mechanisms: cytotoxic granule release, Fas signaling, and reactive oxygen species generation.The main and most well - characterized mechanism utilized in ADCC is the release of perforin and granzymes from effector cell granules. When activated, effector cells such as NK cells polarize their granules in a calcium - dependent manner and undergo exocytosis (de Saint Basile, G. & al. (2010) Nat Rev Immunol. 10:568 - 79, which is incorporated herein by reference in its entirety). Perforin and granzyme B act in concert to induce cell death. Perforin creates pores in the cell membrane that facilitate the entry of granzyme B into target cells, leading to DNA fragmentation and apoptosis. Multiple myeloid - lineage cells can perform ADCC, but in the context of cancer immunotherapy, NK cells appear to be the major effector cell type in vivo. The clinical efficacy of many targeted mAb therapies has been demonstrated to be NK - cell - dependent. NK cells highly express activating FcγRIIIA and do not express inhibitory FcγRIIB, so specifically modifying antibody interactions via FcγRIIIA has been of increasing interest for cancer immunotherapy.

[0064] High - affinity natural killer (haNK) cells: NK - 92 is a NK - like cell line that was first isolated from the blood of a subject suffering from large granular lymphoma and subsequently propagated in cell culture. The NK - 92 cell line has been described (Klingemann & al. (2016) Front Immunol 7:91, which is incorporated herein by reference in its entirety). The determined NK - 92 cells are CD3 - / CD56 +They possess a phenotypic characteristic: they express all known NK cell activating receptors except CD16, but lack all known NK cell inhibitory receptors except NKG2A / CD94 and ILT2 / LIR1, which are expressed at low levels. Furthermore, unlike polyclonal NK cells isolated from blood, NK-92 is a clonal cell line that expresses these receptors in a consistent manner, both in terms of type and cell surface concentration. Similarly, NK-92 cells are not immunogenic and do not induce immune rejection when administered therapeutically to human subjects. In fact, NK-92 cells are well tolerable in humans without known adverse effects on normal tissues.

[0065] haNK cells are derived from the NK-92 cell line and engineered to express the high-affinity CD16 allele. Sequences of high-affinity variants of the Fcγ receptor are well known in the art (see, for example, Bruhns & al. (2009) Blood 113:3716-25, which is incorporated in its entirety herein by reference). Expression of such receptors favorably increases the specific targeting and cytotoxic cell killing of tumor cells when using antibodies specific to the patient's tumor cells. CD16 is most commonly found in a form with relatively low binding affinity to the Fc portion of the IgG molecule. Alternative forms exhibiting higher binding affinity are found in some individuals. The low-affinity and high-affinity forms of CD16 differ only in the substitution of valine (high-affinity) for phenylalanine (low-affinity) at position 157 of the polypeptide chain. The complete sequences of the low-affinity and high-affinity forms can be found in the SwissProt database as entries P08637 and VAR_008801, respectively.

[0066] Transduced NK-92 cells expressing CD16 on their surface (also known as NK-92-CD16, or CD16 / FcεRIγ-NK-92) are referred to herein as haNK cells, and haNK cells expressing antigen-binding scFv are referred to herein as T-haNK cells.

[0067] Checkpoint Inhibitors: Checkpoint inhibitors inhibit the function or activity of molecules that modulate or control, for example, the immune response of immune effector cells, such as T cell function, or inhibit it. Inhibitory molecules, also known herein as checkpoint inhibitors, such as CTLA4 and programmed death 1 (PD-1), can, in some embodiments, reduce the ability of immune effector cells to initiate an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta. For example, inhibition of molecules that modulate or regulate T cell function, such as those that inhibit it, by inhibition at the DNA, RNA, or protein level, can optimize the immune response. In addition to the αCTLA4 antibodies disclosed herein, ipilimumab (also known as MDX-010 and MDX-101, marketed as YERVOY®; Bristol-Myers Squibb) and tremelimumab (an IgG2 monoclonal antibody available from Pfizer, formerly known as tisilimubab, CP-675, 206) also bind to CTLA4.

[0068] Cytotoxic T lymphocyte antigen 4 (CTLA4) is a member of a family of immunoglobulin-associated receptors that play a role in various aspects of T cell immunomodulation. This family includes CD28, CTLA4, and ICOS, as well as other proteins including PD-1, BTLA, and TIGIT. These receptors have both stimulating (CD28, ICOS) and inhibitory (CTLA4, PD-1, BTLA, and TIGIT) roles in T cell function. These pathways are increasingly being targeted as part of immunomodulatory strategies to treat cancer, commonly known as immune checkpoint blockade, and conversely, to treat autoimmunity and CTLA4 deficiency.

[0069] CTLA4 (CD152) and CD28 are CD4 + and CD8 + Both are homologous receptors expressed by T cells and mediate opposing functions in T cell activation. Both receptors share a pair of ligands expressed on the surface of antigen-presenting cells (APCs). CD28 interacts with the CD80 dimer with relatively high affinity and the CD86 monomer with lower affinity, mediating T cell costimulation in conjunction with T cell receptor (TCR) signaling. In contrast, the interaction between ligands and CTLA4 helps inhibit the T cell response, although the exact mechanism is not fully understood. CTLA4 interacts with both ligands with higher affinity and binding activity than CD28, with CTLA4-CD80 forming the most binding-active interaction and CD28-CD86 forming the weakest interaction (Rowshanravan & al. (2018) Blood 131:58-67, the whole is incorporated herein by reference). Among several possibilities, this raises the concept that CTLA4 may compete with CD28 for ligand binding, thereby acting as an antagonist of CD28-mediated costimulation (Thompson & Allison (1997) Immunity 7(4):445-50; Walker & Sansom (2011) Nat Rev Immunol. 11(12):852-63, both incorporated herein in their entirety by reference). These interactions are thought to occur at the immunological synapse between T cells and APCs, where CTLA4 has been shown to recruit CD80, thereby limiting its interaction with CD28 (Yokosuka & al. (2010) Immunity 33(3):326-39, both incorporated herein in their entirety by reference).

[0070] Therefore, in methods for treating cancer, T-haNK cells can be manipulated to express an anti-CTLA4 antigen-binding domain that binds to the CTLA4 molecule with high affinity (Simpson&al.(2013)J.Exp.Med.210(9):1695-710, the entire text is incorporated herein by reference), regThis leads to cell depletion or functional blockade, resulting in T cell activation and enhanced immune response against cancer.

[0071] CTLA4 is T reg It helps mediate the inhibitory function of T reg It has been confirmed to be consistent with the effects function of the CTLA4 described above (Klocke & al. (2016) Proc Natl Acad Sci USA. 113(17):E2383-92, the entire article is incorporated herein by reference).

[0072] In embodiments where autoimmune diseases are treated, T-haNK cells express CTLA4, which leads to the inhibition of the immune response.

[0073] Measurable immune cell activity includes, but is not limited to, (1) cell proliferation by measuring DNA replication; (2) enhancement of cytokine production, including specific measurement of cytokines such as IFN-γ, GM-CSF, or TNF-α; (3) cell-mediated targeted killing or lysis; (4) cell differentiation; (5) immunoglobulin production; (6) phenotypic changes; (7) generation of chemotactic factors or chemotaxis, meaning the ability to respond to chemotactins; (8) immunosuppression by inhibiting the activity of several other immune cell types; and (9) apoptosis, which refers to the fragmentation of activated immune cells under specific circumstances, as an indicator of abnormal activation.

[0074] Recombinant expression vectors and host cells: Generally, the preparation of the fusion protein complexes of the present invention (e.g., components of the TxM complex) can be achieved by the procedures and recognized recombinant DNA techniques disclosed herein.

[0075] Generally, recombinant polypeptides are produced by transforming a suitable host cell with all or part of a nucleic acid molecule or fragment thereof encoding the polypeptide in a suitable expression vehicle. Those skilled in the field of molecular biology will understand that any of the diverse expression systems can be used to provide recombinant proteins. The exact host cell used is not important to the present invention. Recombinant polypeptides can be produced in substantially any eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3 cells, HeLa cells, or preferably COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection in Rockland, Maryland; also see, e.g., Ausubel & al., Current Protocol in Molecular Biology, New York: John Wiley and Sons, 1997 (both are incorporated herein by reference in their entirety)). The method of transfection and the choice of expression vehicle depend on the host system chosen. Transformation methods are described, for example, in Ausubel et al. (cited above), and expression vehicles can be selected from those provided, for example, in Cloning Vectors: A Laboratory Manual (Pouwels et al., 1985, Supp. 1987, the entire manual of which is incorporated herein by reference).

[0076] Various expression systems exist for the production of recombinant polypeptides. Useful expression vectors for producing such polypeptides include, but are not limited to, chromosome vectors, episome vectors, and virus-derived vectors, such as those derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosome elements, viruses such as baculoviruses, papovaviruses such as SV40, vaccinia viruses, adenoviruses, fowlpox viruses, pseudorabies viruses, and retroviruses, as well as vectors derived from combinations thereof.

[0077] Once the recombinant polypeptide is expressed, it can be isolated, for example, using affinity chromatography. For example, an antibody produced against the polypeptide (e.g., manufactured as described herein) can be conjugated to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of the polypeptide-containing cells prior to affinity chromatography can be performed by standard methods (see, for example, Ausubel & al. (previously cited)). Once isolated, the recombinant protein can be further purified, if necessary, by high-performance liquid chromatography (see, for example, Fisher, Laboratory Techniques in Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980, which is incorporated entirely herein by reference).

[0078] Vectors are tools used as a means of shuttle DNA between host cells or express polynucleotide sequences. Insertion of target DNA, such as an anti-CTLA4 binding domain or an scFv encoding a CD16 sequence or fragment, is achieved by ligation techniques and / or mating protocols well known to those skilled in the art. Such DNA is inserted in such a way that its incorporation does not disrupt any necessary components of the vector. In the case of vectors used to express the inserted DNA as a polypeptide, the introduced DNA is operably ligated to vector elements that control its transcription and translation.

[0079] Vectors can be divided into two general classes: Cloning vectors are replication plasmids or phages that have regions not essential for proliferation in suitable host cells, into which foreign DNA can be inserted, and the foreign DNA is replicated and grown as if it were a component of the vector. Expression vectors (e.g., plasmids, yeast, or animal virus genomes) are used to introduce foreign genetic material into host cells or tissues for transcription and translation of the foreign DNA. In expression vectors, the introduced DNA is operably ligated to elements such as promoters that signal the host cell to transcribe the inserted DNA. Some promoters are very useful, such as inducible promoters that regulate gene transcription in response to specific factors. The expression of a gene or its fragment can be controlled by operably ligating an scFv encoding αCTLA4 or a polynucleotide encoding the CTLA4 polypeptide to an inducible promoter. Examples of inducible promoters include those that are tissue-specific and drive expression to specific cell types, steroid-responsive (e.g., glucocorticoids), or heat shock-responsive. Other desirable inducible promoters include those that are not endogenous to the cells into which the construct is introduced, but are responsive to those cells when the inducer is supplied exogenously.

[0080] Vectors have many characteristics. A "plasmid" is a circular double-stranded DNA molecule that can accept additional DNA fragments. Viral vectors can also accept additional DNA segments into the viral genome. Certain vectors can autonomously replicate in host cells (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell genome and replicate as part of the host genome. Generally, useful expression vectors are plasmids and viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), but other expression vectors can also be used.

[0081] Vector selection is determined by the organism or cell being used and the desired fate of the vector. A vector may be capable of one replication within the target cell or it may be a "suicide" vector. Generally, a vector contains a signal sequence, origin of replication, marker gene, enhancer element, promoter, and transcription termination sequence. Vectors often use selection markers to facilitate the identification of the cells incorporating the vector.

[0082] A “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted to result in replication of the inserted segment. Generally, a vector can replicate when associated with an appropriate regulatory element. Suitable vector backbones include, for example, plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs, which are routinely used in the art. The term “vector” includes cloning vectors and expression vectors, as well as viral vectors and embedded vectors. An “expression vector” is a vector containing a regulatory region. Nucleic acid sequences described herein can be expressed using a variety of host / expression vector combinations. Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from bacteriophages, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, Wisconsin), Clontech (Palo Alto, California), Stratagene (La Horrah, California), and Invitrogen / Life Technologies (Carlsbad, California).

[0083] The vector may include, for example, an origin of replication, a scaffolding-binding region (SAR), and / or a marker. The marker gene can confer a selectable phenotype to the host cell. For example, the marker can confer biocide resistance, such as resistance to antibiotics (e.g., kanamycin, G418, bleomycin, or hygromycin). The expression vector may include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG® tag (Kodak, New Haven, Connecticut) sequences, are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including either a carboxyl terminus or an amino terminus.

[0084] Additional expression vectors may include, for example, chromosomal, non-chromosomal, and synthetic DNA sequence segments. Suitable vectors include SV40 and derivatives of known bacterial plasmids, e.g., Escherichia coli plasmids El, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9 and their derivatives; plasmids, e.g., RP4; phage DNA, e.g., numerous derivatives of phage 1, e.g., NM989 and other phage DNA, e.g., M13 and filamentous single-stranded phage DNA; yeast plasmids, e.g., 2μ plasmid or its derivatives; vectors useful in eukaryotic cells, e.g., vectors useful in insect cells or mammalian cells; and vectors derived from combinations of plasmids and phage DNA, e.g., plasmids modified to use phage DNA or other expression regulatory sequences.

[0085] Yeast expression systems can also be used. For example, according to the present invention, to name just two, non-fusion pYES2 vectors (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1 and HindIII cloning sites; Invitrogen) or fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI and HindIII cloning sites, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen) can be used. Yeast two-hybrid expression systems can also be prepared according to the present invention.

[0086] Several delivery methods can be used in in vitro (cell culture) and in vivo (animal and patient) systems. In one embodiment, a lentiviral gene delivery system can be used. Such a system provides stable, long-term presence of genes in dividing and non-dividing cells with broad directivity and ability to insert large DNA. (Dull & al. (1998) J Virol. 72:8463-71, the whole is incorporated herein by reference). In one embodiment, adeno-associated virus (AAV) can be used as a delivery method. AAV is a non-pathogenic single-stranded DNA virus that has recently been actively used to deliver therapeutic genes in in vitro and in vivo systems. (Choi & al. Curr Gene Ther (2005) 5:299-310, the whole is incorporated herein by reference). An exemplary non-viral delivery method can utilize nanoparticle technology. This platform has demonstrated pharmaceutical utility in vivo. Nanotechnology improves drug transcytosis across closely spaced epithelial and endothelial barriers. It provides targeted delivery of its payload to cells and tissues in a specific manner (Allen & Cullis (1998) Science, 303:1818-22, the whole of which is incorporated herein by reference).

[0087] A vector may also include regulatory regions. The term “regulatory region” may refer to a nucleotide sequence that affects the initiation and rate of transcription or translation, as well as the stability and / or mobility of the transcription or translation product. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, induction elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription start sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0088] The term “operably linked” refers to the positioning of regulatory regions and sequences transcribed within a nucleic acid to influence the transcription or translation of such sequences. For example, to place a coding sequence under the control of a promoter, the translation start site of the translational reading frame of a polypeptide is typically located 1 to about 50 nucleotides downstream of the promoter. However, the promoter can be positioned about 5,000 nucleotides upstream of the translation start site or about 2,000 nucleotides upstream of the transcription start site. The promoter typically includes at least a core (basal) promoter. The promoter may also include at least one regulatory element, such as an enhancer sequence, an upstream element, or an upstream activation region (UAR). The selection of the included promoters depends on several factors, including, but not limited to, efficiency, selectivity, inducibility, desired expression level, and cell or tissue preferential expression. Regulating the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence is commonplace for those skilled in the art.

[0089] Examples of vectors include viral vectors (such as adenovirus Ad, AAV, lentivirus, vesicular stomatitis virus (VSV), and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes that can mediate the delivery of polynucleotides to host cells. Vectors may also include other components or functionalities that further modulate gene delivery and / or gene expression or otherwise provide beneficial properties to target cells. As will be described and illustrated in more detail below, such other components include, for example, components that affect cell binding or targeting (including components that mediate cell type or tissue-specific binding); components that affect the uptake of vector nucleic acids by cells; components that affect the localization of polynucleotides within cells after uptake (such as drugs that mediate nuclear localization); and components that affect polynucleotide expression. Such components may also include markers, such as detectable and / or selectable markers, which can be used to detect or select cells that have taken up and are expressing nucleic acids delivered by the vector. Such components may be provided as intrinsic features of the vector (e.g., components that mediate binding and incorporation, or the use of specific viral vectors with functional properties), or the vector may be modified to provide such functionality. Other vectors include those described by Chen & al. (2003) BioTechniques 34:167-71, which are incorporated herein by reference in their entirety. A variety of such vectors are known in the art and are generally available. A “recombinant viral vector” refers to a viral vector containing one or more heterologous gene products or sequences. Because many viral vectors exhibit size constraints related to packaging, heterologous gene products or sequences are typically introduced by substituting one or more portions of the viral genome. Such viruses may be replication-deficient and require the missing functionality to be provided in trans during viral replication and capsid formation (e.g., by using a helper virus or packaging cell line that possesses the gene products necessary for replication and / or encapsulation).Modified viral vectors in which the delivered polynucleotides are supported on the outside of the viral particle are also described (see, for example, Curiel & al. (1991) PNAS 88:8850-54, which is incorporated herein by reference in its entirety).

[0090] Further vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney's mouse leukemia virus and HIV-based viruses. One HIV-based viral vector contains at least two vectors, in which the gag and pol genes are derived from the HIV genome and the env gene is derived from another virus. Examples of DNA viral vectors include pox vectors such as orthopox vectors or avipox vectors, and herpesvirus vectors such as herpes simplex virus I (HSV) vectors [Geller&al.(1995)J.Neurochem,64:487;Lim&al.(1995)in DNA Cloning:Mammalian Systems,D.Glover,Ed.(Oxford Univ.Press);Geller&al.(1993)Proc Natl.Acad.Sci.USA 90:7603;Geller&al.(1990)Proc Natl.Acad.Sci USA 87:1149],Adenovirus Vectors[LaSalle&al.(1993)Science [259:988; Davidson & al. (1993) Nat. Genet. 3:219; Yang & al. (1995) J. Virol. 69:2004] and Adeno-associated Virus Vectors [Kaplitt & al. (1994) Nat. Genet. 8:148] (both are incorporated herein by reference in their entirety).

[0091] Many viral vectors exhibit size constraints related to packaging; therefore, heterologous gene products or sequences are typically introduced by substituting one or more portions of the viral genome. Such viruses may be replication-deficient, requiring the missing function to be provided in trans during viral replication and capsid formation (e.g., by using a helper virus or packaging cell line that possesses the gene products necessary for replication and / or encapsulation). Modified viral vectors in which the delivered polynucleotides are supported on the outside of the viral particle have also been described (see, for example, Curiel, DT, &al. PNAS 88:8850-8854, 1991, which is incorporated herein by reference in its entirety).

[0092] Suitable nucleic acid delivery systems include recombinant viral vectors, typically sequences from at least one of adenovirus-associated viruses (AAV), helper-dependent adenoviruses, retroviruses, or Japanese liposomal hemagglutinating virus (HVJ) complexes. In such cases, the viral vector comprises a potent eukaryotic promoter operably linked to a polynucleotide, such as a cytomegalovirus (CMV) promoter. The recombinant viral vector may contain one or more polynucleotides, preferably about one polynucleotide. In some embodiments, the viral vector used in the method of the present invention contains about 10 8 ~Approx. 5×10 10 It has pfu (plaque-forming units). In embodiments in which polynucleotides are administered together with a non-viral vector, the use of about 0.1 nanograms to about 4000 micrograms, for example, about 1 nanogram to about 100 micrograms, would often be useful.

[0093] Poxvirus vectors introduce genes into the cytoplasm of cells. Tripoxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors may be indicators of several embodiments of the present invention. In some embodiments, adenovirus vectors may result in shorter expression periods (e.g., less than about one month) than adeno-associated viruses, and may exhibit much longer expression periods. Suitable adenovirus vectors are disclosed in International Publication No. 09 / 06479 (Etubics Corp.), the entire contents of which are incorporated herein by reference. The specific vector to be selected depends on the target cell and the condition to be treated. The selection of an appropriate promoter can be easily achieved. An example of a suitable promoter is the 763 base pair cytomegalovirus (CMV) promoter.Other suitable promoters that may be used for gene expression include, but are not limited to, prokaryotic expression vectors such as Rous sarcoma virus (RSV) (Davis, & al., Hum Gene Ther 4:151 (1993), the entire text of which is incorporated herein by reference), the SV40 initial promoter region, the herpesthymidine kinase promoter, the regulatory sequence of the metallothionein (MMT) gene, the β-lactamase promoter, the tac promoter, promoter elements derived from yeast or other fungi, such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter; and animal transcriptional regulatory regions that exhibit tissue specificity and are utilized in transgenic animals: elastase, which is active in pancreatic acinar cells. Examples include the I gene regulatory region, the insulin gene regulatory region active in pancreatic beta cells, the immunoglobulin gene regulatory region active in lymphoid cells, the mouse mammary tumor virus regulatory region active in testes, mammary glands, lymphocytes, and mast cells, the albumin gene regulatory region active in the liver, the alpha-fetoprotein gene regulatory region active in the liver, the alpha-1 antitrypsin gene regulatory region active in the liver, the betaglobin gene regulatory region active in bone marrow cells, the myelin basic protein gene regulatory region active in oligodendrocytes of the brain, the myosin light chain 2 gene regulatory region active in skeletal muscle, and the gonadotropin-releasing hormone gene regulatory region active in the hypothalamus. Certain proteins can be expressed using their innate promoters. Other elements that can enhance expression, such as enhancers or systems that result in high levels of expression, such as the tat gene and tar element, may also be included. This cassette can then be inserted into a vector, such as a plasmid vector including pUC19, pUC118, pBR322, or another known plasmid vector containing, for example, an E. coli (E. coli) origin. See Sambrook, &al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1989), which is incorporated herein by reference in its entirety.Plasmid vectors may also include select markers, such as β-lactamase genes for ampicillin resistance, as long as the marker polypeptide does not adversely affect the metabolism of the organism being treated.

[0094] Another delivery method involves using a single-stranded DNA-producing vector that can produce the expression product within the cell. Polynucleotides can be used in conjunction with micro-delivery vehicles such as cationic liposomes and adenovirus vectors.

[0095] In certain embodiments of the present invention, transfection can be achieved using non-viral vectors. Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, bioristic, virosome, liposome, immunoliposome, polycation or lipid: nucleic acid conjugate, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Patent No. 5,049,386; U.S. Patent No. 4,946,787; and U.S. Patent No. 4,897,355, which are incorporated herein by reference in their entirety, and lipofection reagents are commercially available (e.g., Transfectam and Lipofectin®). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those described in U.S. Patent No. 7,166,298 or U.S. Patent No. 6,890,554, the contents of each of which are incorporated herein by reference in their entirety. Delivery may be to cells (e.g., in vitro or ex vivo administration) or to target tissue (e.g., in vivo administration).

[0096] Synthetic vectors are typically based on cationic lipids or polymers that can form complexes with negatively charged nucleic acids to create particles with a diameter of approximately 100 nm. The complex protects the nucleic acids from degradation by nucleases. Furthermore, cellular and local delivery strategies must address the need for internalization, release, and distribution within appropriate intracellular compartments. Systemic delivery strategies encounter further obstacles, such as strong interactions between cationic delivery vehicles and blood components, uptake by the reticuloendothelial system, renal filtration, toxicity, and the ability of the carrier to target cells of interest. By modifying the surface of cationic nonviral materials, interactions with blood components can be minimized, uptake by the reticuloendothelial system can be reduced, toxicity can be decreased, and binding affinity to target cells can be increased. Plasma protein binding (also called opsonization) is the primary mechanism by which RES recognizes circulating nanoparticles. For example, macrophages such as Kupffer cells in the liver recognize opsonized nanoparticles via scavenger receptors.

[0097] In some embodiments, vector delivery may also be mediated by exosomes. Exosomes are lipid nanovesicles released by many cell types. They mediate intercellular communication by transporting nucleic acids and proteins between cells. Exosomes contain RNA, miRNA, and proteins derived from the endocytosis pathway. They can be taken up by target cells by endocytosis, fusion, or both. Exosomes can be used to deliver nucleic acids to specific target cells.

[0098] The expression constructs of the present invention can also be delivered by nanoclews. Nanoclews are cocoon-like DNA nanocomposites (Sun&al. (2014) J.Am. Chem. Soc. 136:14722-25, the whole thereof is incorporated herein by reference). They can be loaded with nucleic acids for uptake by target cells and release in the cytoplasm of target cells. Methods for constructing nanoclews, loading them, and designing release molecules can be found in Sun&al. (2014) and Sun&al. (2015) Angew. Chem. Int. 2015:12029-33 (the whole thereof is incorporated herein by reference).

[0099] Nucleic acids and vectors may be administered alone or in mixtures in the presence of pharmaceutically acceptable excipients or carriers (e.g., physiological saline). The excipients or carriers are selected based on the mode and route of administration. Suitable pharmaceutically acceptable carriers and their pharmaceutically necessary use in pharmaceutical formulations are described in the well-known references in this field, Remington's Pharmaceutical Sciences (EW Martin) and USP / NF (United States Pharmacopeia and the National Formulary).

[0100] In some embodiments of the present invention, liposomes are used to achieve transfection into cells or tissues. The pharmacology of liposomal formulations of nucleic acids is largely determined by the extent to which the nucleic acid is encapsulated within the liposome bilayer. Encapsulated nucleic acids are protected from nuclease degradation, while those simply bound to the surface of the liposome are not. Encapsulated nucleic acids share the extended circulating life and biodistribution of intact liposomes, while those associated with the surface adopt the pharmacology of naked nucleic acids upon dissociation from the liposome. Nucleic acids can be captured within liposomes using conventional passive loading techniques such as ethanol drop method (as in SALP), reverse-phase evaporation, and ethanol dilution method (as in SNALP).

[0101] Liposomes and polymerosomes may contain multiple solutions and compounds. In certain embodiments, the complexes of the present invention are coupled to or encapsulated within polymerosomes. As a class of artificial vesicles, polymerosomes are small, hollow spheres that encapsulate solutions, fabricated using amphiphilic synthetic block copolymers to form a vesicle membrane. Typical polymerosomes contain an aqueous solution in their core and are useful for encapsulating and protecting sensitive molecules such as drugs, enzymes, other proteins and peptides, as well as DNA and RNA fragments. The polymer membrane provides a physical barrier that isolates the encapsulated material from external materials, such as those found in biological systems. Polymerosomes can be produced from biemulsions by known techniques (see Lorenceau & al. (2005) Langmuir 21(20):9183-86, which is incorporated herein by reference in its entirety).

[0102] In some embodiments of the present invention, nonviral vectors are modified to achieve targeted delivery and transfection. PEGylation (i.e., surface modification with polyethylene glycol) is a primary method used to reduce opsonization and aggregation of nonviral vectors and minimize clearance by the reticuloendothelial system, resulting in an extended circulating life after intravenous (iv) administration. Therefore, PEGylated nanoparticles are often referred to as "stealth" nanoparticles. Nanoparticles that are not rapidly removed from circulation have an opportunity to encounter infected cells.

[0103] In other embodiments, the composition comprises aNK cells transformed or transfected with one or more vectors encoding isolated nucleic acids as embodied herein.

[0104] Transduced cells are prepared for reinjection according to established methods. After approximately 2-4 weeks in culture, the cell count reaches 1 × 10⁶. 6 ~1 × 10 10This may be the case. In this regard, the characteristics of cell proliferation differ from patient to patient and from cell type to cell type. Approximately 72 hours before reinfusion of transduced cells, aliquots are taken for analysis of the phenotype and the percentage of cells expressing the therapeutic agent. For administration, the cells of the present invention are LD of the cell type so as to be applicable to the patient's mass and overall health condition. 50 It can be administered at a rate determined by the side effects of various cell types at different concentrations. Administration can be achieved in single or divided doses.

[0105] Pharmaceutical therapies. In further embodiments, pharmaceutical compositions comprising a soluble fusion protein complex for use as a therapeutic agent are provided herein. The soluble fusion protein complex may be formulated with a pharmaceutically acceptable carrier for preparing the pharmaceutical composition. The pharmaceutical composition may be administered systemically and may be formulated in a pharmaceutically acceptable buffer, such as physiological saline. Preferred routes of administration include, for example, intravesical infusion, subcutaneous, intravenous, intraperitoneal, intramuscular, intratumoral, or intradermal injection, which provide a continuous, sustained, or effective level of the composition in the patient. In particular, the soluble fusion protein complex and the pharmaceutically acceptable carrier may be formulated for parenteral injection, including, but not limited to, subcutaneous, intravenous, intramuscular, intravesicular, intratumoral, or intraperitoneal injection.

[0106] Treatment of human patients or other animals is carried out using therapeutically effective amounts of the therapeutic agents identified herein in physiologically acceptable carriers. Suitable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences by E.W. Martin. The amount of therapeutic agent administered varies depending on the mode of administration, the patient's age and weight, and the clinical manifestation of the neoplasm. Generally, the amount is within the range used for other agents used to treat neoplasms, autoimmune or infectious diseases, although in certain cases, a smaller amount is required due to increased specificity of the soluble fusion protein complex. The soluble fusion protein complex is administered in a dose that enhances the immune response of the target or reduces the proliferation, viability, or infiltration of neoplastic cells, infected cells, or autoimmune cells, as determined by methods known to those skilled in the art.

[0107] Formulation of pharmaceutical compositions. Administration of the fusion protein complex of the present invention for the prevention or treatment of neoplasms or autoimmune diseases is by any suitable means, in combination with other components, to provide a concentration of therapeutic agent effective in improving, reducing or stabilizing the above neoplasms, infectious or autoimmune diseases. The fusion protein complex of the present invention may be contained in any suitable amount in any suitable carrier material, generally present in an amount of 1 to 95% by weight of the total weight of the composition. The composition may be provided in dosage forms suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intravesicular, intratumoral, or intraperitoneal) administration routes. For example, pharmaceutical compositions may be provided in accordance with conventional pharmaceutical practices (e.g., Remington: The Science and Practice of Pharmacy (2020), which is incorporated herein by reference in whole). th It is formulated according to (see ed.), ed. ARGennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JCBoylan, 1988-1999, Marcel Dekker, New York).

[0108] Those skilled in the art will recognize that it is common practice in the art to vary human dosages compared to animal models; therefore, human dosages are initially determined by extrapolating from the amount of soluble fusion protein complex used in mice or non-human primates. For example, dosages may vary between approximately 1 μg soluble fusion protein complex / kg body weight and approximately 5,000 mg complex / kg body weight, or approximately 5 mg / kg body weight and approximately 4,000 mg / kg body weight, or approximately 10 mg / kg body weight and approximately 3,000 mg / kg body weight, or approximately 50 mg / kg body weight and approximately 2,000 mg / kg body weight, or approximately 100 mg / kg body weight and approximately 1,000 mg / kg body weight, or approximately 150 mg / kg body weight and approximately 500 mg / kg body weight. For example, the dosages are approximately 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, and The dosages are approximately 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 mg / kg body weight. Alternatively, the dosage ranges from approximately 5 mg complex / kg body weight to approximately 20 mg complex / kg body weight. In another example, the dosage is approximately 8, 10, 12, 14, 16, or 18 mg / kg body weight. Preferably, the soluble fusion protein complex is administered at approximately 0.5 mg / kg to approximately 10 mg / kg (e.g., approximately 0.5, approximately 1, approximately 3, approximately 5, approximately 10 mg / kg). Of course, this dosage can be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of the specific patient.

[0109] Pharmaceutical compositions are formulated using appropriate excipients to release the therapeutic agent in a controlled manner upon administration. Examples include single or multiple tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes. Preferably, fusion protein complexes are formulated in excipients suitable for parenteral administration.

[0110] Parenteral compositions. Pharmaceutical compositions comprising the fusion protein complex of the present invention may be administered parenterally by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intratumoral, intravesical, intraperitoneal) via conventional non-toxic, pharmaceutically acceptable carriers and adjuvants in dosage forms, formulations, or via suitable delivery devices or implants. Formulations and preparations of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in the aforementioned Remington: The Science and Practice of Pharmacy.

[0111] Compositions comprising the fusion protein complex of the present invention for parenteral use are provided in unit dosage forms (e.g., single-dose ampoules). Alternatively, compositions may be provided in vials containing several doses, and suitable preservatives may be added (see below). Compositions are provided in the form of solutions, suspensions, emulsions, injectors or delivery devices for implantation, or as a dry powder that is reconstituted with water or another suitable vehicle before use. Apart from the active agent for reducing or improving neoplasms, infectious or autoimmune diseases, the compositions include suitable parenterally acceptable carriers and / or excipients. The active therapeutic agent may be incorporated into microspheres, microcapsules, nanoparticles, or liposomes for controlled release. Furthermore, compositions may include suspending agents, solubilizers, stabilizers, pH adjusters, isotonic adjusters and / or dispersants.

[0112] As described above, the pharmaceutical composition comprising the soluble fusion protein complex of the present invention may be in a form suitable for sterile injection. To prepare such a composition, a suitable active therapeutic agent is dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that may be used include water, water adjusted to a suitable pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide, or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl p-hydroxybenzoate, ethyl, or n-propyl). If one of the active therapeutic agents is poorly soluble or only slightly soluble in water, a solubilizer or dissolubilizer may be added, or the solvent may contain 10-60% w / w propylene glycol.

[0113] The present invention provides a method for preventing or treating cancer, neoplasms, or autoimmune diseases or disorders or their symptoms, comprising administering a therapeutically effective amount of a pharmaceutical composition containing a soluble fusion protein complex to a subject in need (e.g., a mammal such as a human). Accordingly, one embodiment is a method for treating a subject who is suffering from or susceptible to cancer, neoplasms, or autoimmune diseases or disorders or their symptoms. In particular, cancers treated by the administration of the pharmaceutical composition include acute leukemia, AIDS-related cancer, breast cancer, bone cancer, brain tumor, head and neck cancer, lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinal cancer, esophageal cancer, gastric cancer, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, pancreatic cancer, sarcoma, Wilms' tumor, cervical cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma, and multidrug-resistant cancer. The method includes administering to a mammal a therapeutic amount of the soluble fusion protein complex described herein or a pharmaceutical composition containing the soluble fusion protein complex sufficient to treat a disease or disorder or its symptoms, under conditions such that the disease or disorder is treated.

[0114] Additionally or alternatively, the soluble fusion protein complex described or a pharmaceutical composition containing the same may be used to prevent or treat cancer in subjects where such treatment is needed. Additionally or alternatively, the soluble fusion protein complex described or a pharmaceutical composition containing the same may be used to prevent or treat autoimmune diseases or disorders in subjects where such treatment is needed.

[0115] The methods described herein involve administering an effective amount of the described soluble fusion protein complex or a pharmaceutical composition containing the same to a subject (including a subject identified as needing such treatment) in order to produce such an effect. Identification of a subject needing such treatment may be at the discretion of the subject or a medical professional and may be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

[0116] The therapeutic methods of the present invention (including prophylactic treatment) generally involve administering a therapeutically effective amount of the soluble fusion protein complex described herein to a subject (e.g., animal, human) in need of it, including mammals, particularly humans, wherein the IL-15 peptide of the first domain is sequence-identical (e.g., at least 85% sequence-identical) to the IL-15 peptide of the first domain, the IL-15 peptide of the second domain is sequence-identical (e.g., at least 85% sequence-identical) to the IL-15 peptide of the second domain. Such treatment is appropriately administered to subjects, particularly humans, who are suffering from, have, are susceptible to or at risk of suffering from, neoplasms, infectious diseases, autoimmune diseases, disorders or symptoms thereof. The determination of these "at-risk" subjects can be made by any objective or subjective determination based on diagnostic tests or the opinion of the subject or healthcare provider (e.g., genetic testing, enzyme or protein markers, markers (as defined herein), family history, etc.). The fusion protein complex of the present invention may be used to treat any other disorder for which an increase in the immune response is desired.

[0117] The present invention also provides a method for monitoring the progress of treatment. This method includes determining the level of a diagnostic marker (marker) (e.g., any target described herein, modulated by a soluble fusion protein complex, protein, or indicator thereof, etc.) or a diagnostic measurement (e.g., screening, assay) in a subject who is suffering from or susceptible to a disorder or symptoms associated with a neoplasm, wherein the subject is administered a therapeutic dose of the described soluble fusion protein complex sufficient to treat the disease or symptoms thereof. The level of the marker determined by this method can be compared to a known level of the marker in either a healthy normal control or another affected patient to establish the disease state of the subject. In some cases, a second level of the marker in the subject is determined at a later point in time than the determination of the first level, and the two levels are compared to monitor the course of the disease or the effectiveness of the treatment. In certain embodiments, the pre-treatment level of the marker in the subject is determined before initiating treatment according to the present invention, and this pre-treatment level of the marker can then be compared to the level of the marker in the subject after initiation of treatment to determine the effectiveness of the treatment.

[0118] Combination therapy. The soluble fusion protein complexes described herein may be administered in combination with any other standard treatment, such methods are known to those skilled in the art and are described in Remington's Pharmaceutical Sciences by E.W. Martin. Where necessary, the fusion protein complexes of the present invention may be administered in combination with any conventional antineoplasm therapy, including, but not limited to, immunotherapy, therapeutic antibodies, targeted therapy, surgery, radiotherapy, or chemotherapy.

[0119] Kits or pharmaceutical systems. Pharmaceutical compositions comprising the fusion protein complex of the present invention may be assembled into kits or pharmaceutical systems for use in the treatment of neoplasms or autoimmune diseases. Kits or pharmaceutical systems according to this aspect of the present invention include carrier means such as boxes, cartons, or tubes that tightly enclose one or more container means such as vials, tubes, ampoules, or bottles. Kits or pharmaceutical systems of the present invention may also include relevant instructions for using the fusion protein complex and / or cells of the present invention. [Examples]

[0120] Example 1 - Preparation of molecules shown in Figures 1-3 By transfecting mammalian cells and expressing various molecules, we created molecules as shown in Figures 1 to 3. Those skilled in the art are well aware of the methods for creating and expressing fusion protein complexes in mammalian cells. Briefly, the DNA sequence encoding the soluble fusion protein complex was cloned into a mammalian expression vector under a CMV promoter containing an SV40 polyadenylated sequence. The plasmid contained ampicillin and puromycin resistance markers. The mammalian expression vector was transformed into chemically qualified Escherichia coli (E. coli). The transformed E. coli were cultured in ampicillin-containing medium under standard conditions. The plasmid was isolated and purified from the E. coli culture using a Qiagen Maxiprep® column according to the manufacturer's protocol.

[0121] Chinese hamster ovary-S (CHO-S) cells were suspended and cultured in a shaking flask in CD-CHO medium supplemented with 8 mM L-glutamine at 37°C and 8% CO2, shaking at 125 rpm. For transfection, exponentially growing cells were pelletized by centrifugation (1,400 rpm for 10 minutes), resuspended in 10 mL of electroporation buffer, and repelled (1,400 rpm for 5 minutes). The cell pellet was then mixed with a plasmid containing the target DNA sequence at a DNA concentration of 150 μg / mL in electroporation buffer, with 2 × 10⁶ cells per pellet. 8The cells were resuspended at a density of cells / mL. Cells were transfected using the OC-400 processing assembly in the Maxcyte® ExPERT ATx Transfection System. Transfected cells were incubated at 37°C and 5% CO2 for 30 minutes, then approximately 4–6 × 10⁻¹⁶ cells were incubated. 6 Cells were resuspended in an Efficient Feed A cocktail (CHO-CD EfficientFeed® A + 0.2% Pluronic F-68 + 1% HT supplement + 1% L-glutamine) at a density of cells / mL. Cells were incubated overnight on a shaker at 37°C, 5% CO2, and 125 rpm. 1 mM sodium butyrate was added, and the culture was incubated for a further 13 days at 32°C, 3% CO2, and 125 rpm. Maxcyte® Feed Cocktail (13.9% CD hydrolysate, 69.5% CHO CD EfficientFeed® A, 6.2% glucose, 6.9% FunctionMax® Titer Enhancer, and 3.5% L-glutamine) was added at 10% of the culture volume on days 3 and 8. The culture was harvested on day 14.

[0122] The titer of the fusion protein was determined by obtaining a small aliquot of the cell culture supernatant after Maxcyte® transfection. Titer was measured using a Protein A biosensor (Sartorius) on an OctetRed96e instrument (Sartorius). The "% main peak" was determined after titer measurement. The final elution pool after Protein A column purification or second cation exchange column purification was analyzed using a size exclusion (SE) column (Sepax Zenix-C SEC-300) on an Agilent HPLC (Agilent) equilibrated with 50 mM sodium phosphate, pH 6.8, and 250 mM NaCl. The "% peak area" under the curve was calculated using Agilent HPLC software. Purity analysis results are shown in Table 1 below.

[0123] [Table 1]

[0124] Example 2 - Bonding Evaluation Surface plasmon resonance (SPR) studies were performed using purified fusion proteins to evaluate their binding affinity to relevant targets. SPR kinetic assays were carried out by immobilizing a rabbit α-human IgG Fc antibody (Thermo) onto a PCH sensor (Sartorius) as a capture molecule. All target molecules were then captured on the sensor surface via α-IgG1-Fc and Fc domain interactions. Binding kinetics to IL-2Rβ, human CTLA4, and mouse CTLA4 were determined by OneStep injection at 40 nM and 20 nM of each analyte. The results of these studies are shown in Table 2 below. As these data show, the binding affinity of αCTLA4 / TxM-C to CTLA4 is reduced compared to forms A and B, indicating that the proximity of scFv at the C-terminus of the Fc domain is detrimental to structural integrity. αCTLA4-scFv has a higher affinity for CTLA4 than when the binding domain is in the IgG form. All scFvs are formed in a VL-VH configuration, but the orientation of VH and VL relative to the linker does not appear to affect the bond affinity.

[0125] [Table 2]

[0126] Example 3 - Stability Evaluation To evaluate the stability of various fusion protein configurations, purified fusion proteins were analyzed by differential scanning fluorescence (DSF). 20 μL of 1 mg / mL ACEIgG1Fc was mixed with 10 μL of SYPRO orange in a suitable well or plate. The plates were scanned at 25°C to 70°C (in 0.5°C increments) using a CFX96 real-time system (BioRad). The results are shown in Figure 4. The molecule in Figure 3 appears to be intrinsically more stable than the molecule in Figure 2.

[0127] Example 4 - Evaluation of Off-Target Effects A useful therapeutic candidate must not only bind specifically to the target but also exhibit no off-target effects. Various molecular compositions were investigated according to several assays for off-target effects. A general method for determining off-target effects can be found in Jain & al. (2017) Proc. Natl. Acad. Sci. USA 114:944-949, which is incorporated herein by reference in its entirety. Cetuximab was included in the test as a negative control, while αIL-18 antibodies (43-12b) served as the positive control antibody. The results are shown in Table 3 below. The MSD multireactivity assay for nonspecific adhesion (Column A) often shows faster clearance in monoclonal antibodies. Hydrophobic interaction chromatography (HIC) (Column B) measures the hydrophobicity of monoclonal antibodies, which likely correlates with the tendency to aggregate. Clonal self-interaction using biolayer interferometry (CSI-BLI) (Column C) measures the tendency of monoclonal antibodies to self-aggregate, which often indicates low solubility. Stability acceleration (column D) measures the long-term aggregation tendency of the monoclonal antibody. Based on these results, both αCTLA4 / TxM-B and αCTLA4 / IL-15 fusions are strong candidates.

[0128] [Table 3]

[0129] Example 5 - B16.F10 Melanoma Tumor Model For the preclinical evaluation of these candidate immunotherapies, a syngeneic B16.F10 melanoma tumor model was used via tumor (it) administration. C57BL / 6 mice were purchased from Jackson Laboratories (Maine, USA). The B16.F10 mouse melanoma cell line was maintained as a monolayer in cultures using RPMI medium supplemented with 10% thermoinactivated fetal bovine serum (FBS), 1% non-essential amino acids, 10 mM HEPES, 1% sodium pyruvate, and 1% penicillin / streptomycin. A single-cell suspension of the B16.F10 monolayer was prepared and placed in 50 μL of PBS at a rate of 1.3 × 10⁶ cells. 5 The cells were intradermally transplanted into the shaved flanks of each of the two C57BL / 6 mice. The tumors were allowed to grow to an average diameter of 5–7 mm (approximately 8 days), at which point the mice were randomly enrolled in the treatment group. For each mouse, the larger of the two tumors was injected with a total volume of 50 μL of the indicated treatment on days 0, 2, and 4 after enrollment. The it treatment involves PBS (as a negative control) or four B16.F10 neoepitopes, 25 μg of anti-OX40 antibody, and 5 yeast units (YU, 1 YU = 10¹¹) of Saccharomyces cerevisiae yeast lysate expressing one of the following: 25 μg αCTLA4-7; 25 μg αCTLA4-7 + 25 μg NAI(N-803); 25 μg αCTLA4 / TxM-B(N-844-2); 25 μg IL-15 / αCTLA4(N844-4); or 25 μg αCTLA4 / IL-15(N-844-5). 7 It consisted of cells. Both treated (injected) and untreated (distal) tumors were measured at periodic intervals using digital calipers, and the longest diameter (A) and the shortest diameter (B) were used to determine V = (AB 2 The tumor volume was calculated as () / 2. Tumor growth was monitored over time, and when the tumor grew to ≥1000 mm 3Mice were euthanized when the tumor volume reached a certain level. The aggregated results are shown in Figure 5. Tumor growth in individual mice is shown in Figures 6A to 6E. The efficacy of the treatment in injected tumors and distal tumors at day 11, as measured by AUC calculations, is shown in Figures 7A and 7B, respectively. The results indicate that administration of the soluble fusion protein complex significantly reduces tumor volume. Figure 7C shows that the described soluble fusion protein complex is generally tolerable up to 18 days in the mouse melanoma model.

[0130] Example 6 - pSTAT5 assay The function of the listed soluble fusion protein complexes is measured by an in vitro staining assay. IL-2 deficient aNK cells (4 × 10⁶ cells per well of a 96-well plate) 5 Cells (per well) are stimulated with a dilution series of IL-18 / IL-15 / IL-12 superkines (eight 10-fold dilutions starting from 100 nM). After stimulation, cells are fixed, permeabilized, and stained with PE-labeled αpSTAT5. PE-stained aNK cells are detected and quantified by intracellular flow cytometry. Data are processed with GraphPad Prism to determine the EC of superkine stimulation. 50 To decide.

[0131] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as they are incorporated herein by reference, to the same extent that each reference is incorporated by reference individually and specifically.

[0132] The use of the term “at least one” followed by a list of one or more items (e.g., “at least one of A and B”) should be interpreted as meaning one item selected from the enumerated items (A or B) or any combination of two or more enumerated items (A and B), unless otherwise specifically indicated herein or unless clearly inconsistent with the context. The enumeration of ranges of values ​​herein is intended solely as a convenient way to refer individually to each distinct value contained within the range, unless otherwise indicated herein, and each distinct value is incorporated herein as if it were individually enumerated herein. All methods described herein may be performed in any preferred order unless otherwise indicated herein or unless clearly inconsistent with the context. The use of any examples or exemplary language provided herein (e.g., “etc.”) is intended solely to better illustrate the invention and does not limit the scope of the invention unless specifically claimed. No language herein should be interpreted as indicating that an unclaimed element is essential for the practice of the invention.

[0133] Specific embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Variations of these specific embodiments may become apparent to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will appropriately use such variations, and the inventors intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter enumerated in the appended claims, as permitted by applicable law. Furthermore, unless otherwise indicated herein or unless clearly inconsistent with the context, any combination of the elements described above in all possible variations thereof is incorporated into the Invention. The present invention includes, for example, the following embodiments: [1] A soluble fusion protein complex comprising: (a) a first domain comprising an interleukin-15 (IL-15) peptide having at least 85% sequence identity with SEQ ID NO: 17; (b) a second domain comprising a fusion polypeptide comprising an IL-15 receptor alpha-Sushi (IL15RαSu) peptide and an αCTLA4 antibody heavy chain, wherein the αCTLA4 antibody heavy chain has at least 85% sequence identity with SEQ ID NO: 10; and (c) a third domain comprising an αCTLA4 antibody light chain having at least 85% sequence identity with SEQ ID NO: 3, wherein the IL-15 peptide of the first domain binds to the IL-15RαSu peptide of the second domain to form a soluble fusion protein complex. [2] The soluble fusion protein complex according to [1], wherein the αCTLA4 antibody heavy chain has at least 90% sequence identity with SEQ ID NO: 10 and includes SEQ ID NOs: 11-13, and the αCTLA4 antibody light chain has at least 90% sequence identity with SEQ ID NO: 3 and includes SEQ ID NOs: 14-16. [3] The soluble fusion protein complex according to [1] or [2], wherein the αCTLA4 antibody heavy chain has at least 95% sequence identity with SEQ ID NO: 10 and includes SEQ ID NOs: 11-13, and the αCTLA4 antibody light chain has at least 95% sequence identity with SEQ ID NO: 3 and includes SEQ ID NOs: 14-16. [4] The soluble fusion protein complex according to [3], wherein the αCTLA4 antibody heavy chain comprises the sequence of SEQ ID NO: 10, and the αCTLA4 antibody light chain comprises the sequence of SEQ ID NO: 3. [5] The IL15RαSu peptide is a soluble fusion protein complex according to any one of [1] to [4], having at least 85% sequence identity with SEQ ID NO: 8. [6] The IL-15 peptide is a soluble fusion protein complex according to any one of [1] to [5], comprising SEQ ID NO: 17. [7] The IL-15 peptide is a soluble fusion protein complex according to any one of [1] to [6], comprising SEQ ID NO: 1. [8] The fusion polypeptide further comprises a peptide linker between the amino terminus of the IL15RαSu peptide and the carboxyl terminus of the αCTLA4 antibody heavy chain, as described in any of [1] to [7]. [9] The fusion polypeptide comprises at least 85% sequence identity with SEQ ID NO: 5, a soluble fusion protein complex according to any one of [1] to [8].

[10] The fusion polypeptide comprises SEQ ID NO: 5, the soluble fusion protein complex according to [9].

[11] The fusion polypeptide further comprises a peptide linker between the carboxyl terminus of the IL15RαSu peptide and the amino terminus of the αCTLA4 antibody heavy chain, as described in any of [1] to

[10] .

[12] The fusion polypeptide comprises at least 85% sequence identity with SEQ ID NO: 4, the soluble fusion protein complex according to any one of [1] to

[11] .

[13] The fusion polypeptide is a soluble fusion protein complex according to any one of [1] to

[12] , comprising SEQ ID NO: 4. A pharmaceutical composition comprising a soluble fusion protein complex described in any of

[14] [1] to

[13] and a pharmaceutically acceptable carrier.

[15] The pharmaceutical composition according to

[14] , which is formulated for parenteral injection.

[16] A pharmaceutical composition according to

[14] or

[15] , formulated for subcutaneous, intravenous, intramuscular, intravesicular, intratumorial, or intraperitoneal injection.

[17] A pharmaceutical composition according to any one of

[14] to

[16] , formulated for intravenous injection.

[18] A method for preventing or treating cancer in a subject in need thereof, comprising administering to the subject a soluble fusion protein complex described in any of [1] to

[13] or a pharmaceutical composition described in any of

[14] to

[17] .

[19] The cancer is selected from the group consisting of acute leukemia, AIDS-related cancer, breast cancer, bone cancer, brain tumor, head and neck cancer, lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinal cancer, esophageal cancer, gastric cancer, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer, pancreatic cancer, sarcoma, Wilms' tumor, cervical cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma, and multidrug-resistant cancer, according to

[18] .

[20] A method for preventing or treating an autoimmune disease or disorder in a subject in need thereof, comprising administering to the subject a soluble fusion protein complex described in any of [1] to

[13] or a pharmaceutical composition described in any of

[14] to

[17] .

[21] Use of a soluble fusion protein complex according to any of [1] to

[13] or a pharmaceutical composition according to any of

[14] to

[17] for the prevention or treatment of cancer in a person in need thereof.

[22] Use of a soluble fusion protein complex according to any of [1] to

[13] or a pharmaceutical composition according to any of

[14] to

[17] for the prevention or treatment of an autoimmune disease or disorder in a person in need thereof.

[0134] Table 4

[0135] Table 5

[0136] Table 6

Claims

1. A soluble fusion protein complex, (a) A first domain comprising an interleukin-15 (IL-15) peptide having at least 85% sequence identity with SEQ ID NO: 17, (b) A fusion polypeptide comprising an IL-15 receptor alpha-Sushi (IL15RαSu) peptide and an αCTLA4 antibody heavy chain, having a second domain having at least 85% sequence identity with either SEQ ID NO: 4 or 5, (c) A third domain comprising an αCTLA4 antibody light chain having at least 85% sequence identity with SEQ ID NO: 3 A soluble fusion protein complex comprising the IL-15 peptide of the first domain, wherein the IL-15 peptide of the second domain is non-covalently bonded to the IL-15RαSu peptide of the second domain to form a soluble fusion protein complex.

2. The soluble fusion protein complex according to claim 1, wherein the αCTLA4 antibody heavy chain has at least 90% sequence identity with SEQ ID NO: 10 and includes SEQ ID NOs: 11 to 13, and the αCTLA4 antibody light chain has at least 90% sequence identity with SEQ ID NO: 3 and includes SEQ ID NOs: 14 to 16.

3. The soluble fusion protein complex according to claim 1, wherein the αCTLA4 antibody heavy chain has at least 95% sequence identity with SEQ ID NO: 10 and includes SEQ ID NOs: 11 to 13, and the αCTLA4 antibody light chain has at least 95% sequence identity with SEQ ID NO: 3 and includes SEQ ID NOs: 14 to 16.

4. The soluble fusion protein complex according to claim 3, wherein the αCTLA4 antibody heavy chain comprises the sequence of SEQ ID NO: 10, and the αCTLA4 antibody light chain comprises the sequence of SEQ ID NO:

3.

5. The soluble fusion protein complex according to claim 1, wherein the IL15RαSu peptide has at least 85% sequence identity with SEQ ID NO:

8.

6. The soluble fusion protein complex according to claim 1, wherein the IL-15 peptide comprises SEQ ID NO:

17.

7. The soluble fusion protein complex according to claim 1, wherein the IL-15 peptide comprises SEQ ID NO:

1.

8. The soluble fusion protein complex according to claim 1, wherein the fusion polypeptide further comprises a peptide linker between the amino terminus of the IL15RαSu peptide and the carboxyl terminus of the αCTLA4 antibody heavy chain.

9. The soluble fusion protein complex according to claim 1, wherein the fusion polypeptide has at least 95% sequence identity with SEQ ID NO:

5.

10. The soluble fusion protein complex according to claim 9, wherein the fusion polypeptide comprises SEQ ID NO:

5.

11. The soluble fusion protein complex according to claim 1, wherein the fusion polypeptide further comprises a peptide linker between the carboxyl terminus of the IL15RαSu peptide and the amino terminus of the αCTLA4 antibody heavy chain.

12. The soluble fusion protein complex according to claim 1, wherein the fusion polypeptide has at least 95% sequence identity with SEQ ID NO:

4.

13. The soluble fusion protein complex according to claim 1, wherein the fusion polypeptide comprises SEQ ID NO:

4.

14. A pharmaceutical composition comprising a soluble fusion protein complex according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, formulated for parenteral injection.

16. The pharmaceutical composition according to claim 14, which is formulated for subcutaneous, intravenous, intramuscular, intravesicular, intratumoral, or intraperitoneal injection.

17. The pharmaceutical composition according to claim 14, formulated for intravenous injection.

18. The pharmaceutical composition according to claim 14 for use in a method for preventing or treating cancer in a subject in need thereof, the method comprising administering the soluble fusion protein complex to the subject.

19. The pharmaceutical composition according to claim 18, wherein the cancer is selected from the group consisting of acute leukemia, AIDS-related cancer, breast cancer, bone cancer, brain tumor, head and neck cancer, lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinal cancer, esophageal cancer, gastric cancer, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer, pancreatic cancer, sarcoma, Wilms' tumor, cervical cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma, and multidrug-resistant cancer.

20. The pharmaceutical composition according to claim 14 for use in a method for preventing or treating an autoimmune disease or disorder in a subject in need thereof, the method comprising administering the soluble fusion protein complex to the subject.

21. Use of a soluble fusion protein complex according to any one of claims 1 to 13 in the manufacture of a pharmaceutical product for the prevention or treatment of cancer in a person in need thereof.

22. Use of a soluble fusion protein complex according to any one of claims 1 to 13 in the manufacture of a pharmaceutical product for the prevention or treatment of an autoimmune disease or disorder in a person in need thereof.

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