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 TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 302,044, filed January 22, 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 and nucleic acid sequences submitted contemporaneously herewith as a Sequence Listing, ST.26 XML file entitled "000112wopoa_SequenceListing.XML" (file size 29.8 kilobytes (KB), created Jan. 10, 2023). The above Sequence Listing is hereby incorporated by reference in its entirety pursuant to 37 C.FR § 1.52(e)(5).

[0003] The present 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 similar 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, whereas CD28 transmits stimulatory signals.

[0005] Interleukin-15 (IL-15) inhibits CD8 + / CD4 + Stimulation of T cells and natural killer (NK) cells can promote both innate and adaptive immune responses, but T regulatory (T reg) cells or induce activation-associated death of effector T cells and NK cells. IL-15 is therefore a promising molecule for antitumor immunotherapy. Bessard&al.(2009)Mol Cancer Ther.8(9):2736-45.

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

[0007] Disclosed herein is a soluble fusion protein complex comprising a first domain comprising an IL-15 peptide or variant thereof, a second domain comprising a fusion polypeptide comprising an IL-15RαSushi peptide (IL15RαSu) and an αCTLA4 antibody heavy chain, and a third domain comprising an αCTLA4 antibody light chain. The IL-15 peptide or variant binds to the IL15RαSu peptide to form a soluble fusion protein complex. The soluble fusion protein complex and pharmaceutical compositions thereof modulate immune responses. Also disclosed herein are methods for preventing or treating cancer, in which the immune response is enhanced, and methods for preventing or treating autoimmune diseases or disorders, in which the immune response is reduced.

[0008] In some embodiments, the soluble fusion protein complex comprises (a) a first domain having an IL-15 peptide having sequence identity (e.g., at least about 85% sequence identity) to SEQ ID NO: 17; (b) a second domain having a fusion polypeptide comprising an IL15RαSu peptide and an αCTLA4 antibody heavy chain having sequence identity (e.g., at least about 85% sequence identity) to SEQ ID NO: 10; and (c) a third domain having an αCTLA4 antibody light chain having sequence identity (e.g., at least about 85% sequence identity) to SEQ ID NO: 3. In some embodiments, the IL15RαSu peptide has sequence identity (e.g., at least about 85% sequence identity) to SEQ ID NO: 8. The first domain may comprise an IL-15 variant comprising an N72D mutation (IL-15N72D). Additionally, the IL-15 peptide in the first domain may comprise or lack a leader sequence.

[0009] In some embodiments, the soluble fusion protein complex may comprise a complementarity 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 comprise a linker between the IL15RαSu peptide and the αCTLA4 antibody heavy chain. The fusion polypeptide may have sequence identity (e.g., at least about 85% sequence identity) to SEQ ID NO:4 or SEQ ID NO:5.

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

[0011] Also described is a method of preventing or treating cancer, comprising administering to a subject in need thereof a soluble fusion protein complex as described herein, 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 may be formulated into a pharmaceutical composition suitable for administration to a subject to prevent or treat cancer.

[0012] Also described are methods of preventing or treating an autoimmune disease or disorder, comprising administering to a subject in need thereof a soluble fusion protein complex as described herein, comprising a first domain having an IL-15 peptide or 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 may be formulated into a pharmaceutical composition suitable for administration to a subject to prevent or treat an autoimmune disease or disorder.

[0013] Various objects, features, aspects and advantages will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0014] [Figure 1] 2 and 3, as well as two exemplary molecules illustrated according to this key. [Diagram 2] Three "TxM" configurations are shown: Configuration A (TxM-A, also called N-844-1) has the antibody domain conjugated to an IL15RαSu peptide; Configuration B (TxM-B, also called N-844-2) has the antibody domain conjugated to an IL-15 peptide; Configuration C (TxM-C, also called N-844-3) has the antibody domain conjugated to the CH3 half of an Fc domain. [Diagram 3]Shown are 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] FIG. 4 is a graphical representation of differential scanning fluorimetry Tm analysis of various molecules shown in FIG. 2 and FIG. 3 (nogapendine alfa invaxcept (also known as NAI or 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)). [Diagram 5] FIG. 1 is a graphical representation of the average tumor growth in mice inoculated with B16.F10 melanoma tumor cells in both flanks. Treatment was administered directly to the tumor in one flank ("injected tumor"). Tumor growth was monitored in both the injected tumor and the non-injected tumor in the other flank ("distant tumor"). [Figure 6A] 6A shows a graphical representation of tumor growth in individual mice summarized 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 summarized in Figure 5. Figure 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 summarized in Figure 5. Figure 6C shows tumors grown in individual mice treated with αOX40 antibody + IL-15-α-CTLA4 (N-844-4). [Figure 6D] 6A shows a graphical representation of tumor growth in individual mice summarized 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 6E] Graphical representation of tumor growth in individual mice summarized in Figure 5. Figure 6E shows tumors grown in individual mice treated with αOX40 antibody + αCTLA4-7 + NAI (N-803). [Figure 7A-7B] 7A and 7B are graphical representations of the efficacy of treatment in injected and distant tumors on day 11 based on area under the curve (AUC) calculations. Figures 7A and 7B show the efficacy of treatment in injected and distant tumors, respectively. Figure 7C shows the tolerability of treatment through day 18. [Figure 7C] Figure 7C is a graphical representation of the efficacy of treatment in injected and distant tumors on day 11 based on area under the curve (AUC) calculations. Figure 7C shows the tolerability of treatment through day 18. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Definitions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terms are used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0016] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., up to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Thus, the recitation of "a cell," for example, includes a plurality of cells of the same type. Furthermore, to the extent the terms "comprising," "including," "having," "having," "comprising," or variations thereof are used anywhere in the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0017] As used in this specification and the appended claims, "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise.

[0018] As used herein, "about" when referring to a measurable value, such as an amount, duration, etc., is meant to encompass variations that are rounded off from the value beyond the last significant figure. For example, a designation of "about 2.5" would read as 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 "ameliorate" means to reduce, inhibit, attenuate, reduce, halt or stabilize the onset or progression of a disease.

[0020] "Antibody" or "immunoglobulin" includes both polyclonal and monoclonal antibodies. Preferred antibodies are monoclonal antibodies that react with an antigen. The term "antibody" also includes a mixture of two or more antibodies reactive with an antigen (e.g., a cocktail of different types of monoclonal antibodies reactive with the antigen). Furthermore, "antibody" includes whole antibodies, biologically functional fragments thereof, single chain antibodies, and genetically engineered antibodies, such as chimeric antibodies containing portions from two or more species, bifunctional antibodies, antibody conjugates, humanized antibodies, and fully human antibodies. Biologically functional antibody fragments that can also be used are peptide fragments derived from an antibody sufficient to bind to an antigen. As used herein, "antibody" is meant to include whole antibodies as well as any antibody fragments (e.g., F(ab')2, Fab', Fab, Fv) that can bind to an epitope, antigen, or antigenic fragment of interest. "Binding" to a molecule means having a physicochemical affinity for that molecule.

[0021] As used herein, the term "cancer" refers to a disease characterized by unregulated cell growth or replication, as known in the art. "Cancer" includes colorectal cancer and AIDS-related cancers, such as leukemias, for example acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic 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, oligodendroglioma, pituitary tumor, Schwannoma and metastatic brain tumor; head and neck cancers, including various lymphomas, such as mantle cell lymphoma, non-Hodgkin's lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinal cancers, such as retinoblastoma, esophageal cancer, gastric cancer, multiple myeloma, pulmonary arterial cancer ... 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 carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer endometrial sarcoma, multidrug resistant cancer; as well as proliferative diseases such as angiogenesis associated with tumor angiogenesis, macular degeneration (e.g., wet / dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration, and other proliferative diseases and conditions such as restenosis and polycystic kidney disease, as well as other cancers or proliferative diseases that may respond to modulation of their environment, alone or in combination with other therapies.

[0022] The term "disease" refers to any condition or disorder that impairs or prevents 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 cells and age-related diseases.

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

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

[0025] In the context of two or more nucleic acid or polypeptide sequences, "identical" or percent "identity" refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window. For purposes of this disclosure, the degree of amino acid or nucleic acid sequence identity is determined using the BLAST algorithm as described in Altschul & al. (199) J. Mol. Biol. 215:403-10, which is incorporated herein by reference. 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 (HSPSs) 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 meet some positive-valued threshold score T. T is referred to as the neighborhood word score threshold (Altschul & al., supra). Initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. The cumulative score is calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residuals; always >0) and N (penalty score for mismatched residuals; always <0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. The extension of the word hit in each direction is stopped when the cumulative alignment score falls below its maximum achieved value by an amount X, the cumulative score falls below 0 due to the accumulation of one or more negatively scoring residue alignments, or the end of either sequence is reached. The default parameters of the BLAST program can be used to determine the percent identity of amino acid or nucleic acid sequences. For the analysis of amino acid sequences, the defaults for BLASTP are as follows: word length (W), 3; expectation (E), 10; and the BLOSUM62 score matrix.For analysis of nucleic acid sequences, the BLASTN program defaults are a wordlength (W) of 11; expectation (E) of 10; M=5; N=-4; and a comparison of both strands. The TBLASTN program, which queries a nucleotide sequence database using a protein sequence, uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM 62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915, incorporated herein by reference).

[0026] In addition to calculating percent sequence identity, 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 indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01.

[0027] As used herein, the term "immune effector cell" refers to a cell involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include T cells, e.g., 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 phagocytes. "Immune effector function or immune effector response," as that term is used herein, refers to, e.g., a function or response of an immune effector cell that enhances or promotes immune attack of a target cell. For example, immune effector function or response refers to a property of a T or NK cell that promotes killing or inhibiting growth or proliferation of a target cell. For T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0028] As used herein, in relation to administering a treatment to a subject, the term "in combination" refers to the use of two or more treatments for therapeutic benefit.In relation to administration, the term "in combination" can also refer to the prophylactic use of a treatment to a subject when used with at least one additional treatment.The use of the term "in combination" does not limit the order in which treatments (e.g., a first and a second treatment) are administered to a subject. The treatment can 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), simultaneously or subsequently (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) 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 treatments are administered to a subject within a series of time intervals that provide a greater benefit than if they were administered otherwise. Any additional treatments can be administered in any order with the other additional treatments.

[0029] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques.

[0030] "Patient" or "individual" or "subject" are used interchangeably herein and refer to a mammalian subject to be treated, and interchangeably refer 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, cows, pigs, sheep). In certain embodiments, the subject may be a human (e.g., adult male, adult female, adolescent male, adolescent female, boy, girl) under the care of a physician or other medical practitioner. In certain embodiments, the subject may not be under the care of a physician or other medical practitioner.

[0031] "Pharmaceutically acceptable" ingredients / carriers, etc. are suitable for use in humans and / or animals without undue adverse side effects (toxicity, irritation, allergic response, etc.) commensurate with a reasonable benefit / risk ratio.

[0032] "Prevent", "preventing" and "prevention" refer to the administration of an agent or composition to a clinically asymptomatic individual who is susceptible or prone to a particular adverse condition, disorder or disease, and thus relates to the prevention of the onset of symptoms and / or their underlying causes.

[0033] By "reduce" is meant a negative alteration of a condition, disorder or disease compared to a "healthy" or "suitable" control subject not having the condition, disorder or disease. A negative alteration can 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] By "specifically binds" it is meant that a molecule, protein, peptide, antibody or antibody fragment recognizes and binds to a polypeptide of the invention, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, that may contain a polypeptide of the invention.

[0035] As used herein, "treat", "treating" and "treatment" refer to the administration of an agent or formulation to a clinically symptomatic individual suffering from an adverse condition, disorder or disease to result in a reduction in the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying causes, and / or promote amelioration or repair of damage. Treatment may include administration of a CTLA4 and IL-15 fusion molecule or an analog thereof. Although not excluded, it will be understood that treating a disorder or condition does not require that the associated disorder, condition or symptoms be completely eliminated.

[0036] Treatment of 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 be present after a known tumor is removed by a first line of treatment (e.g., surgery), thereby preventing possible cancer recurrence; neoadjuvant therapy, given before surgery 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 is achieved; maintenance therapy, given in lower or less frequent doses to help extend remission; first line treatment (also called standard treatment); second line (or third, fourth, etc.) therapy (also called salvage therapy) is given if the disease does not respond or recur after first line therapy; palliative therapy (also called supportive care) to address symptom management without the hope of significantly reducing the cancer.

[0037] "Tumor" refers to a mass of transformed cells characterized by neoplastic, uncontrolled cell proliferation and at least partially containing angiogenic vasculature. The abnormal neoplastic cell growth is rapid and continues after the stimulus that initiated the new growth has ceased. The term "tumor" is used broadly to include tumor parenchymal cells as well as the supporting stroma, including angiogenic blood vessels that infiltrate the tumor parenchymal cell mass. Tumors are generally malignant tumors, i.e., cancers that have the ability to metastasize (i.e., metastatic tumors), although tumors can be non-malignant (i.e., non-metastatic tumors). Tumors are the hallmark of cancer, a neoplastic disease whose natural history is fatal.

[0038] Certain methodologies of the invention include steps that involve comparing a value, level, feature, characteristic, property, etc., to a "suitable control," referred to interchangeably herein as a "suitable control." A "suitable control" or "suitable control" is a control or standard familiar to one of skill in the art that is useful for comparison purposes. In one embodiment, a "suitable control" or "suitable control" is a value, level, feature, characteristic, property, etc., that is determined prior to performing the treatment and / or drug administration methodology described herein. For example, transcription rates, mRNA levels, translation rates, protein levels, biological activity, cellular traits or characteristics, genotype, phenotype, etc., can be determined prior to introducing the treatment and / or drug of the invention into a subject. In another embodiment, a "suitable control" or "suitable control" is a value, level, feature, characteristic, property, etc., that is determined in a cell or organism, e.g., a control or normal cell or organism, that exhibits, for example, a normal or healthy (e.g., non-disease) trait. In yet another embodiment, a "suitable control" or "suitable 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 genes or gene products from a particular species are disclosed, it is understood that the disclosure is intended to be illustrative only and should not be construed as limiting unless the context in which it clearly appears indicates. Thus, for example, for genes or gene products disclosed herein, it is intended to encompass homologous and / or orthologous genes and gene products from other species.

[0040] GenBank and NCBI submissions identified by accession numbers cited herein are hereby incorporated by reference.

[0041] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0042] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0043] Interleukin-15. Human IL-15 (huIL-15) has a high binding affinity (equilibrium dissociation constant (K D ) about 10 -11huIL-15 is a member of the small four alpha-helical bundle family of cytokines that associates with the huIL-15 receptor alpha chain (huIL-15Rα) at ​​the IL-2 / IL-15 receptor β / common γ chain (huIL-15RβγC) complex that is presented on the surface of T cells and NK cells. This cytokine / receptor interaction leads to the expansion and activation of effector T and NK cells that play a key role in eradicating virally infected and malignant cells. Normally, huIL-15 and huIL-15Rα are co-produced in dendritic cells, form a complex intracellularly, and are then secreted and presented on the cell surface as a heterodimeric molecule. Thus, the nature of the huIL-15 and huIL-15Rα interaction suggests that these interchain binding domains may function as human-derived immunostimulatory scaffolds to generate soluble dimeric molecules capable of target-specific binding.

[0044] In certain embodiments of the soluble fusion protein complexes of the present invention, the IL-15 peptide may be an IL-15 variant having an amino acid sequence different from the native IL-15 peptide. huIL-15 and variants thereof are referred to using the native amino acid, its position in the mature sequence and the variant amino acid. For example, huIL15N72D refers to human IL-15 containing a substitution of N to D at position 72. In one embodiment, the IL-15 variant functions as an IL-15 agonist, e.g., as demonstrated 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, e.g., as demonstrated by decreased binding activity to the IL-15RβγC receptor compared to the native IL-15 sequence.

[0045] IL-15:IL-15Rα complex. The formation of IL-15:IL-15Rα complex, in which both IL-15 peptide and IL-15 receptor α chain are co-expressed in the same cells, can stimulate immune effector cells bearing IL-2βγC receptors via a transpresentation mechanism. Furthermore, when IL-15 peptide is bound to IL-15Rα, the affinity of IL-15 peptide for IL-2Rβ increases approximately 150-fold compared to free IL-15. A superagonist mutant of IL-15 peptide (IL-15N72D) has increased IL-2Rβ binding ability (4-5-fold higher than native IL-15). Taking advantage of the strong interaction of IL-15N72D with soluble IL-15Rα, we created an IL-15 superagonist complex with IL-15N72D bound to IL-15RαSu. As an example, the superagonist complex can be an IL-15 derivative linked to an IL-15Rα / IgG1 Fc fusion protein, also known as nogapendin alfa invaxcept (NAI). NAI can 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, which describes NAI, is incorporated herein by reference in its entirety. Clinical trials involving N-803 are described in NCT04385849, which is incorporated herein by reference in its entirety.

[0046] Fusion protein complexes. The present disclosure provides soluble fusion protein complexes. The soluble fusion complexes described herein comprise a first domain (e.g., 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 scope of "IL-15 peptide")), a second domain comprising a fusion polypeptide, and a third domain. The IL-15 peptide in the first domain preferably comprises an amino acid sequence different from the native (or wild-type) IL-15 protein. The IL-15 peptide preferably binds to an IL-15Rα peptide and functions as an IL-15 agonist or antagonist. Preferably, an IL-15 peptide with agonist activity in the first domain may have superagonist activity. The IL-15 peptide may function as an IL-15 agonist or antagonist independent of association with IL-15Rα. An IL-15 agonist is exemplified by a similar or increased biological activity compared to wild-type IL-15. An IL-15 antagonist is exemplified by a decreased biological activity compared to wild-type IL-15 or the ability to inhibit an IL-15-mediated response. In some examples, the IL-15 peptide in the first domain binds with increased or decreased activity to the IL-15RβγC receptor. Optionally, the sequence of the IL-15 peptide in the first domain has at least one amino acid change, e.g., a substitution or deletion, compared to a native (or wild-type) human IL-15 peptide, such that such change results in IL-15 agonist or antagonist activity. Preferably, the amino acid substitution / deletion is 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 binding to the IL-15Rα polypeptide or the ability to produce the IL-15 peptide. Suitable amino acid substitutions / deletions for generating an IL-15 peptide in the first domain can be identified by rational or random mutagenesis and functional assays as provided herein or other empirical methods based on comparison with putative or known IL-15 structures, with molecules homologous to the IL-15 peptide (e.g., IL-2), with known structures.Furthermore, suitable 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 comprise 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 (where "D8" refers to the amino acid and residue position in the native mature human IL-15 sequence and "N" refers to the substituted amino acid residue at that position in the IL-15 variant), I6S, D8A, D61A, N65A, N72R, V104P or Q108A substitutions result in IL-15 peptides with antagonist activity, and N72D substitutions result in IL-15 peptides with agonistic activity. Alternative IL-15 peptides include those disclosed in US Pat. Nos. 8,163,879 and 9,255,141, both of which are incorporated by reference in their entireties.

[0047] In some embodiments, the first domain may comprise a first biologically active polypeptide covalently linked to an interleukin-15 (IL-15) peptide or a functional fragment thereof, and the second domain may comprise a second biologically active polypeptide covalently linked to a soluble interleukin-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof (e.g., IL15RαSu peptide), and the IL-15 peptide of the first domain binds to the soluble IL-15Rα peptide or a functional fragment thereof 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, the 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 the immunoglobulin Fc peptides. The soluble fusion protein complex may comprise a first domain comprising 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 comprising 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 comprise an antibody or a functional fragment thereof. For example, one of the domains may comprise 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 WO 2021 / 250594, the entire contents of which are incorporated herein by reference. In another example, the first or second domain may comprise an αCTLA4 scFv or a disease antigen-specific antibody or a functional derivative thereof. Examples of scFvs are disclosed in U.S. Pat. No. 11,105,188, the entire contents of which are incorporated herein by reference. Soluble fusion protein complexes comprising covalently linked IL-15 peptide and IL-15Rα domains have several important applications. For example, a soluble fusion protein complex comprising αCTLA4 scFv on the first or second domain may be used to deliver the IL-15:IL-15Rα complex to a particular cell, such as a NK cell. Alternatively, the NK cell may comprise a high affinity NK cell engineered to express the fusion protein, as described below. Thus, the protein fusion or conjugate complex provides a means to selectively damage or kill cells that contain the ligand. Examples of cells or tissues that may be damaged or killed by the protein fusion or conjugate complex include tumors as well as virally or bacterially infected cells that express one or more ligands. Cells or tissues 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 comprise a third domain that may comprise another antibody or a functional fragment thereof. For example, the third domain may comprise, for example, an antibody light chain (e.g., an antibody fragment that does not include an Fc region). In certain instances, the third domain comprises an αCTLA4 antibody light chain or a peptide having sequence identity (e.g., at least about 85% sequence identity) to an αCTLA4 antibody light chain.

[0050] The IL-15 and IL-15Rα peptides of the present invention suitably correspond in amino acid sequence to naturally occurring IL-15 and IL-15Rα molecules, e.g., human, mouse or other rodents or other mammalian IL-15 and IL-15Rα molecules. The sequences of these polypeptides and encoding nucleic acids are known in the literature, including human interleukin-15 (IL-15) mRNA-GenBank: U14407.1 (herein incorporated by reference in its entirety), mouse interleukin-15 (IL-15) mRNA-GenBank: U14332.1 (herein incorporated by reference in its entirety), human interleukin-15 receptor alpha chain precursor (IL-15Rα) mRNA-GenBank: U31628.1 (herein incorporated by reference in its entirety), mouse interleukin-15 receptor, alpha chain-GenBank: BC095982.1 (herein incorporated by reference in its entirety).

[0051] In some situations, it may be useful to make the protein fusion or conjugate complex of the invention multivalent, for example to increase 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 to generate a multivalent complex. Furthermore, multivalent fusion proteins can be made by covalently or non-covalently linking one protein to four proteins (same or different), for example, by using standard biotin-streptavidin labeling techniques or by conjugation to a suitable solid support such as latex beads. Chemically cross-linked proteins (e.g., cross-linked to dendrimers) are also suitable multivalent species. For example, proteins can be modified by including a modifiable tag sequence, such as a biotinylated BirA tag, or a sequence encoding an amino acid residue with a chemically reactive side chain, such as Cys or His. Such amino acid tags or chemically reactive amino acids can be placed at various positions in the fusion protein, preferably distal to the active site of the biologically active polypeptide or effector molecule. For example, the C-terminus of the soluble fusion protein complex can be covalently linked to a tag or other fusion protein that contains such reactive amino acids. Suitable side chains can be included to chemically link two or more fusion proteins to suitable dendrimers or other nanoparticles to obtain multivalent molecules. Dendrimers are synthetic chemical polymers that can have any one of several different functional groups on their surface (D. Tomalia, Aldrichimica Acta, 26:91:101 (1993), incorporated herein by reference in its entirety). Exemplary dendrimers for use in accordance with 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 comprise an immunoglobulin domain. Alternatively, the protein binding domain-IL-15 fusion protein may be further linked to an immunoglobulin domain. The immunoglobulin domain comprises a region that allows interaction with other immunoglobulin domains to form a multi-chain protein as provided above. For example, IgG1 C H 2-C H Immunoglobulin heavy chain regions such as Fc heavy chain region 3 can stably interact to create an Fc region. Preferred immunoglobulin domains that contain an Fc domain also contain a region with effector function, including Fc receptor or complement protein binding activity, and / or a region with a glycosylation site. In some embodiments, the immunoglobulin domain of the fusion protein complex contains a mutation that reduces or enhances Fc receptor or complement binding activity, or glycosylation or dimerization, thereby affecting the biological activity of the resulting protein. For example, an immunoglobulin domain that contains a mutation that reduces binding to an Fc receptor can be used to generate a fusion protein complex of the invention that has lower binding activity to cells bearing Fc receptors, which may be advantageous for reagents designed to recognize or detect specific antigens.

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

[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 to SEQ ID NO: 17, which does not include a leader sequence. The IL-15 peptide may comprise SEQ ID NO: 17. An exemplary nucleotide sequence encoding SEQ ID NO: 17 is provided herein as SEQ ID NO: 18. Alternatively, the IL-15 peptide may further comprise a leader sequence and 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 to SEQ ID NO: 1, which includes a leader sequence. The IL-15 peptide may comprise SEQ ID NO: 1. An exemplary nucleotide sequence encoding SEQ ID NO: 1 is provided herein as SEQ ID NO: 2.

[0055] The IL15RαSu 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 to SEQ ID NO: 8. The IL15RαSu peptide may comprise SEQ ID NO: 8. The αCTLA4 antibody heavy 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 to SEQ ID NO: 10. The αCTLA4 antibody heavy chain may comprise 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 to SEQ ID NO: 3. The αCTLA4 antibody light chain may comprise SEQ ID NO:3.

[0056] In particular, the soluble fusion protein complex may comprise a first domain having an IL-15 peptide having at least about 85% sequence identity to SEQ ID NO: 17, a second domain having an IL15RαSu peptide and an αCTLA4 antibody heavy chain having at least about 85% sequence identity to SEQ ID NO: 10, and a third domain having an αCTLA4 antibody light chain having at least 85% sequence identity to SEQ ID NO: 3. The IL15RαSu peptide and the αCTLA4 antibody heavy chain may be part of a 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 to SEQ ID NO: 8. Additionally or alternatively, the heavy and light chains of the αCTLA4 antibody may comprise complementarity determining regions (CDRs). The CDR region of the αCTLA4 antibody heavy 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 one or more of SEQ ID NOs: 11-13. Alternatively, the CDR region of the αCTLA4 antibody heavy chain may comprise each of SEQ ID NOs: 11-13. The CDR region of 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 one or more of SEQ ID NOs: 14-16. Alternatively, the CDR region of the αCTLA4 antibody light chain may comprise each of SEQ ID NOs: 14-16. In particular, the soluble fusion protein complex may comprise an αCTLA4 antibody heavy chain having at least about 90% sequence identity to SEQ ID NO: 10, and may further comprise an αCTLA4 antibody light chain having about 90% sequence identity to SEQ ID NO: 11-13 and SEQ ID NO: 3, and SEQ ID NO: 14-16. Alternatively, the soluble fusion protein complex may comprise an αCTLA4 antibody heavy chain having at least about 95% sequence identity to SEQ ID NO: 10, and may further comprise an αCTLA4 antibody light chain having about 95% sequence identity to SEQ ID NO: 11-13 and SEQ ID NO: 3, and SEQ ID NO: 14-16.Alternatively, the αCTLA4 antibody heavy chain of the soluble fusion protein complex comprises SEQ ID NO:10 and the αCTLA4 antibody light chain comprises SEQ ID NO:3.

[0057] The IL-15 peptide of the soluble fusion protein complex may or may not include a leader sequence. For example, a soluble fusion protein complex may include an IL-15 peptide lacking a leader sequence, where the IL-15 peptide comprises SEQ ID NO: 17. Alternatively, a soluble fusion protein complex may include an IL-15 peptide including a leader domain, where the IL-15 peptide comprises SEQ ID NO: 1.

[0058] The αCTLA4 antibody (e.g., the αCTLA4 antibody heavy chain) may be directly conjugated to the 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 the αCTLA4 antibody (e.g., the αCTLA4 antibody heavy chain), and in other embodiments, the IL-15 peptide or IL15RαSu peptide may be conjugated to the Fc terminus of the αCTLA4 antibody heavy chain. The αCTLA4 antibody may be a complete antibody or only a Fab fragment, while in other embodiments, the αCTLA4 also includes an Fc domain. In certain embodiments, the αCTLA4 molecule is an IgA, IgE, IgG, or IgM antibody.

[0059] Additionally or alternatively, the soluble fusion protein complex may further comprise a peptide linker between one or more domains of the complex. For example, when the fusion polypeptide of the second domain of the soluble fusion protein complex comprises an IL15RαSu peptide and an 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 carboxy 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 comprise SEQ ID NO:5.

[0060] Alternatively, a peptide linker may be between the carboxy 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 to SEQ ID NO:4. Alternatively, the fusion polypeptide may comprise SEQ ID NO:4.

[0061] Amino acid mutations: There are several ways in which researchers have mutated the amino acid sequence of IgG Fc regions to modulate FcγR-based effector functions. Some examples include point mutations, design algorithms, yeast display, and asymmetric engineering. Results from each of these different techniques have resulted in a large number of mutations that modify Fc-FcγR interactions and the resulting effector functions. 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 that has 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, incorporated herein by reference in its entirety). Combining some of the identified mutations (e.g., S298A / E333A / K334A) enhanced ADCC compared to IgG1. A series of Fc mutants with optimized Fcγ receptor affinity using computational design algorithms and high-throughput screening identified S239D / I332E and S239D / I332E / A330L as two mutants with enhanced ADCC activity. The crystal structure of an Fc fragment with the mutations S239D / A330L / I332E was solved, and modeling studies suggested that additional hydrogen bonds, hydrophobic contacts, and / or electrostatic interactions result in enhanced binding to FcγRIIIa. Addition of G236A to the S239D / I332E mutations 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 greater than 100-fold increase in ADCC activity (Stavenhagen & al. (2007) Cancer Res. 67:8882-90, incorporated herein by reference in its entirety). Antibody variants with asymmetrically engineered Fc domains, created by introducing different amino acid changes in each Fc domain, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A changes in one Fc domain and D270E / K326D / A330M / K334E in the other Fc domain, demonstrated a greater than 2000-fold increase in affinity for FcγRIIIa F158 and a greater than 1000-fold increase in affinity for FcγRIIIa V158 (Mimoto & al. (2013) MAbs. 5:229-36, incorporated herein by reference in its entirety).

[0062] Glycoengineering: IgG contains a conserved glycosylation site at amino acid N297 in the CH2 domain. The glycan core structure is composed of N-acetylglucosamine (GlcNAc) and mannose, and further modifications can include bisecting GlcNAc, fucose, galactose, and sialic acid. One of the first reports linking glycoengineering with enhanced Fc effector function demonstrated that an IgG1 antibody produced in a Chinese hamster ovary (CHO) cell line expressing β(1,4)-N-acetylglucosaminyltransferase III expressed bisecting GlcNAc, which resulted in enhanced ADCC activity compared to IgG1 (Umana & al. (1999) Nat Biotechnol. 17:176-80, incorporated herein by reference in its entirety). Furthermore, IgG1 lacking fucose had up to 50-fold increased FcγRIIIa binding and enhanced ADCC compared to IgG1 (Shields & al. (2002) J Biol Chem. 277:26733-40, incorporated herein by reference in its entirety). It was subsequently demonstrated that fucose-deficient antibodies have improved ADCC function compared to antibodies containing a bisecting GlcNAc (Shinkawa & al. (2003) J Biol Chem. 278:3466-73, incorporated herein by reference in its entirety). Amino acid N162 in FcγRIIIa contains a glycan, and the absence of fucose allows for greater carbohydrate-carbohydrate interactions with Fc, which increases overall binding strength.

[0063] Antibody-Dependent Cellular Cytotoxicity (ADCC): ADCC is a process in which antibodies coat target cells and recruit effector cells to induce target cell death via a non-phagocytic mechanism. Antibodies bind to their specific antigens on the target cell surface via their antigen-binding fragment (Fab) portion and can interact with effector cells via their fragment crystallizable region (Fc) portion, thereby acting as a bridge linking the effector to the target. Several classes of human antibodies can mediate ADCC, including IgG, IgA, and IgE, but IgG1 is the most used subclass for cancer therapeutic antibodies (Zahavi & al. (2018) Antibody Therapeutics 1(1):7-12, incorporated herein by reference in its entirety). In order for effector cells to carry out ADCC, they must express an Fc receptor (FcR) that binds the antibody. Known classes of FcR include FcγR, which binds IgG, FcαR, which binds IgA, and FcεR, which binds IgE. Fcγ R is most important for tumor cell clearance by myeloid cells and is composed of activating Fcγ RI (CD64), Fcγ RIIA (CD32A), Fcγ RIIIA (CD16A) and inhibitory Fcγ RIIB (CD32B) receptors. When Fcγ R binds to antibodies, it triggers receptor cross-linking and downstream signal propagation. They activate Fcγ R signals through their immunoreceptor tyrosine-based activation motifs, while they activate inhibitory Fcγ R signals through their immunoreceptor tyrosine-based inhibitory motifs. Many effector cells also express other receptor types, such as inhibitory killer inhibitory receptors (e.g., KIR) and activating NKG2D receptors on natural killer (NK) cells. The delicate balance between activating and inhibitory pathway signaling ultimately determines the effector cell response. Myeloid cells that can act as ADCC effectors are NK cells, monocytes, macrophages, neutrophils, eosinophils and dendritic cells. Once activated, these effector cells mediate target cell death through three key mechanisms: cytotoxic granule release, Fas signaling, and generation of reactive oxygen species.The main and best characterized mechanism utilized in ADCC is the release of perforin and granzymes from effector cell granules. Upon activation, effector cells such as NK cells polarize and exocytose their granules in a calcium-dependent manner (de Saint Basile, G. & al. (2010) Nat Rev Immunol. 10:568-79, incorporated herein by reference in its entirety). Perforin and granzyme B cooperate 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. Although multiple myeloid lineage cells are capable of ADCC, 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. Because NK cells highly express activating Fcγ RIIIA and do not express inhibitory Fcγ RIIB, modifying antibody interactions specifically through Fcγ RIIIA is of growing interest for cancer immunotherapy.

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

[0065] haNK cells are derived from the NK-92 cell line and engineered to express a high affinity CD16 allele. Sequences of high affinity variants of Fcγ receptors are well known in the art (see, e.g., Bruhns & al. (2009) Blood 113:3716-25, incorporated herein by reference in its entirety). Expression of such receptors advantageously increases the specific targeting and cytotoxic cell killing of tumor cells when using antibodies specific for the patient's tumor cells. CD16 is most commonly found in a form that has a relatively low binding affinity for the Fc portion of IgG molecules. Alternative forms that exhibit higher binding affinity are found in some individuals. The low 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 and high affinity forms can be found in the SwissProt database as entries P08637 and VAR_008801, respectively.

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

[0067] Checkpoint inhibitors: Checkpoint inhibitors inhibit the function or activity of molecules that regulate or control, e.g., inhibit, immune responses of immune effector cells, e.g., T cell function. Inhibitory molecules, e.g., CTLA4, programmed death 1 (PD-1), also referred to herein as checkpoint inhibitors, can, in some embodiments, reduce the ability of immune effector cells to mount immune effector responses. 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. Inhibition of molecules that modulate or regulate, e.g., inhibit, T cell function, e.g., by inhibition at the DNA, RNA, or protein level, can optimize immune responses. In addition to the αCTLA4 antibodies disclosed herein, ipilimumab (also called MDX-010 and MDX-101, commercially available as YERVOY™; Bristol-Myers Squibb) and tremelimumab (an IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, 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 various aspects of T-cell immunoregulation. This family includes other proteins including CD28, CTLA4, and ICOS, as well as PD-1, BTLA, and TIGIT. These receptors have both stimulatory (CD28, ICOS) and inhibitory (CTLA4, PD-1, BTLA, and TIGIT) roles in T-cell function. These pathways are increasingly being targeted as part of immune-modulatory strategies to treat cancer, commonly referred to as immune checkpoint blockade, and conversely to treat autoimmunity and CTLA4 deficiency.

[0069] CTLA4 (CD152) and CD28 are expressed in CD4 + and CD8 + CD28 is a homologous receptor expressed by both CD80 and CD86 on T cells, and mediates 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 CD80 dimers with relatively high affinity and CD86 monomers with lower affinity, mediating T cell costimulation in conjunction with T cell receptor (TCR) signals. In contrast, the interaction of the ligand with CTLA4 serves to inhibit T cell responses, although the exact mechanism is not fully understood. CTLA4 interacts with both ligands with higher affinity and avidity than CD28, with CTLA4-CD80 forming the highest avidity interaction and CD28-CD86 forming the weakest interaction (Rowshanravan & al. (2018) Blood 131:58-67, incorporated herein by reference in its entirety). Among other 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 of which are incorporated herein by reference in their entirety). These interactions are thought to occur at the immune 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, which is incorporated herein by reference in its entirety).

[0070] Thus, in a method of treating cancer, T-haNK cells can be engineered to express an anti-CTLA4 antigen binding domain that binds with high affinity to the CTLA4 molecule (Simpson & al. (2013) J. Exp. Med. 210(9):1695-710, incorporated herein by reference in its entirety), and T regThis results in the depletion or functional blocking of cells, leading to enhanced T cell activation and immune response against cancer.

[0071] CTLA4 is a T reg Indeed, early experiments showed that the major autoimmune phenotype of CTLA4-deficient mice could be prevented by the presence of CTLA4-expressing cells in mixed chimeras (Bachmann & al. (1999) J Immunol. 163(3):1128-31, incorporated herein by reference in its entirety). This observation has been corroborated by many other experiments (Walker & Sansom (2011)), and recently a series of conditional and inducible CTLA4 deletion experiments have demonstrated that the major phenotype is prevented by T reg This is consistent with the effector function of CTLA4 described above (Klocke & al. (2016) Proc Natl Acad Sci USA. 113(17):E2383-92, incorporated herein by reference in its entirety).

[0072] In embodiments in which autoimmune disease is treated, T-haNK cells express CTLA4, which results in inhibition of the immune response.

[0073] Immune cell activities that may be measured include, but are not limited to, (1) cell proliferation by measuring DNA replication; (2) enhanced cytokine production, including specific measurement of cytokines such as IFN-γ, GM-CSF, or TNF-α; (3) cell-mediated target killing or lysis; (4) cell differentiation; (5) immunoglobulin production; (6) phenotypic change; (7) production of chemotactic factors or chemotaxis, meaning capable of responding to chemotactins that have chemotactic properties; (8) immunosuppression by inhibition of the activity of several other immune cell types; and (9) apoptosis, which refers to the fragmentation of activated immune cells under certain circumstances as an indicator of aberrant activation.

[0074] Recombinant Expression Vectors and Host Cells: In general, preparation of the fusion protein complexes of the invention (e.g., components of the TxM complex) can be accomplished by procedures disclosed herein and recognized recombinant DNA techniques.

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

[0076] A variety of expression systems exist for the production of recombinant polypeptides. Expression vectors useful for producing such polypeptides include, but are not limited to, chromosomal, episomal and virus-derived vectors, such as those derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculovirus, papovaviruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retroviruses, and combinations thereof.

[0077] Once the recombinant polypeptide is expressed, it is isolated, for example, using affinity chromatography. In one example, an antibody raised against the polypeptide (e.g., produced as described herein) can be bound to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of cells bearing the polypeptide prior to affinity chromatography can be carried out by standard methods (see, e.g., Ausubel & al., supra). Once isolated, the recombinant protein can be further purified, if desired, by, for example, high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980, which is incorporated herein by reference in its entirety).

[0078] A vector is a tool used to shuttle DNA between host cells or as a means to express polynucleotide sequences. Insertion of a DNA of interest, such as an anti-CTLA4 binding domain or an scFv encoding a CD16 sequence or fragment, is accomplished by ligation techniques and / or mating protocols well known to those skilled in the art. Such DNA is inserted such that its incorporation does not destroy 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 linked to vector elements that control its transcription and translation.

[0079] Vectors can be divided into two general classes: cloning vectors are replicating plasmids or phages with regions that are not essential for propagation in a suitable host cell into which foreign DNA can be inserted and which replicates and propagates 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 linked to an element such as a promoter that signals the host cell to transcribe the inserted DNA. Some promoters are very useful, such as inducible promoters that control gene transcription in response to a specific factor. Expression of the gene or a fragment thereof can be controlled by operably linking the scFv encoding αCTLA4 or the polynucleotide encoding the CTLA4 polypeptide to an inducible promoter. Examples of inducible promoters include those that are tissue specific and drive expression to a particular cell type, steroid responsive (e.g., glucocorticoid) or heat shock responsive. Other desirable inducible promoters include those that are not endogenous to the cells into which the construct has been introduced, but are responsive in those cells when an inducing agent is exogenously provided.

[0080] Vectors have many indications. 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 replicate autonomously in a host cell (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell and replicate as part of the host genome. In general, useful expression vectors are plasmids and viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), although other expression vectors can also be used.

[0081] The choice of vector is determined by the organism or cell used and the desired fate of the vector. The vector can be replicated once in the target cell or can be a "suicide" vector. In general, the vector comprises a signal sequence, an origin of replication, a marker gene, an enhancer element, a promoter and a transcription termination sequence. The vector often uses a selection marker to facilitate the identification of the cells that have incorporated the vector.

[0082] A "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted to effect replication of the inserted segment. Generally, a vector is capable of replicating when associated with the appropriate control elements. Suitable vector backbones include those routinely used in the art, such as, for example, plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs. The term "vector" includes cloning and expression vectors as well as viral and integrating vectors. An "expression vector" is a vector that includes a regulatory region. A variety of host / expression vector combinations may be used to express the nucleic acid sequences described herein. Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen / Life Technologies (Carlsbad, CA).

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

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

[0085] Yeast expression systems can also be used. For example, according to the invention, 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, ProBond resin purified, enterokinase cleaved N-terminal peptide; Invitrogen), to name just two, can be used. Yeast two-hybrid expression systems can also be prepared according to the invention.

[0086] Several delivery methods may be utilized for in vitro (cell culture) and in vivo (animal and patient) systems. In one embodiment, a lentiviral gene delivery system may be utilized. Such a system provides stable long-term presence of genes in dividing and non-dividing cells with broad tropism and capacity for large DNA inserts. (Dull & al. (1998) J Virol. 72:8463-71, incorporated herein by reference in its entirety). In one embodiment, adeno-associated virus (AAV) may be utilized as a delivery method. AAV is a non-pathogenic single-stranded DNA virus that has been actively used in recent years to deliver therapeutic genes in in vitro and in vivo systems (Choi & al. Curr Gene Ther (2005) 5:299-310, incorporated herein by reference in its entirety). An exemplary non-viral delivery method may utilize nanoparticle technology. This platform has demonstrated pharmaceutical utility in vivo. Nanotechnology has improved drug transcytosis across tight 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, incorporated herein by reference in its entirety).

[0087] A vector may also include a regulatory region. The term "regulatory region" may refer to a nucleotide sequence that influences 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, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0088] The term "operably linked" refers to the positioning of regulatory regions and sequences to be transcribed within a nucleic acid so as to affect 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 translation reading frame of the polypeptide is typically located 1 to about 50 nucleotides downstream of the promoter. However, the promoter can be located about 5,000 nucleotides upstream of the translation start site or about 2,000 nucleotides upstream of the transcription start site. A promoter typically includes at least a core (basal) promoter. A promoter may also include at least one control element, such as an enhancer sequence, an upstream element, or an upstream activation region (UAR). The choice of promoter to include depends on several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell or tissue preferential expression. It is routine for one of skill in the art to regulate expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.

[0089] Vectors include, for example, viral vectors (such as adenoviruses Ad, AAV, lentiviruses and vesicular stomatitis viruses (VSV) and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes that can mediate delivery of polynucleotides to host cells. Vectors can also include other components or functionalities that further regulate gene delivery and / or gene expression or otherwise provide beneficial properties to target cells. As described and exemplified in more detail below, such other components include, for example, components that affect binding or targeting to cells (including components that mediate cell type or tissue specific binding); components that affect uptake of vector nucleic acid by cells; components that affect localization of polynucleotides within cells after uptake (such as agents that mediate nuclear localization); and components that affect expression of polynucleotides. Such components can also include markers, such as detectable and / or selectable markers, that can be used to detect or select cells that have taken up and are expressing nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (such as the use of certain viral vectors that have components or functionalities that mediate binding and uptake) or the vector may be modified to provide such functionalities. Other vectors include those described by Chen & al. (2003) BioTechniques 34:167-71, which is incorporated herein by reference in its entirety. A variety of such vectors are known in the art and are generally available. "Recombinant viral vector" refers to a viral vector that contains one or more heterologous gene products or sequences. Because many viral vectors exhibit size constraints related to packaging, the heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses can be replication-deficient, requiring that the missing functions be provided in trans during viral replication and encapsidation (e.g., by using a helper virus or packaging cell line that carries gene products necessary for replication and / or encapsidation).Modified viral vectors in which the polynucleotide to be delivered is carried on the outside of the viral particle have also been described (see, e.g., Curiel & al. (1991) PNAS 88:8850-54, incorporated herein by reference in its entirety).

[0090] Further vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney Murine Leukemia Virus and HIV-based viruses. An 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 virus vectors include pox vectors such as orthopox vectors and avipox vectors, and herpes virus 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], and 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] (all of which are incorporated by reference in their entireties).

[0091] Since many viral vectors exhibit size constraints related to packaging, heterologous gene products or sequences are typically introduced by replacing one or more parts of the viral genome. Such viruses can be replication-deficient and require the missing functions to be provided in trans during viral replication and encapsidation (e.g., by using a helper virus or packaging cell line carrying gene products necessary for replication and / or encapsidation). Modified viral vectors in which the polynucleotide to be delivered is carried on the outside of the viral particle have also been described (see, e.g., Curiel, DT, & al. PNAS 88:8850-8854, 1991, the entirety of which is incorporated herein by reference).

[0092] Suitable nucleic acid delivery systems include recombinant viral vectors, typically sequences from at least one of adenovirus-associated virus (AAV), helper-dependent adenovirus, retrovirus, or the hemagglutinin virus of Japan (HVJ) complex. In such cases, the viral vector comprises a strong eukaryotic promoter, such as a cytomegalovirus (CMV) promoter, operably linked to a polynucleotide. The recombinant viral vector can comprise one or more polynucleotides therein, preferably about one polynucleotide. In some embodiments, the viral vector used in the methods of the present invention can comprise about 10 8 ~Approx. 5×10 10 In embodiments in which the polynucleotide is administered with a non-viral vector, it will often be useful to use from about 0.1 nanograms to about 4000 micrograms, for example from about 1 nanogram to about 100 micrograms.

[0093] Poxvirus vectors introduce genes into the cytoplasm of cells. Avian poxvirus vectors provide only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be indicative of some embodiments of the present invention. Adenovirus vectors, in some embodiments, provide shorter-term expression (e.g., less than about one month) than adeno-associated virus, and can exhibit much longer-term expression. Suitable adenovirus vectors are disclosed in WO 09 / 06479 (Etubics Corp.), the entire contents of which are incorporated herein by reference in their entirety. The particular vector selected will depend on the target cell and the condition being treated. Selection of an appropriate promoter can be easily accomplished. 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 the Rous sarcoma virus (RSV) (Davis, & al., Hum Gene Ther 4:151 (1993), incorporated herein by reference in its entirety), the SV40 early promoter region, the herpes thymidine kinase promoter, the regulatory sequences 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 control regions that exhibit tissue specificity and have been utilized in transgenic animals: elastase, which is active in pancreatic acinar cells. I gene control region, insulin gene control region active in pancreatic beta cells, immunoglobulin gene control region active in lymphoid cells, mouse mammary tumor virus control region active in testis, breast, lymphocytes and mast cells, albumin gene control region active in liver, alpha-fetoprotein gene control region active in liver, alpha 1-antitrypsin gene control region active in liver, beta globin gene control region active in bone marrow cells, myelin basic protein gene control region active in brain oligodendrocytes, myosin light chain 2 gene control region active in skeletal muscle, gonadotropin releasing hormone gene control region active in hypothalamus. Certain proteins can be expressed using their native promoter. Other elements that can enhance expression, such as enhancers or systems that result in high levels of expression, such as tat genes and tar elements, can also be included. This cassette can then be inserted into a vector, such as a plasmid vector, such as pUC19, pUC118, pBR322, or other known plasmid vectors that contain, for example, an E. coli origin of replication. See Sambrook, & al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989), which is incorporated herein by reference in its entirety.The plasmid vector may also contain a selection marker, such as the β-lactamase gene for ampicillin resistance, so long as the marker polypeptide does not adversely affect the metabolism of the organism being treated.

[0094] Another delivery method is to use single-stranded DNA-producing vectors that can produce expression products intracellularly. Polynucleotides can be used with microdelivery 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 of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions and drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Pat. No. 5,049,386; U.S. Pat. No. 4,946,787; U.S. Pat. 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. Pat. No. 7,166,298 or U.S. Pat. No. 6,890,554, the contents of each of which are incorporated by reference in their entirety. Delivery can be to a cell (eg, in vitro or ex vivo administration) or to a target tissue (eg, in vivo administration).

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

[0097] In some embodiments, the delivery of vectors can 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 endocytic pathway. They can be taken up by target cells by endocytosis, fusion or both. Exosomes can be utilized 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, incorporated herein by reference in its entirety). 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, incorporated herein by reference in their entirety.

[0099] The nucleic acids and vectors may be administered alone or in admixture in the presence of a pharma- ceutically acceptable excipient or carrier (e.g., saline). The excipient or carrier is selected based on the mode and route of administration. Suitable pharmaceutical carriers and medical requirements for use in pharmaceutical formulations are described in Remington's Pharmaceutical Sciences (EW Martin) and USP / NF (United States Pharmacopeia and the National Formulary), which are well-known references in the field.

[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 liposomal bilayer. Encapsulated nucleic acids are protected from nuclease degradation, whereas those simply bound to the surface of the liposome are not. Encapsulated nucleic acids share the extended circulation life and biodistribution of intact liposomes, whereas those associated with the surface adopt the pharmacology of naked nucleic acids upon dissociation from the liposome. Nucleic acids can be entrapped within liposomes using conventional passive loading techniques such as ethanol drop (for SALP), reverse phase evaporation, and ethanol dilution (for SNALP).

[0101] Liposomes and polymersomes can contain multiple solutions and compounds. In certain embodiments, the complexes of the present invention are coupled to or encapsulated in polymersomes. As a class of artificial vesicles, polymersomes are small hollow spheres that enclose a solution, made using amphiphilic synthetic block copolymers to form the vesicle membrane. A typical polymersome contains an aqueous solution in its core and is useful for encapsulating and protecting sensitive molecules such as drugs, enzymes, other proteins and peptides, and 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. Polymersomes can be produced from double emulsions 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, non-viral vectors are modified to achieve targeted delivery and transfection. PEGylation (i.e., surface modification with polyethylene glycol) is the primary method used to reduce opsonization and aggregation of non-viral vectors and minimize clearance by the reticuloendothelial system, resulting in extended circulation life after intravenous (iv) administration. Therefore, PEGylated nanoparticles are often referred to as "stealth" nanoparticles. Nanoparticles that are not rapidly removed from circulation have the opportunity to encounter infected cells.

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

[0104] Transduced cells are prepared for reinfusion according to established methods. After approximately 2-4 weeks in culture, cell numbers reach 1 x 10 6 ~1×10 10In this regard, cell growth characteristics vary from patient to patient and cell type to cell type. Approximately 72 hours prior to re-infusion of the transduced cells, an aliquot is taken for analysis of the phenotype and percentage of cells expressing the therapeutic agent. For administration, the cells of the invention are grown in a LD of the cell type as applied to the patient's mass and overall health. 50 and various concentrations can be administered at rates determined by the side effects of the cell type. Administration can be accomplished by single or divided doses.

[0105] Pharmaceutical treatment. In a further embodiment, provided herein is a pharmaceutical composition comprising the soluble fusion protein complex for use as a therapeutic agent. The soluble fusion protein complex may be formulated with a pharma- ceutical acceptable carrier to prepare a pharmaceutical composition. The pharmaceutical composition may be administered systemically and may be formulated in a pharma- ceutical acceptable buffer, such as physiological saline. Preferred routes of administration include, for example, intravesical instillation, 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 a pharma- ceutical 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 a therapeutically effective amount of the therapeutic agents identified herein in a physiologically acceptable carrier. Suitable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences by EW Martin. The amount of therapeutic agent administered will vary depending on the mode of administration, the age and weight of the patient, and the clinical symptoms of the neoplasm. In general, the amount will be within the range of amounts used for other agents used to treat other diseases associated with neoplasms, autoimmune or infectious diseases, although in certain instances, lesser amounts may be required due to the increased specificity of the soluble fusion protein complex. The soluble fusion protein complex is administered at a dosage that enhances the immune response of the subject or reduces the proliferation, survival or invasiveness of neoplastic, infected or autoimmune cells, as determined by methods known to those skilled in the art.

[0107] Formulation of Pharmaceutical Compositions. Administration of the fusion protein complexes of the invention for the prevention or treatment of neoplasia or autoimmune disease is by any suitable means that results in a concentration of therapeutic agent effective in combination with other ingredients to ameliorate, reduce or stabilize said neoplasia, infectious or autoimmune disease. The fusion protein complexes of the invention may be contained in any suitable amount in any suitable carrier substance, and are generally present in an amount of 1-95% by weight of the total weight of the composition. The compositions may be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intravesicular, intratumoral or intraperitoneal) administration routes. For example, pharmaceutical compositions may be prepared in accordance with conventional pharmaceutical practice (e.g., Remington: The Science and Practice of Pharmacy (2006), incorporated herein by reference in its entirety). th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0108] Those skilled in the art will recognize that modifying human dosages compared to animal models is routine in the art, and 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 can vary between about 1 μg soluble fusion protein complex / kg body weight to about 5000 mg complex / kg body weight, or about 5 mg / kg body weight to about 4,000 mg / kg body weight, or about 10 mg / kg body weight to about 3,000 mg / kg body weight, or about 50 mg / kg body weight to about 2000 mg / kg body weight, or about 100 mg / kg body weight to about 1000 mg / kg body weight, or about 150 mg / kg body weight to about 500 mg / kg body weight. For example, the dose may be about 1, about 5, about 10, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1,000, about 1,050, about 1,100, about 1,150, about 1,200, about 1,250, about 1,300, about 1,350, about 1,400, about 1,450, about 1,500, about 1,600, about 1,700, about 1,800, about 1,900, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500 or about 5,000 mg / kg body weight. Alternatively, the dosage ranges from about 5 mg conjugate / kg body weight to about 20 mg conjugate / kg body weight. In another example, the dosage is about 8, about 10, about 12, about 14, about 16 or about 18 mg / kg body weight. Preferably, the soluble fusion protein complex is administered at about 0.5 mg / kg to about 10 mg / kg (e.g., about 0.5, about 1, about 3, about 5, about 10 mg / kg). Of course, this dosage may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of initial clinical trials and the needs of a particular patient.

[0109] The pharmaceutical composition is formulated with suitable excipients into a pharmaceutical composition that releases the therapeutic agent in a controlled manner upon administration. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches and liposomes. Preferably, the fusion protein complex is formulated in an excipient suitable for parenteral administration.

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

[0111] Compositions comprising the fusion protein complexes of the invention for parenteral use are provided in unit dosage form (e.g., single-dose ampoules). Alternatively, the compositions are provided in vials containing several doses, and suitable preservatives may be added (see below). The compositions are in the form of solutions, suspensions, emulsions, injection devices or implantable delivery devices, or provided as dry powders to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates neoplastic, infectious or autoimmune diseases, the compositions comprise suitable parenterally acceptable carriers and / or excipients. The active therapeutic agent may be incorporated into microspheres, microcapsules, nanoparticles, liposomes for controlled release. Furthermore, the compositions may comprise suspending agents, solubilizing agents, stabilizing agents, pH adjusting agents, isotonicity adjusting agents and / or dispersing agents.

[0112] As described above, pharmaceutical compositions containing the soluble fusion protein complexes of the present invention may be in a form suitable for sterile injection. To prepare such compositions, a suitable active therapeutic agent is dissolved or suspended in a parenterally acceptable liquid vehicle. Among the acceptable vehicles and solvents that may be used are 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. Aqueous formulations may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate). If one of the active therapeutic agents is poorly soluble or only slightly soluble in water, a solubility enhancer or solubilizer may be added, or the solvent may contain 10-60% w / w propylene glycol.

[0113] The present invention provides a method of preventing or treating cancer, a neoplasm, or an autoimmune disease or disorder, or a symptom thereof, comprising administering to a subject (e.g., a mammal, such as a human) in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a soluble fusion protein complex. Thus, one embodiment is a method of treating a subject suffering from or susceptible to cancer, a neoplasm, or an autoimmune disease or disorder, or a symptom thereof. In particular, cancers that may be treated by administration of the pharmaceutical composition include acute leukemia, AIDS-related cancers, breast cancer, bone cancer, brain cancer, 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 cancer, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma, and multidrug resistant cancer. The method comprises administering to the mammal a therapeutic amount of a soluble fusion protein complex or a pharmaceutical composition comprising a soluble fusion protein complex as described herein sufficient to treat the disease or disorder or a symptom thereof under conditions such that the disease or disorder is treated.

[0114] Additionally or alternatively, the described soluble fusion protein complexes or pharmaceutical compositions containing same may be used to prevent or treat cancer in a subject in need thereof. Additionally or alternatively, the described soluble fusion protein complexes or pharmaceutical compositions containing same may be used to prevent or treat autoimmune diseases or disorders in a subject in need thereof.

[0115] The methods herein include administering to a subject (including a subject identified as in need of such treatment) an effective amount of the described soluble fusion protein complex or a pharmaceutical composition containing same to produce such an effect. Identification of a subject in need of 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] Therapeutic methods (including prophylactic treatments) of the invention generally involve administration of a therapeutically effective amount of a soluble fusion protein complex as described herein, for example a soluble fusion protein complex having a first domain having an IL-15 peptide having sequence identity (e.g., at least 85% sequence identity) to SEQ ID NO: 17, a second domain comprising a fusion polypeptide comprising an IL15RαSu peptide and an αCTLA4 antibody heavy chain having sequence identity (e.g., at least 85% sequence identity) to SEQ ID NO: 10, and a third domain having an αCTLA4 antibody light chain having sequence identity (e.g., at least 85% sequence identity) to SEQ ID NO: 3, to a subject (e.g., animal, human), including a mammal, particularly a human, in need thereof, where the IL-15 peptide of the first domain binds to the IL15RαSu peptide of the second domain. Such treatments are suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a neoplasm, infectious disease, autoimmune disease, disorder, or a symptom thereof. The determination of these "at risk" subjects can be made by any objective or subjective determination, by diagnostic test or the opinion of the subject or health care provider (e.g., genetic testing, enzyme or protein markers, Markers (as defined herein), family history, etc.) The fusion protein complexes of the present invention may be used in the treatment of any other disorder in which an increased immune response is desired.

[0117] The present invention also provides a method of monitoring the progress of a treatment. The method includes determining the level of a diagnostic marker (marker) (e.g., any target delineated herein that is modulated by a soluble fusion protein complex, protein or indicator thereof, etc., described herein) or diagnostic measurement (e.g., screening, assay) in a subject suffering from or susceptible to a neoplasia-related disorder or a symptom thereof, and the subject is administered a therapeutic amount of the described soluble fusion protein complex sufficient to treat the disease or a symptom thereof. The level of the marker determined in this manner can be compared to known levels of the marker in either healthy normal controls or other affected patients to establish the disease status of the subject. In some cases, a second level of the marker in the subject is determined at a time point subsequent to the determination of the first level, and the two levels are compared to monitor the progress of the disease or the effectiveness of the treatment. In certain embodiments, a pre-treatment level of the marker in the subject is determined before initiating treatment according to the present invention, and then this pre-treatment level of the marker can 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 can be administered in combination with any other standard treatment, such methods being known to those skilled in the art and described in Remington's Pharmaceutical Sciences by EW Martin. If necessary, the fusion protein complexes of the present invention can be administered in combination with any conventional anti-neoplastic therapy, including but not limited to immunotherapy, therapeutic antibodies, targeted therapy, surgery, radiation therapy, or chemotherapy.

[0119] Kit or Pharmaceutical System. A pharmaceutical composition comprising the fusion protein complex of the present invention may be assembled into a kit or pharmaceutical system for use in ameliorating a neoplasm or autoimmune disease. A kit or pharmaceutical system according to this aspect of the present invention comprises a carrier means, such as a box, carton, tube, etc., having one or more container means, such as vials, tubes, ampoules, bottles, etc., in intimate confinement therein. A kit or pharmaceutical system of the present invention may also include associated instructions for using the fusion protein complex and / or cells of the present invention. EXAMPLES

[0120] Example 1 – Preparation of the molecules shown in Figures 1-3 Mammalian cells were transfected to express various molecules to produce molecules as shown in Figures 1-3. Those skilled in the art are well aware of how to produce and express fusion protein complexes in mammalian cells. Briefly, DNA sequences encoding soluble fusion protein complexes were cloned into a mammalian expression vector under a CMV promoter with an SV40 polyadenylation sequence. The plasmid contained ampicillin and puromycin resistance markers. The mammalian expression vector was transformed into chemically competent E. coli. The transformed E. coli was cultured under standard conditions in ampicillin-containing medium. Plasmids were isolated and purified from E. coli cultures using Qiagen Maxiprep® columns according to the manufacturer's protocol.

[0121] Chinese Hamster Ovary-S (CHO-S) cells were cultured in suspension in shake flasks in CD-CHO medium supplemented with 8 mM L-glutamine at 37°C and 8% CO2 and shaken at 125 rpm. For transfection, exponentially growing cells were pelleted by centrifugation (1,400 rpm for 10 min), resuspended in 10 mL of electroporation buffer, and repelleted (1,400 rpm for 5 min). The cell pellet was diluted with 2 × 10 6 cells in electroporation buffer mixed with a plasmid carrying the DNA sequence of interest at a DNA concentration of 150 μg / mL. 8The cells were transfected using the OC-400 processing assembly in the Maxcyte® ExPERT ATx Transfection System. The transfected cells were incubated at 37°C and 5% CO2 for 30 min and then transfected at a density of approximately 4-6 × 10 6 The cells were resuspended in Efficient Feed A cocktail (CHO-CD EfficientFeed™ A + 0.2% Pluronic F-68 + 1% HT supplement + 1% L-glutamine) at a density of 1000 cells / mL. The cells were incubated overnight on a shaker at 37°C, 5% CO2 and 125 rpm. 1 mM sodium butyrate was added and the cultures were incubated for an additional 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 cultures were harvested on day 14.

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

[0123] [Table 1]

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

[0125] [Table 2]

[0126] Example 3 – Stability assessment To assess the stability of the various configurations of fusion proteins, purified fusion proteins were analyzed by differential scanning fluorimetry (DSF). 20 μL of 1 mg / mL ACE IgG1Fc was mixed with 10 μL of SYPRO orange in the appropriate wells or plates. Plates were scanned from 25 °C to 70 °C (0.5 °C increments) on a CFX96 real-time system (BioRad). The results are shown in Figure 4. It can be seen that the molecules in Figure 3 are inherently more stable than those in Figure 2.

[0127] Example 4 – Assessment of off-target effects Useful therapeutic candidates must not only bind specifically to the target, but also must not exhibit off-target effects. Various molecular configurations were examined following 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, incorporated herein by reference in its entirety. Cetuximab is included in the study as a negative control, while αIL-18 antibody (43-12b) serves as a positive control antibody. The results are shown in Table 3 below. The MSD polyreactivity assay (column A) tests for nonspecific adhesion, which often indicates faster clearance in monoclonal antibodies. Hydrophobic interaction chromatography (HIC) (column B) measures the hydrophobicity of monoclonal antibodies, which likely correlates with aggregation tendency. Clonal self-interaction using biolayer interferometry (CSI-BLI) (column C) measures the tendency of monoclonal antibodies to self-aggregate, which often indicates poor solubility. Accelerated stability (column D) measures the long-term aggregation tendency of a monoclonal antibody. Based on these results, both the αCTLA4 / TxM-B and αCTLA4 / IL-15 fusions are strong candidates.

[0128] [Table 3]

[0129] Example 5 – B16.F10 melanoma tumor model A syngeneic B16.F10 melanoma tumor model was used for preclinical evaluation of these candidate immunotherapies by tumor (it) administration. C57BL / 6 mice were purchased from Jackson Laboratories (Maine, USA). The B16.F10 murine melanoma cell line was maintained as a monolayer in culture using RPMI medium supplemented with 10% heat-inactivated 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 generated and 1.3 × 10 cells were cultured in a volume of 50 μL of PBS. 5 Cells were implanted intradermally into each of the left and right shaved flanks of C57BL / 6 mice. Tumors were allowed to grow until they reached an average diameter of 5-7 mm (approximately 8 days), at which point mice were randomly enrolled into treatment groups. 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. It treatments consisted of PBS (as a negative control) or 5 yeast units (YU, 1YU=10) of Saccharomyces cerevisiae yeast lysate expressing one of the four B16.F10 neoepitopes, 25 μg of anti-OX40 antibody, and 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 Both treated (injected) and untreated (distal) tumors were measured at periodic intervals using digital calipers, and the longest diameter (A) and the shorter diameter (B) were used to calculate V=(AB 2 Tumor volume was calculated as 0.01% (0.1%) / 2. Tumor growth was monitored over time and tumors >1000 mm 3Mice were euthanized when tumor volume reached 100 μg / kg. Aggregate results are shown in Figure 5. Tumor growth in individual mice is shown in Figures 6A-6E. Efficacy of treatment in injected and distant tumors on day 11, as measured by AUC calculation, is shown in Figures 7A and 7B, respectively. The results show that administration of soluble fusion protein complexes significantly reduces tumor volume. Figure 7C shows that the described soluble fusion protein complexes are generally tolerated in a mouse melanoma model for up to 18 days.

[0130] Example 6 –pSTAT5 Assay The functionality of the listed soluble fusion protein complexes is measured by an in vitro staining assay. IL-2-depleted aNK cells (4 × 10 in each well of a 96-well plate) were cultured in a 2× 4-well plate. 5 Cells / well) are stimulated with a dilution series of IL-18 / IL-15 / IL-12 superkines (8 10-fold dilutions starting at 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 in GraphPad Prism to calculate the EC 50 Determine.

[0131] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0132] 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 construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "etc.") provided herein is intended merely to better clarify the invention and does not limit the scope of the invention unless specifically claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0133] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these specific embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors expect that such variations will be employed by those of skill in the art as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, the invention encompasses any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

[0134] [Table 4]

[0135] [Table 5]

[0136]

Table 6

Claims

1. A soluble fusion protein complex comprising: (a) a first domain comprising an interleukin-15 (IL-15) peptide having at least 85% sequence identity to 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, said αCTLA4 antibody heavy chain having at least 85% sequence identity to SEQ ID NO: 10; (c) a third domain comprising an αCTLA4 antibody light chain having at least 85% sequence identity to SEQ ID NO:3; wherein said IL-15 peptide of said first domain binds to said IL-15RαSu peptide of said second domain to form a soluble fusion protein complex.

2. The soluble fusion protein complex of claim 1, wherein the αCTLA4 antibody heavy chain has at least 90% sequence identity with SEQ ID NO: 10 and includes SEQ ID NOs: 11-13, 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 of claim 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 of 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 of any one of claims 1 to 4, wherein the IL15RαSu peptide has at least 85% sequence identity with SEQ ID NO:

8.

6. The soluble fusion protein complex of any one of claims 1 to 5, wherein the IL-15 peptide comprises SEQ ID NO:

17.

7. The soluble fusion protein complex of any one of claims 1 to 6, wherein the IL-15 peptide comprises SEQ ID NO:

1.

8. The soluble fusion protein complex of any one of claims 1 to 7, wherein the fusion polypeptide further comprises a peptide linker between the amino terminus of the IL15RαSu peptide and the carboxy terminus of the αCTLA4 antibody heavy chain.

9. The soluble fusion protein complex of any one of claims 1 to 8, wherein the fusion polypeptide comprises at least 85% sequence identity to SEQ ID NO:

5.

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

5.

11. The soluble fusion protein complex of any one of claims 1 to 10, wherein the fusion polypeptide further comprises a peptide linker between the carboxy terminus of the IL15RαSu peptide and the amino terminus of the αCTLA4 antibody heavy chain.

12. The soluble fusion protein complex of any one of claims 1 to 11, wherein the fusion polypeptide comprises at least 85% sequence identity to SEQ ID NO:

4.

13. The soluble fusion protein complex of any one of claims 1 to 12, wherein the fusion polypeptide comprises SEQ ID NO:

4.

14. A pharmaceutical composition comprising the soluble fusion protein complex of any one of claims 1 to 13 and a pharma- ceutically acceptable carrier.

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

16. 16. The pharmaceutical composition of claim 14 or 15, formulated for subcutaneous, intravenous, intramuscular, intravesicular, intratumoral or intraperitoneal injection.

17. The pharmaceutical composition according to any one of claims 14 to 16, which is formulated for intravenous injection.

18. A method for preventing or treating cancer in a subject in need thereof, comprising administering to said subject a soluble fusion protein complex according to any one of claims 1 to 13 or a pharmaceutical composition according to any one of claims 14 to 17.

19. 19. The method of claim 18, wherein the cancer is selected from the group consisting of acute leukemia, AIDS-related cancer, breast cancer, bone cancer, brain cancer, 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 cancer, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma, and multidrug resistant cancer.

20. A method for preventing or treating an autoimmune disease or disorder in a subject in need thereof, comprising administering to said subject a soluble fusion protein complex of any one of claims 1 to 13 or a pharmaceutical composition of any one of claims 14 to 17.

21. Use of a soluble fusion protein complex according to any one of claims 1 to 13 or a pharmaceutical composition according to any one of claims 14 to 17 for the prevention or treatment of cancer in a subject in need thereof.

22. Use of a soluble fusion protein complex according to any one of claims 1 to 13 or a pharmaceutical composition according to any one of claims 14 to 17 for the prevention or treatment of an autoimmune disease or disorder in a subject in need thereof.

Images