IL-12 / IL-18 Supercaine

Abstract

A novel soluble fusion protein complex comprising interleukin-18 (IL-18) and interleukin-12 (IL-12) polypeptide domains is provided herein. Also provided are compositions comprising the soluble fusion protein complex and methods for producing memory-like cytokine-enhanced natural killer (M-CENK) cells using the soluble fusion protein complex.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 493,443, filed March 31, 2023, under 35 U.S.SC § 119(e). The full disclosure of U.S. Provisional Patent Application No. 63 / 493,443 is incorporated herein by reference.

[0002] Sequence listing reference This application includes a sequence listing submitted electronically as an ST.26 XML file. The XML file, named "PAT005342US002.xml", is 17,000 bytes in size and was recorded on 18 March 2024. The information contained in the XML file is incorporated herein by reference in its entirety in accordance with 37 CFR §1.52(e)(5). [Background technology]

[0003] Therapeutic heteromeric protein complexes containing the interleukin-15 (IL-15) superagonist N-803 have been found to modulate immune responses in the treatment of various cancers. N-803 is a multimeric protein complex containing an IL-15 mutant that binds to the IL-15 receptor alpha (α) and then to an immunoglobulin G1 (IgG1) crystallizable fragment (Fc). N-803 has been shown to exhibit improved pharmacokinetic properties, longer persistence in lymphoid tissues, and enhanced antitumor activity compared to the natural, uncomplexed IL-15. [Overview of the project] [Problems that the invention aims to solve]

[0004] The continuous evaluation of the pharmacokinetic properties of therapeutic heteromer protein conjugates such as N-803, as well as the methods for producing these conjugates, are crucial for assessing clinical efficacy and manufacturing efficiency. [Means for solving the problem]

[0005] One embodiment is an isolated soluble fusion protein complex comprising two soluble proteins, comprising: (a) an interleukin-18 (IL-18) polypeptide domain; an interleukin-15 receptor alpha subunit binding domain (IL-15RαSu); an immunoglobulin (Ig) crystallizable fragment (Fc) domain; and a first soluble fusion protein comprising an IL-12 domain comprising p40 and p35 subunits; and (b) a second soluble protein comprising IL-15 (IL-15); wherein the IL-15-RαSu domain of the first soluble fusion protein binds to the IL-15 of the second soluble protein to form a soluble fusion protein complex; and the immunoglobulin Fc domain of the first soluble protein complex is covalently linked by a disulfide bond to the Fc domain of the second soluble protein complex, relating to a soluble fusion protein complex. In one aspect of this embodiment, the first soluble fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0006] Another embodiment relates to an isolated soluble fusion protein complex comprising a soluble fusion protein comprising an interleukin-18 (IL-18) polypeptide domain, an immunoglobulin crystallizable fragment (Fc) domain, and an IL-12 polypeptide domain comprising p40 and p35 subunits, wherein the Fc domain of the first soluble protein is covalently linked by a disulfide bond to the Fc domain of the second soluble protein. In one aspect of this embodiment, the soluble fusion protein complex comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0007] Another embodiment is a method of producing memory-like cytokine enhanced natural killer (M-CENK) cells, comprising obtaining a plurality of mononuclear cells and contacting the plurality of mononuclear cells with a corticosteroid; incubating the plurality of mononuclear cells in the presence of the corticosteroid to enrich the mononuclear cells within NK cells; and inducing the enriched NK cells with a soluble fusion protein complex isolated and containing two soluble proteins, a first soluble fusion protein containing an interleukin-18 (IL-18) polypeptide domain, an IL-15 receptor alpha subunit binding domain (IL-15RαSu), an immunoglobulin (Ig) crystallizable fragment (Fc) domain, and an IL-12 polypeptide domain containing p40 and p35 subunits, and a second soluble protein containing IL-15 (IL-15), wherein the IL-15-RαSu domain of the first soluble fusion protein binds to the IL-15 of the second soluble protein to form a soluble fusion protein complex; and the immunoglobulin Fc domain of the first soluble protein complex is covalently bound by a disulfide bond to the Fc domain of the second soluble protein complex, or a soluble fusion protein complex, or IL-15 or a derivative thereof and a soluble fusion protein complex isolated and containing a soluble fusion protein containing an interleukin-18 (IL-18) polypeptide domain, an Ig crystallizable fragment (Fc) domain, and an IL-12 polypeptide domain containing p40 and p35 subunits; wherein the Fc domain of the first soluble protein is covalently bound by a disulfide bond to the Fc domain of the second soluble protein, to produce M-CENK cells.

[0008] In one aspect of the method, the corticosteroid is hydrocortisone.

[0009] In another aspect of the method, the IL-15 derivative is N-803.

[0010] In any one embodiment of the embodiments, the IL-15 polypeptide domain is an IL-15 variant (IL-15N72D) that includes a mutation from asparagine to aspartic acid at amino acid position 72.

[0011] In any one embodiment, the IL-18 domain is linked to the IL-15RαSu domain by a mobile polypeptide linker.

[0012] In any one embodiment of the model, the IL-18 polypeptide domain is linked to the Ig Fc domain by a mobile polypeptide linker.

[0013] In one embodiment of the model, the p40 and p35 subunits of the IL-12 domain are linked by a mobile polypeptide linker.

[0014] In any one embodiment of the model, the IL-12 p40 subunit is linked to the Ig Fc domain by a mobile polypeptide linker. [Brief explanation of the drawing]

[0015] [Figure 1A-C] These are schematic diagrams of the soluble fusion protein complexes disclosed herein, such as the known IL-12 / 15 / 18 superkine (SK) (ALTOR BIOSCIENCE®) (IL-12 / 15 / 18 SK (Altor); Figure 1A), the novel IL-12 / 15 / 18 SK (new) (Figure 1B), the novel IL-18 / 15 / 12 SK (Figure 1C), the novel IL-18 / 12 SK (Figure 1D), and the novel IL-12 / 18 SK (Figure 1E). [Figure 1D-E]These are schematic diagrams of the soluble fusion protein complexes disclosed herein, such as the known IL-12 / 15 / 18 superkine (SK) (ALTOR BIOSCIENCE®) (IL-12 / 15 / 18 SK (Altor); Figure 1A), the novel IL-12 / 15 / 18 SK (new) (Figure 1B), the novel IL-18 / 15 / 12 SK (Figure 1C), the novel IL-18 / 12 SK (Figure 1D), and the novel IL-12 / 18 SK (Figure 1E). [Figure 1F] Figures 1A to 1E show graphs illustrating the results of cell line activity assays reported for IL-18, using HEK-BLUE® IL-18 cells (INVIVOGEN®) along with the SK protein constructs shown. [Figure 2A] HPLC chromatograms for superkine protein complexes such as IL-12 / 15 / 18 (Figure 2A), IL-18 / 15 / 12 (Figure 2B), and IL-18 / 12 (Figure 2C) are shown. [Figure 2B] HPLC chromatograms for superkine protein complexes such as IL-12 / 15 / 18 (Figure 2A), IL-18 / 15 / 12 (Figure 2B), and IL-18 / 12 (Figure 2C) are shown. [Figure 2C] HPLC chromatograms for superkine protein complexes such as IL-12 / 15 / 18 (Figure 2A), IL-18 / 15 / 12 (Figure 2B), and IL-18 / 12 (Figure 2C) are shown. [Figure 3A] The results of an IL-18 reporter assay show that the IL-18 / 12 superkine design exhibited superior IL-18 activity compared to a novel IL-18 / 15 / 12 superkine and a known IL-12 / 15 / 18 superkine protein complex known as HCW9201 (see Figure 3B for a schematic diagram of HCW9201). The schematic diagram and EC50 value of HCW9201 were obtained from Becker-Hapak, MK, Cancer Immunol Res, 9(9).2020. [Figure 3B]The results of an IL-18 reporter assay show that the IL-18 / 12 superkine design exhibited superior IL-18 activity compared to a novel IL-18 / 15 / 12 superkine and a known IL-12 / 15 / 18 superkine protein complex known as HCW9201 (see Figure 3B for a schematic diagram of HCW9201). The schematic diagram and EC50 value of HCW9201 were obtained from Becker-Hapak, MK, Cancer Immunol Res, 9(9).2020. [Figure 4A] This graph shows the percentage of IFN-γ+CD56+ cells induced by either the IL-18 / 12 superkine protein complex + N-803 (Figure 4A) or the IL-18 / 15 / 12 superkine protein complex (Figure 4B). As can be seen from the graph, the IL-18 / 12 superkine protein complex + N-803 showed superior M-CENK activation compared to the IL-18 / 15 / 12 superkine protein complex. [Figure 4B] This graph shows the percentage of IFN-γ+CD56+ cells induced by either the IL-18 / 12 superkine protein complex + N-803 (Figure 4A) or the IL-18 / 15 / 12 superkine protein complex (Figure 4B). As can be seen from the graph, the IL-18 / 12 superkine protein complex + N-803 showed superior M-CENK activation compared to the IL-18 / 15 / 12 superkine protein complex. [Figure 5]The graph shows the results of the pSTAT-5 FLOW assay with the novel IL-18 / 15 / 12 superkine protein complex disclosed herein, compared to a known IL-12 / 15 / 18 protein complex (i.e., HCW9201 - see Figure 3B). The novel IL-18 / 15 / 12 superkine protein complex exhibits higher IL-15 activity than the known IL-12 / 15 / 18 protein complex. (EC50 values ​​for HCW9201 were obtained from Becker-Hapak, MK, Cancer Immunol Res, 9(9).2020. For the pSTAT-5-FLOW assay, IL2 aNK cells were stimulated for 20 minutes with each of eight constructs at different concentrations. For FLOW decoding, cells were immobilized / permeabilized and stained with PE-anti-pSTAT5.) [Modes for carrying out the invention]

[0016] After reading this description, it will be obvious to those skilled in the art how the disclosure is implemented in various alternative embodiments and applications. However, not all of the various embodiments of the invention are described herein. It will be understood that the embodiments presented herein are presented only as examples and are not limiting. As such, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the disclosure as described herein.

[0017] Before the disclosure and description of this technology, it should be understood that the following embodiments are not limited, as they may naturally vary in terms of specific compositions, methods for preparing such compositions, or uses thereof. It should also be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to limit them.

[0018] For the convenience of the reader, detailed explanations are divided into various sections, and disclosures found in any section may be combined with disclosures in other sections. For the convenience of the reader, titles or subtitles may be used herein and are not intended to affect the scope of this disclosure.

[0019] definition Unless otherwise defined, all scientific and technical terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure belongs. In this specification and in the following claims, several terms will be referenced to those which are defined as follows:

[0020] The terminology used herein is intended solely to describe specific embodiments and is not intended to be limiting. Where used herein, the singular forms "a," "an," and "the" are also intended to include the plural forms unless the context clearly indicates otherwise.

[0021] "Optional" or "optional" means that the event or situation described thereafter may or may not occur, and that the description includes both cases in which the event or situation occurs and cases in which it does not occur.

[0022] The term "approximately," when used before a numerical specification, such as temperature, time, quantity, concentration, or other such terms that include a range, indicates an approximate value that may vary by (+) or (-) 10%, 5%, 1%, or any sub-range or sub-value between them. Preferably, when the term "approximately" is used in relation to a quantity, it means that the quantity may vary by + / - 10%.

[0023] "Comprising" or "Comprises" is intended to mean that a composition and method includes the elements listed, but does not exclude anything else. "Essentially consisting of" when used to define a composition and method means excluding any other elements that are essentially important to the combination for the presented purpose. Thus, a composition essentially consisting of elements as defined herein does not exclude other materials or steps that do not substantially affect the basic and novel features of the claimed invention. "Consists of" means excluding elements and substantial method steps that are in greater quantities than the other components. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0024] An “antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragment thereof that specifically binds to and recognizes an antigen. The recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which, in turn, define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen-binding region of an antibody plays a significant role in determining binding specificity and affinity. In some embodiments, the antibody or antibody fragment may originate from different organisms, including humans, mice, rats, hamsters, and camels. The antibodies of the present invention may include antibodies that have been modified or mutated at one or more amino acid positions to improve or modulate a desired function of the antibody (e.g., glycosylation, expression, antigen recognition, effector function, antigen binding, specificity, etc.).

[0025] Antibodies are large, complex molecules with a complex internal structure (approximately 150,000 molecular weight or approximately 1,320 amino acids). Natural antibody molecules contain two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and each heavy chain, in turn, consists of two regions: a variable ("V") region involved in binding to the target antigen, and a constant ("C") region that interacts with other components of the immune system. The light and heavy chain variable regions coexist in a three-dimensional space such that they form the variable region that binds to the antigen (e.g., a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acid lengths) called complementarity-determining regions ("CDRs"). The six CDRs within the antibody variable domain (three from the light chain and three from the heavy chain) fold together in three-dimensional space to form the actual antibody-binding site that docks onto the target antigen. The location and length of the CDR are precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services, 1983, 1987. The variable region not included within the CDR is called the framework ("FR") and forms the environment for the CDR.

[0026] Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids, primarily involved in antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0027] As used herein, the terms “Fc domain” or “crystallizable domain fragment” are used according to their simple, ordinary meanings and refer to either a recombinant or naturally occurring form of the “base” or tail-terminal region (C-terminus) of an antibody. An Fc domain typically consists of two heavy chains contributing to two or three constant domains, depending on the class of the antibody. The Fc region comprises two heavy chain constant Ig domains in IgG, IgA, and IgD antibodies, and three heavy chain constant Ig domains in IgE and IgM antibodies.

[0028] The term "Fc" refers to a non-antigen-binding fragment of an antibody. Such "Fc" can be in monomeric or polymeric form. The original immunoglobulin source of natural Fc is preferably of human origin and may be any immunoglobulin. In some embodiments, Fc is IgG1 or IgG2 Fc. In the case of natural Fc, it is formed from a monomeric polypeptide that may be linked to a dimeric or polymeric form by covalent (i.e., disulfide) and non-covalent associations. The number of intermolecular disulfide bonds between monomeric subunits of a natural Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). An example of natural Fc is a disulfide-bonded dimer obtained from papain digestion of IgG (see Ellison et al. (1982), Nucleic Acids Res. 10:4071-9). The term "Fc" as used herein is common to monomeric, dimeric, and polymeric forms.

[0029] In several embodiments, the term "Fc" refers to a molecule or sequence that, even after modification from native Fc, still contains a receptor binding site. As with modified Fc and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from the whole antibody or produced by recombinant gene expression or other means.

[0030] In embodiments, Fc (or Fc domain) is directly or indirectly bound to an IL-15Rα or IL-15 domain. In embodiments, Fc (or Fc domain) is covalently and / or genetically fused to an IL-15Rα or IL-15 domain. In further embodiments, Fc (or Fc domain) from one soluble fusion protein complex disclosed herein is covalently bound by a disulfide bond to Fc (or Fc domain) of a second soluble protein complex containing IL-15. In one embodiment, the soluble fusion protein complex comprises a first soluble protein containing an IL-18 polypeptide domain, an IL15RαSu domain, an Fc domain, and an IL-12 domain containing p40 and p35 subunits; and a second soluble protein containing IL-15. In yet another embodiment, the soluble fusion protein complex comprises a soluble fusion protein comprising an IL-18 polypeptide domain, an Fc domain, and an IL-12 polypeptide domain including p40 and p35 subunits, where the Fc domain of the first soluble protein is covalently bonded to the Fc domain of the second soluble protein by a disulfide bond.

[0031] Antibodies exist, for example, as intact immunoglobulins or as several well-characterized fragments produced by digestion by various peptidases. For example, pepsin digests antibodies that lack disulfide bonds in the hinge region, producing F(ab)'2, a dimer of Fab, which is itself a light chain linked to VH-CH1 by disulfide bonds. F(ab)'2 can decrease under mild conditions, such as by disrupting the disulfide bonds in the hinge region, thereby converting the F(ab)'2 dimer back into a Fab monomer. A Fab' monomer is essentially an antigen-binding moiety that has part of a hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)). While various antibody fragments are defined in terms of digestion of intact antibodies, those skilled in the art will understand that such fragments can be newly synthesized chemically or by recombinant DNA methods. Thus, when the term antibody is used herein, it also includes antibody fragments produced by modification of whole antibodies, or those newly synthesized by recombinant DNA methods (e.g., single-stranded Fv) or identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0032] Single-stranded variable fragments (scFv) are typically fusion proteins of the variable regions (VH) and light chains (VL) of immunoglobulins, linked to a short linker peptide of approximately 10 to 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility. The linker can either link the N-terminus of the VH to the C-terminus of the VL, or vice versa.

[0033] An mAb epitope is the region of the antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antigen. That is, as measured in a competitive binding assay, a 1x, 5x, 10x, 20x, or 100x excess of one antibody inhibits the binding of the other by at least 30%, but preferably 50%, 75%, 90%, or even 99% (see, for example, Junghans et al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if several amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0034] The phrases "specifically (or selectively) bind to an antibody" or "specifically (or selectively) immunoreactive with an antibody" refer, when referring to a protein or peptide, to a binding reaction in which the presence of the protein is decisive, often within a heterogeneous population of proteins and other biologics. Therefore, under specified immunoassay conditions, a particular antibody will bind to a particular protein at least twice the background level, and more typically, 10 to 100 times the background level. Specific binding to an antibody under such conditions requires an antibody selected for its specificity to a particular protein. For example, by selecting polyclonal antibodies, one can obtain only a subset of antibodies that are specifically immunoreactive with a selected antigen and not specifically immunoreactive with other proteins. This selection can be achieved by subtracting antibodies that cross-react with other molecules. Various immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that exhibit specific immunoreactivity with a particular protein (see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to measure specific immunoreactivity).

[0035] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a part thereof, is modified, substituted, or replaced so as to be linked to a constant region of a different or modified class, effector function, and / or species, or to an entirely different molecule that gives the chimeric antibody new characteristics, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a part thereof, is modified, substituted, or replaced with a variable region having a different or modified antigen specificity.

[0036] "Protein complex" or "complex" as used herein refers to two or more polypeptides that associate simultaneously. The complex may be constructed via protein-protein binding and / or binding between a receptor and a ligand. The proteins may associate via non-covalent protein-protein interactions, but furthermore, specific polypeptides in the complex may be covalently bound directly or indirectly, for example, via a chemical linker, bond, or another protein. For example, the heterotetrameric IL-15 / IL-15RαSu complex comprises two IL-15 domains non-covalently bound to two IL-15RαSu domains, where the two IL-15RαSu domains are covalently bound by a disulfide bond.

[0037] "Detectable means" or "detectable moiety" refers to a composition, substance, sequence, or compound; or a portion thereof, that is detectable by suitable means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means.

[0038] For example, detectable means include , , , 94 , 62 , 89 , 52 , 125 , 86 , 45 , 123 , 68 , 32 , 111 , 67 ,

[0038] , 105 , 142 , 99m , 64 , 59 , 131 ,

[0037] , 94 , 47 , 124 ,<0000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​143 Mr. 149 PM 153 Sm、 154-1581 God 161 Tb 166 Dy 166 Ho 169 Err 175 sun 177 sun 186 Re 188 Re 189 Re 194 Ir 198 I 199 I 211 And 211 Pb、 212 Hello 212 Pb、 213 Hello 223 Ra 225 Ac、Cr、V、Mn、Fe、Co、Ni、Cu、La、Ce、Pr、Nd、Pm、Sm、Eu、Gd、Tb、Dy、Ho、Er、Tm、Yb、Lu、 32P, fluorophores (e.g., fluorescent dyes), high electron density reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultra-small superparamagnetic iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO nanoparticle aggregates, monochrystalline iron oxide nanoparticles, monochrystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery media containing gadolinium chelate ("Gd-chelate") molecules, gadolinium, radioisotopes, radionuclides (e.g., carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g., labeled fluorine-18), any gamma-ray emitting radionuclides, positron-emitting radionuclides, radiolabeled glucose, The present invention relates to radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g., microbubble shells containing albumin, galactose, lipids, and / or polymers; including microbubble gas cores containing air, heavy gases, perfluorocarbons, nitrogen, octafluoropropane, perflexane lipid microspheres, perflutren, etc.), iodinating contrast agents (e.g., iohexol, iodixanol, iobersol, iopamidol, ioxiran, iopromide, diatrizoic acid, metrizoic acid, ioxaglucic acid), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins or other entities, which can be detected, for example, by incorporating a radiolabel into a peptide or antibody that is specifically reactive with the target peptide. The detectable means may be a monovalent detectable activator or a detectable activator that can form a bond with another composition.

[0039] As used herein, the term “conjugate” refers to an association between atoms or molecules. The association may be direct or indirect. For example, a conjugate between an antigen-binding domain and a peptide compound may be direct, for example, by a covalent bond (e.g., a disulfide bond), or indirect, for example, by a non-covalent bond (e.g., electrostatic interactions (e.g., ionic, hydrogen, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effect), hydrophobic interactions, etc.). In some embodiments, conjugates are formed using conjugate chemistry, including, but not limited to, nucleophilic substitution (e.g., reactions of amines and alcohols with acyl halides and activated esters), electrophilic substitution (e.g., enamine reactions), and addition to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are discussed, for example, in March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982.

[0040] "Contact" is used in its simple, ordinary sense to refer to a process that allows at least two different species (e.g., compounds including biomolecules or cells) to become close enough to react, interact, or physically come into contact. However, it should be understood that the resulting reaction product may be produced directly from a reaction between additive reagents or from an intermediate formed from one or more additive reagents that may be produced in the reaction mixture.

[0041] The term “contact” may also include enabling two species to react, interact, or come into physical contact, where the two species may be antibodies and fusion proteins, biological samples, etc., as described herein.

[0042] A "control" sample or value refers to a sample that serves as a reference, usually a known reference, in comparison to a test sample. For example, a test sample may be taken from test conditions (e.g., in the presence of the test compound) and compared to a sample from known conditions (e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control)). A control may also represent the mean value collected from several tests or results. Those skilled in the art will understand that controls can be designed to evaluate any number of parameters. For example, a control may be designed to determine the background level of a signal (negative control) or the expected level of a signal (e.g., a calibration curve or positive control). Those skilled in the art will understand which control is useful under given circumstances and will be able to analyze the data based on comparison with the control value. Controls are also useful in determining the significance of the data. For example, if the value of a given parameter varies significantly in the control, the variation in the test sample will not be considered significant.

[0043] "Biological sample" or "sample" refers to material obtained from or derived from a subject or patient. Biological samples include tissue sections such as biopsy and autopsy samples, as well as frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or preparations (e.g., serum, plasma, platelets, red blood cells), sputum, tissues, cultured cells (e.g., primary cultures, explants, and transformed cells), feces, urine, synovial fluid, joint tissue, synovial tissue, synovial cells, fibroblast-like synovial cells, macrophage-like synovial cells, immune cells, hematopoietic cells, fibroblasts, macrophages, and T cells. Biological samples are typically obtained from eukaryotes such as mammals, e.g., primates, e.g., chimpanzees or humans; cows; dogs; cats; rodents, e.g., guinea pigs, rats, mice; rabbits; or birds; reptiles; or fish.

[0044] The term "polymer" refers to a molecule containing repeating subunits (e.g., a polymer monomer). For example, polymer molecules may be based on polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), poly[amino(1-oxo-1,6-hexanediyl)], poly(oxy-1,2-ethanediyloxycarbonyl-1,4-phenylenecarbonyl), tetraethylene glycol (TEG), polyvinylpyrrolidone (PVP), poly(xylene), or poly(p-xylylene). For example, see “Chemistry of Protein Conjugation and Cross-Linking” Shan S. Wong CRC Press, Boca Raton, Fla., USA, 1993; “BioConjugate Techniques” Greg T. Hermanson Academic Press, San Diego, Calif., USA, 1996; “Catalog of Polyethylene Glycol and Derivatives for Advanced PEGylation, 2004” Nektar Therapeutics Inc, Huntsville, Ala., USA (all of these are incorporated by reference for any purpose).

[0045] When "interleukin-15 protein" or "IL-15" is used herein, it includes either a recombinant or naturally occurring form of the interleukin-15 (IL-15) protein or its variant or homologue that maintains IL-15 protein activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the IL-15 protein). In some embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the naturally occurring IL-15 protein, either through the entire sequence or a portion thereof (e.g., a 50, 100, 150, or 200 consecutive amino acid portion). In some embodiments, the IL-15 protein is substantially identical to the protein identified by UniProt reference number P40933 or a variant or homologue substantially identical thereto.

[0046] When "interleukin-15 receptor subunit alpha protein" or "IL-15Rα" is used herein, it includes either the interleukin-15 receptor subunit alpha (IL-15Rα) protein or a recombinant or naturally occurring form of its variant or homologue that maintains IL-15Rα protein activity (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to IL-15Rα protein). In some embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to naturally occurring IL-15Rα protein, through the entire sequence or a portion thereof (e.g., a 50, 100, 150, or 200 consecutive amino acid portion). In several embodiments, the IL-15Rα protein is substantially identical to the protein identified by UniProt reference number Q13261, or a variant or homolog thereof that is substantially identical.

[0047] As used herein, "domain" refers to a conserved portion of a protein that functions independently of the rest of the protein sequence. Domains can form stable three-dimensional structures that exist as functional units independent of the rest of the protein. For example, the IL-15RαSu domain is a portion of IL-15Rα that retains IL-15 binding activity.

[0048] As used herein, “IL-15 domain” refers to a polypeptide comprising at least a portion of the sequence of the IL-15 protein. In some embodiments, the IL-15 domain comprises at least a portion of the sequence of the IL-15 protein and contains one or more amino acid substitutions or deletions within the amino acid sequence of the IL-15 protein. In some embodiments, the IL-15 domain is an IL-15 variant containing a different amino acid sequence compared to the IL-15 protein. In some embodiments, the IL-15 domain binds to the IL-15Rα protein or a fragment thereof. In some embodiments, the IL-15 domain is bound to the IL-15Rα protein or a fragment thereof. In some embodiments, the sequence of the IL-15 domain has at least one amino acid change, e.g., substitution or deletion, compared to the IL-15 protein. In some embodiments, the amino acid substitution / deletion is located within a portion of IL-15 that interacts with IL-15Rβ and / or γC. In some embodiments, the amino acid substitution / deletion does not affect the ability to bind to the IL-15Rα polypeptide or to produce the IL-15 domain. In several embodiments, the amino acid substitutions may be conserved or non-conservative changes, and further amino acid insertions, compared to the IL-15 protein. In several embodiments, the IL-15 domain contains 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 IL-15 protein sequence. In several embodiments, the IL-15 domain contains the N72D substitution of the IL-15 protein sequence (IL15N72D: SEQ ID NO: 3 (amino acid sequence) and SEQ ID NO: 4 (nucleic acid sequence)).

[0049] The term "sucoiden," as used herein, refers to a common motif in proteins containing a β-sandwich sequence. Sucoiden are common in protein-protein interactions and typically contain four cysteine ​​molecules forming two disulfide bonds in 1-3 and 2-4 patterns. For example, the region of IL-15Rα that binds to IL-15 contains a sucoiden.

[0050] In several embodiments, the IL-15Rα sucoid domain includes an amino acid sequence containing the sequence of SEQ ID NO: 7. In several embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the sequence of SEQ ID NO: 7, either through the entire sequence or a portion thereof (e.g., a continuous amino acid portion of 50, 100, 150, or 200). In several embodiments, the IL-15Rα sucoid domain associates with the IL-15 protein. In several embodiments, the IL-15Rα sucoid domain associates with the IL-15 domain.

[0051] Soluble fusion proteins, compositions, and methods In one embodiment, an isolated soluble fusion protein complex comprising two soluble proteins is provided, wherein the soluble fusion protein complex comprises (a) a first soluble fusion protein comprising an interleukin-18 (IL-18) polypeptide domain; an IL-15 receptor alphasus-binding domain (IL-15RαSu); an immunoglobulin crystallizable fragment (Fc) domain; and an IL-12 domain comprising p40 and p35 subunits; and (b) a second soluble protein comprising IL-15 (IL-15), wherein the IL-15-RαSu domain of the first soluble fusion protein binds to IL-15 of the second soluble protein to form a soluble fusion protein complex; and the immunoglobulin Fc domain of the first soluble protein complex is covalently bonded to the Fc domain of the second soluble protein complex by a disulfide bond. In one embodiment, the first soluble fusion protein of the soluble fusion protein complex comprises the amino acid sequence described in Sequence ID No. 5.

[0052] In another embodiment, a soluble fusion protein is provided comprising an interleukin-18 (IL-18) polypeptide domain, an immunoglobulin crystallizable fragment (Fc) domain, and an IL-12 polypeptide domain comprising p40 and p35 subunits; where the Fc domain of the first soluble protein is covalently bonded to the Fc domain of the second soluble protein by a disulfide bond, an isolated soluble fusion protein complex is provided. In one embodiment, the soluble fusion protein complex comprises the amino acid sequence described in Sequence ID No. 1.

[0053] As provided herein, the IL-15 polypeptide domain is an IL-15 variant (IL-15N72D) containing the N72D mutation.

[0054] Furthermore, as provided herein, the IL-18 polypeptide domain is linked to the IL-15RaSu domain by a mobile linker, or to the IgFc domain by a mobile polypeptide linker.

[0055] Furthermore, as provided herein, the p40 and p35 subunits of the IL-12 domain are linked by a mobile polypeptide linker. In addition, the IL-12 p40 subunit is linked to the homodimer Ig Fc domain by a mobile polypeptide linker.

[0056] Furthermore, provided herein are compositions comprising the soluble fusion protein complex disclosed herein.

[0057] In another embodiment, a method is provided for producing memory-like cytokine-enhanced natural killer (M-CENK or M-ceNK) cells. The method comprises obtaining a plurality of mononuclear cells and contacting the plurality of mononuclear cells with a corticosteroid; incubating the plurality of mononuclear cells in the presence of a corticosteroid to enrich the mononuclear cells within the NK cells; and inducing the enriched NK cells with a soluble fusion protein complex disclosed herein or IL-15 or a derivative thereof and a soluble fusion protein complex disclosed herein to produce M-CENK cells. In one embodiment, the corticosteroid is hydrocortisone.

[0058] It should be obvious to those skilled in the art that many further modifications are possible, other than those already described, without departing from the concept of the invention as herein. Therefore, the subject matter of the invention should not be limited except to the scope of the appended claims. Furthermore, when interpreting both the specification and the claims, all terms should be interpreted in the broadest possible form that is consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted to refer to an element, component, or step in a non-exclusive manner, indicating that the referenced element, component, or step may be presented or used, or may be combined with other elements, components, or steps not explicitly referenced. Where the specification or claims refer to at least one of several selected from the group consisting of A, B, C…. and N, the text should be interpreted to require exclusively one element from a group such as A+N or B+N.

[0059] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes from thereto will be proposed to those skilled in the art, and should be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes. [Examples]

[0060] Those skilled in the art will understand that the descriptions of preparing and using the particles described herein are for illustrative purposes only and that this disclosure is not limited by such examples.

[0061] Example 1: MAXCYTE® Transient Transfection For transient expression of IL-18 / 12 SK and IL-18 / 15 / 12 SK by MAXCYTE® transfection, CHO-S cells were cultured in a suspension in CD-CHO medium supplemented with 8 mM L-glutamine in a shaker flask at 37°C, 125 rpm, and 8% CO2. For transfection, cells in the logarithmic growth stage were pelletized by centrifugation at 1,400 rpm for 10 minutes, resuspended in 10 mL of electroporation buffer, and repelled at 1,400 rpm for 5 minutes. The cell pellet was divided into 2 × 10⁶ cells. 8 The cells were resuspended in electroporation buffer at a density of cells / mL, mixed with a plasmid containing either the IL18 / 12 SK or IL18 / 15 / 12 SK sequence at a concentration of 150 μg / mL, and 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 minutes, then approximately 4–6 × 10⁶ cells were produced. 6The cells were resuspended at a density of cells / mL in Efficient Feed A Cocktail (CHO-CD EFFICIENTFEED® A + 0.2% Pluronic F-68 + 1% HT supplement + 1% L-glutamine). The cell culture was incubated overnight at 37°C, 5% CO2, and 125 rpm, then 1 mM sodium butyrate (buryrate) was added, and the culture was further incubated at 32°C, 3% CO2, and 125 rpm for 13 days; during this incubation period, MAXCYTE® Feed Cocktail (13.9% CD hydrolysate, 69.5% CHO CD EFFICIENTFEED® A, 6.2% glucose, 6.9% FUNCTIONMAX® titer enhancer, 3.5% L-glutamine) was added at 10% of the culture volume on days 4 and 8.

[0062] Purification of IL18 / 12 SK and IL18 / 15 / 12 SK MAXCYTE® transfection cell medium was centrifuged and filtered through a 0.22 μm filter to remove cells and debris, and then loaded onto a HITRAP® MABSELECT SURE® column (CYTIVA®) on an AKTA Pure system pre-equilibriumated with pH 7.0, 10 mM sodium phosphate, and 150 mM NaCl. After loading, the column was washed with 10 column volumes of the same buffer. Proteins were eluted with 100 mM sodium acetate, pH 3.6, and immediately neutralized with 2 M Tris at pH 8.0. The eluted fractions were pooled and dialyzed in 10 mM HEPES and 150 mM sodium chloride at pH 7.4.

[0063] Table 1 shows a comparison of production between the IL-12 / 15 / 18 SK (Altor) (ALTOR BIOSCIENCE®) design and the IL-18 / 15 / 12 SK and IL-18 / 12 SK designs, as described above, using the MAXCYTE® transient expression system and subsequent purification with a Protein A column. The IL-12 / 15 / 18 SK (Altor) (ALTOR BIOSCIENCE®) production yielded a very low titer of approximately 50 μg / ml (0.047 g / L at collection) and a recovery rate of only 1.9% (see the bolded amounts in the "IL-12 / 15 / 18 SK (Altor)" column in Table 1).

[0064] [Table 1]

[0065] Example 2 - Production of M-CENK cells Fresh or cryopreserved / thawed apheresis products (patient-derived mononuclear cells) are expanded in NK-GM medium (NK-MACS® basal medium containing NK-MACS® supplement and 10% human AB serum) supplemented with N-803 (0.8 nM) and hydrocortisone (1 μM). Once the culture has grown to at least 1 × 10⁶ 6When the cells reach at least 85% CD56-positive cell density by day 20 expansion, add a constant concentration of 1) N-803 (100-300 ng / mL), IL-12 (1-100 ng / mL), and IL-18 (5-250 ng / mL), or 2) N-803 (100-300 ng / mL) and IL12 / 18 SK (5 μg / mL) to the culture. Stimulate the cells with the above cytokines for 14-16 hours to induce a memory-like phenotype in CD56-positive cells. After completion of the M-CENK induction step, wash the cells using a Sepax C-Pro instrument. Most typically, 5% albumin (human) solution is used as the washing and resuspension solution. For cryopreservation, prepare M-CENK cells in a medium containing 5% albumin (human) USP:CryoStor 10 (CS10) (1:1). M-CENKs possess an enhanced ability to kill cancer cell targets through their increased IFN-γ production. Furthermore, these cells are phenotypically CD56+, CD25+, DNAM-1+, and NKP30+, NKG2D+, NKG2A+, and CD3-.

[0066] Example 3 - Stimulation of CENK with a cytokine cocktail (human IL-12, IL-18, and N-803) The NANT001 bioreactor platform system (IMMUNITYBIO®, INC.) is a self-contained bioreactor that executes pre-programmed protocols that direct automated procedures and real-time monitoring throughout the entire manufacturing process, including recovery, concentration, expansion, and M-CENK induction stages. Programmable process parameters include pH monitoring, cell imaging, temperature, and locking parameters. The NANT001 bioreactor includes a constant temperature compartment, touchscreen user interface, barcode reader, pH estimation unit, integrated imaging system, and gas flow control. The components are a single-use closed system design that is easy to load for safe cGMP-compliant cell processing, and include a waist bag up to 4L, sterile disconnector, collection bottle, two auxiliary bags up to 100mL each, sterile connector, and 636cm². 2The system includes cell culture flasks, culture medium bags up to 3 L, and buffer bags up to 3 L. Exemplary systems suitable for use herein are described, for example, in U.S. Patent No. 10,801,005 and U.S. Patent Application Publication No. 2017 / 0037357, which are incorporated herein by reference.

[0067] Once the NANT001 bioreactor culture of the apheresis product has been expanded, ≥1 × 10 6 When the cell density reaches ≥85% CD56-positive cells by day 20 expansion at a cell / mL density, a constant concentration of 1) N-803 (100-300 ng / mL), IL-12 (1-100 ng / mL), and IL-18 (5-250 ng / mL) or 2) N-803 (100-300 ng / mL) and IL12 / 18 superkine (5 μg / mL) is added to the culture in fresh NK growth medium (NK-GM) until the final total culture volume is approximately 650 mL. Stimulating the cells with cytokines for 14-16 hours induces the memory-like phenotype (M-CENK) of CD56-positive cells contained within the NANT001 bioreactor. This cytokine stimulation in the bioreactor is then terminated by large-volume cell harvesting of the culture using the automated transport feature of NANT001. Samples are collected for mycoplasma testing before culture harvesting.

[0068] array: IL-18 / 12 superkine protein complex SEQ ID NO: 1 (IL-18 / 12 superkine; amino acid) - Leader peptide is not included (see SEQ ID NO: 8). [ka] Sequence ID 2 (IL-18 / 12 superkine; nucleic acid) - does not include a leader sequence (see Sequence ID 9). [ka]

[0069] IL-18 / 15 / 12 Supercaine SEQ ID NO: 3 (IL15N72D; amino acid) leader peptide is not included (see SEQ ID NO: 8). NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Sequence ID 4 (IL15N72D; nucleic acid) [ka] Sequence ID 5 (IL18 / IL15Ra / IL12; amino acid) [ka] Sequence ID 6 (IL18 / IL15Ra / IL12; nucleic acid) [ka] Sequence ID 7 IL-15Rα sucrose domain amino acid sequence ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR Sequence ID 8 (IL-18 / 12 superkine and IL15N72D leader peptide sequence; amino acids) MEWSWVFLFFLSVTTGVHS Sequence ID 9 (IL-18 / 12 superkine reader sequence; nucleic acid) atggaatggagctgggtctttctcttcttcctgtcagtaaccaccggtgtccactcc

Claims

1. An isolated soluble fusion protein complex containing two soluble proteins, a. Interleukin-18 (IL-18) polypeptide domain; IL-15 receptor alpha-susu binding domain (IL-15RαSu); Immunoglobulin (Ig) crystallizable fragment (Fc) domain; and IL-12 polypeptide domain containing p40 and p35 subunits A first soluble fusion protein containing; and b. A second soluble protein containing IL-15 (IL-15); This includes, where the IL-15-RαSu domain of the first soluble fusion protein binds to the IL-15 of the second soluble protein to form the soluble fusion protein complex; and The immunoglobulin Fc domain of the first soluble protein complex is covalently bonded to the Fc domain of the second soluble protein complex by a disulfide bond. A soluble fusion protein complex.

2. The soluble fusion protein complex according to claim 1, wherein the IL-15 polypeptide domain is an IL-15 variant (IL-15N72D) containing a mutation from asparagine to aspartic acid at amino acid position 72.

3. The soluble fusion protein complex according to claim 1, wherein the IL-18 polypeptide domain is linked to the IL-15RαSu domain by a mobile polypeptide linker.

4. The soluble fusion protein complex according to claim 1, wherein the p40 and p35 subunits of the IL-12 domain are linked by a mobile polypeptide linker.

5. The soluble fusion protein complex according to claim 1, wherein the IL-12 p40 subunit is linked to the Ig Fc domain by a mobile polypeptide linker.

6. The soluble fusion protein complex according to claim 1, wherein the first soluble fusion protein comprises the amino acid sequence described in SEQ ID NO:

5.

7. An isolated soluble fusion protein complex comprising an interleukin-18 (IL-18) polypeptide domain, an Ig crystallizable fragment (Fc) domain, and an IL-12 polypeptide domain containing p40 and p35 subunits; wherein the Fc domain of the first soluble protein is covalently bonded to the Fc domain of the second soluble protein by a disulfide bond.

8. The soluble fusion protein complex according to claim 7, wherein the IL-15 polypeptide domain is an IL-15 variant (IL-15N72D) containing the N72D mutation.

9. The soluble fusion protein complex according to claim 7, wherein the IL-18 polypeptide domain is linked to the IgFc domain by a mobile polypeptide linker.

10. The soluble fusion protein complex according to claim 7, wherein the IL-12 p40 subunit is linked to the Ig Fc domain by a mobile polypeptide linker.

11. A soluble fusion protein complex according to claim 7, comprising the amino acid sequence described in SEQ ID NO:

1.

12. A method for producing memory-like cytokine-enhanced natural killer (M-CENK) cells, a. Obtaining multiple mononuclear cells and bringing the multiple mononuclear cells into contact with a corticosteroid; b. Incubating the plurality of mononuclear cells in the presence of the corticosteroid to enrich the mononuclear cells within the NK cells; c. The enriched NK cells are induced with i) the soluble fusion protein complex described in claim 1 or ii) IL-15 or a derivative thereof and the soluble fusion protein complex described in claim 7 to produce the M-CENK cells. A method that includes this.

13. The method according to claim 12, wherein the corticosteroid is hydrocortisone.

14. The method according to claim 12, wherein the IL-15 derivative is N-803.

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