Compositions and methods related to car-t cells

Abstract

A solution to the problem of inefficient immunotherapy due to reduced Natural Killer Cell cytolytic function and a lack of direct drug delivery methods for NK-based therapy is addressed by engineering a super fusion activator (SFA) that modifies NK cell activity in the tumor microenvironment (TME).

Description

COMPOSITIONS AND METHODS RELATED TO CAR-T CELLSRELATED APPLICATION

[0001] This Application claims priority to US Provisional Patent Application 63 / 674,382 filed July 23, 2024 which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] None.REFERENCE TO SEQUENCE LISTING

[0003] A sequence listing required by 37 CFR 1.821-1.825 is being submitted electronically with this application. The sequence listing is incorporated herein by reference. The sequence listing that is contained in the file named "UTSKP0570WO" which is 13 KB (as measured in Microsoft Windows®) and was created on 7 / 23 / 2025.FIELD OF THE INVENTION

[0004] Embodiments of the invention are directed generally to the field of medicine and immunotherapy, specifically to compositions and methods for enhancing immune responses against cancer.BACKGROUND

[0005] Adoptive cellular immunotherapy (ACI) is a promising approach in cancer treatment that harnesses the power of the immune system to combat cancer cells. Unlike traditional cancer treatments such as chemotherapy and radiation therapy, which directly target cancer cells, ACI involves modifying or enhancing a patient’s own immune cells to recognize and attack cancer cells more effectively with greater precision and potentially fewer off-target effects.

[0006] The process typically begins by isolating immune cells, such as T cells or Natural Killer (NK) cells, from the patient’s blood. These cells are then modified in the laboratory to specifically target cancer cells. This can be achieved through various methods, including genetic engineering to express chimeric antigen receptors (CARs) on T cells or by boosting the activity of NK cells, natural killer cells, or tumor-infiltrating lymphocytes through the expression of activating ligands or cytokines.

[0007] Once modified, the immune cells are expanded in number and infused back into the patient. Upon encountering cancer cells, these engineered cells recognize specific markers on the cancer cell surface, such as tumor-associated antigens, and initiate a targeted immune response, leading to tumor destruction.

[0008] ACI has shown remarkable success in treating certain hematologic malignancies, such as leukemia and lymphoma, resulting in durable remissions and, in some cases, cures. However, its efficacy in solid tumors remains limited due to challenges such as the immunosuppressive tumor immune microstructure (TIME), which inhibits immune cell infiltration and function.

[0009] NK cells play a critical role in innate immune defense against malignant cells, making them promising candidates for ACI. However, challenges such as difficulties in ex vivo cell expansion, variability in NK cell activity among patients, and suppression of NK cell cytotoxicity in the TIME due to mechanisms like PD-L1 expression or TGF-[3 signaling limit their therapeutic potential.

[0010] Despite its promise, ACI faces challenges including high costs, logistical complexities, and potential adverse side effects such as cytokine release syndrome and neurotoxicity. Moreover, the lack of targeted delivery methods and robust NK cell activators hinders the efficacy of NK-based therapies, particularly for solid tumors. There remains a critical need for novel compositions and methods to enhance NK cell function and overcome immunosuppressive barriers in the TIME to improve immunotherapy outcomes.SUMMARY

[0011] Embodiments described herein provide a solution to the problem of inefficient immunotherapy due to reduced Natural Killer (NK) cell cytolytic function and a lack of direct drug delivery methods for NK-based therapy. Certain embodiments address the challenge of suppressed NK cell activity in the tumor immune microenvironment (TIME), particularly in solid tumors, by providing compositions and methods to enhance NK cell cytotoxicity and target tumor cells effectively. The issue of dysregulated NK cell activity in the tumor microenvironment (TME) or tumor immune microenvironment (TIME) is addressed by providing improved modalities and optimize NK cell killing function. In addition, a direct delivery method, such as intratumoral injection or nanoparticle-based targeting, prevents randomcirculation in the bloodstream, reducing side effects. Embodiments are directed to an engineered super fusion activator (SFA) RSP03-MICA that activates NK cells in the TIME and suppresses tumor growth in both hematologic malignancies and solid tumors, and methods using the same.

[0012] Certain embodiments are directed to super fusion activators (SFA) comprising a polypeptide having an amino terminal R-spondin protein segment (e.g., Rspo3) polypeptide segment operatively coupled a carboxy terminal MHC class I polypeptide-related sequence (MICA) polypeptide segment. The amino terminal polypeptide can be linked to the carboxy terminal polypeptide by a peptide linker. In certain embodiments a representative super fusion activator has the amino acid sequence of SEQ ID NO:2. The super fusion activator can be encoded by the nucleic acid sequence of SEQ ID NO: 1. In certain embodiments the amino terminal Rspo3 polypeptide segment has the amino acid sequence of SEQ ID NO:3. In certain embodiments the carboxy terminal MICA polypeptide segment is has the amino acid sequence of SEQ ID NO:4.

[0013] Other embodiments are directed to a host cell expressing an SFA described herein. In certain embodiments, the host cell is a T cell. The T cell can be a chimeric antigen receptor (CAR-T) cell expressing a chimeric antigen receptor (CAR).

[0014] Certain embodiments are directed to a chimeric antigen receptor T (CAR-T) cell expressing a chimeric antigen receptor and an SFA activator described herein. In certain embodiments, the chimeric antigen receptor targets GPC3 (for example see SEQ ID NO:6 and SEQ ID NO: 7).

[0015] Other embodiments are directed to a polynucleotide encoding an SFA described herein.

[0016] Other embodiments are directed to methods of treating cancer comprising administering an SFA or an SFA-expressing host cell as described herein to a patient having a tumor.

[0017] Certain embodiments include compositions comprising an SFA combined with additional polypeptide segments, such as IL-15, IL-21, or anti-PD-Ll, to further enhance NK cell activation and overcome immunosuppression in the TIME, particularly for solid tumors. These compositions may also include targeting moieties, such as anti-EGFR or anti-HER2 antibody fragments, to improve tumor-specific delivery.

[0018] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0019] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0020] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0021] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0022] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and / or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps), but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and / or method.

[0023] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0024] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and / or method that includes materials,steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel character! stic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.DEFINITIONS

[0026] As used herein, certain terms are defined to provide clarity and consistency in the interpretation of this specification and the claims. These definitions are intended to supplement the ordinary and customary meanings as understood by those skilled in the art.

[0027] The term “super fusion activator” (SFA) refers to a recombinant polypeptide comprising an amino terminal Rspo3 polypeptide segment operatively coupled to a carboxy terminal MICA polypeptide segment, designed to enhance Natural Killer (NK) cell activation and cytotoxicity within the tumor immune microenvironment (TIME). The SFA may include additional polypeptide segments, such as IL-15, IL-21, or anti-PD-Ll, and may be linked by a peptide linker, such as SEQ ID NO: 5.

[0028] The term “tumor immune microenvironment” (TIME) refers to the cellular and molecular environment surrounding a tumor, including immune cells, stromal cells, and signaling molecules, which can suppress immune responses and promote tumor growth. The TIME is particularly relevant to solid tumors, where immunosuppressive mechanisms, such as PD-L1 expression or TGF- signaling, hinder immune cell function.

[0029] The term “Natural Killer (NK) cell” refers to a type of innate immune cell capable of recognizing and killing malignant cells without prior sensitization. NK cells are characterized by their expression of markers such as NK1.1 and DX5 and their ability to produce cytolytic granules (e.g., granzyme B, perforin) and cytokines (e.g., IFN-y).

[0030] The term “chimeric antigen receptor T (CAR-T) cell” refers to a T cell genetically engineered to express a chimeric antigen receptor (CAR), which enables targeted recognition oftumor-associated antigens, such as glypican-3 (GPC3), and may co-express an SFA to enhance anti-tumor activity.

[0031] The term “solid tumor” refers to a malignant neoplasm characterized by a mass of abnormal tissue, including but not limited to carcinomas, sarcomas, and melanomas, such as hepatocellular carcinoma, pancreatic cancer, or lung cancer, which typically lacks a fluid-filled cavity and poses immunosuppressive barriers in the TIME.

[0032] The term “tumor-associated antigen” refers to a molecule, such as a protein or glycoprotein, expressed at higher levels on tumor cells than on normal cells, making it a suitable target for immunotherapy. Examples include, but are not limited to, glypican-3 (GPC3), epidermal growth factor receptor (EGFR), and human epidermal growth factor receptor 2 (HER2).

[0033] The term “targeting moiety” refers to a molecule or fragment thereof, such as an antibody, antibody fragment, or peptide, that specifically binds to a tumor-associated antigen to direct therapeutic agents, such as SFAs or SFA-expressing cells, to the tumor site, enhancing specificity and efficacy within the TIME.

[0034] The term “intratumoral injection” refers to the direct administration of a therapeutic agent, such as an SFA polypeptide, SFA-expressing host cell, or delivery vehicle (e.g., nanoparticle or viral vector), into the tumor mass to achieve high local concentrations within the TIME while minimizing systemic exposure.

[0035] The term “immune checkpoint inhibitor” refers to a therapeutic agent, typically an antibody or antibody fragment, that blocks inhibitory pathways in the immune system, such as programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), or cytotoxic T- lymphocyte-associated protein 4 (CTLA-4), to enhance immune cell activity against tumors.

[0036] The term “nanoparticle-based carrier” refers to a nanoscale delivery system, typically ranging from 1 to 1000 nanometers in size, designed to encapsulate or conjugate therapeutic agents, such as SFA polypeptides or polynucleotides, for targeted delivery to tumor cells, often functionalized with targeting moieties to improve tumor penetration.

[0037] The term “viral vector” refers to a genetically engineered virus, such as an adeno- associated virus (AAV) or lentivirus, used to deliver a nucleic acid sequence, such as an SFA- encoding polynucleotide, into target cells, often modified to express targeting ligands for tumor specificity.

[0038] The term “polypeptide” refers to a chain of amino acids joined by peptide bonds, which may be modified by natural or chemical processes, including but not limited to acetylation, glycosylation, or phosphorylation, and may include synthetic or modified amino acids.

[0039] The term “polynucleotide” refers to a polymeric form of nucleotides of any length, including DNA or RNA, which may be single-stranded or double-stranded and may include modified nucleotides or non-nucleotide components.

[0040] The term “host cell” refers to a prokaryotic or eukaryotic cell, such as a T cell or NK cell, that has been genetically modified to express a recombinant nucleic acid, such as an SFA- encoding polynucleotide or a CAR, and includes the progeny of such cells.DESCRIPTION OF THE DRAWINGS

[0041] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0042] FIG. 1. Schematic diagram of a super fusion activator (SFA) RSPO3-MICA, illustrating its structure and mechanism of enhancing Natural Killer (NK) cell function in the tumor immune microenvironment (TIME).

[0043] FIG. 2. Diagram summarizing experimental methods used to evaluate NK cell function in response to SFA expression, including assays for cell maturation and cytotoxicity.

[0044] FIG. 3. Flow cytometry analysis showing the effect of SFA exogenous expression in the TIME on NK cell maturation, with SFA exogenous expression in the TIME increases mature NK cells assessed by CD27 and CD1 lb surface markers in tumor-infiltrating NK cells.

[0045] FIG. 4. Bar charts quantifying the percentage of cytolytic granules (granzyme B, perforin) and cytokine IFN-y in tumor-infiltrating NK cells following SFA exogenous expression in the TIME.

[0046] FIG. 5. Graphs and images showing tumor growth curves and representative tumor samples in immunocompetent (C57BL / 6) and immunodeficient (NRG) mice inoculated with B16F10 cells expressing empty vector, mbMICA, mbRspo3, or SFA, demonstrating that SFA exogenous expression in the TIME reduces tumor growth in solid tumor models.

[0047] FIG. 6. Line graph summarizing the percentage of NK cell cytotoxicity against YAC- 1 target cells at various effector-to-target ratios, illustrating that SFA exogenous enhances NK cytoxicity in the TIME.DESCRIPTION

[0048] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment, and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0049] Immunotherapy efficiently utilizes a patient’s immune system to fight cancer with fewer side effects, particularly for solid tumors and hematologic malignances. Clinically, immunotherapy is the last line of therapy for cancer patients whose immune systems have already been severely damaged. In addition, the hostile tumor immune microenvironment (TIME) continues to impede immune cell activity and reduce therapeutic efficacy by mechanisms such as PD-L1 expression and TGF 0 signaling. Natural Killer (NK) cells are the front-line responders in immunosurveillance, which can kill cancer cells and interact with other immune cells. It is well-documented that NK cell functions are highly suppressed in the TIME, leading to a patient’s poor response to conventional T-cell therapy. To address this, the inventors have developed a novel super fusion activator (SFA) RSPO3-MICA that enhances NK cell maturation and cytotoxicity in the TIME, as demonstrated in experimental models (see Examples,

[0105] —

[0114] ). One example of a super fusion activator is encoded by the SFA cDNA having the following nucleic acid sequence: ATGCACTTGCGACTGATTTCTTGTTTTTTTATCATTTTGAACTTTATGGAATACATTG GCAGCCAAAACGCCTCCCGAGGAAGGCGCCAGCGAAGAATGCATCCTAATGTCAGT CAAGGCTGCCAAGGAGGCTGTGCAACGTGTTCAGATTACAATGGCTGTTTGTCATGT AAGCCCAGACTGTTTTTTGTTCTGGAAAGGATTGGCATGAAGCAGATAGGAGTGTGT CTCTCTTCGTGTCCAAGTGGATATTACGGAACTCGATATCCAGATATAAATAAATGTACAAAATGCAAAGTTGACTGTGATACCTGTTTCAACAAAAATTTCTGCACAAAGTGTAAAAGTGGATTTTACTTACACCTTGGAAAGTGCCTTGACAGTTGCCCAGAAGGGTTAGAAGCCAACAATCATACTATGGAATGTGTCAGTATTGTACACTGTGAGGCCAGTGAATGGAGTCCATGGAGTCCATGTATGAAGAAAGGAAAAACATGTGGCTTCAAAAGGGGGACTGAAACACGGGTCCGAGATATACTACAGCATCCTTCAGCCAAGGGTAACCTGTGCCCCCCAACCAGCGAGACAAGAACTTGTATAGTACAAAGAAAGAAGTGTTCAAAGGGAGAGCGAGGAAAAAAGGGAAGAGAGAGAAAACGAAAAAAACTGAATAAAGAAGAAAGAAAGGAAACAAGCTCCTCCTCTGACAGCAAAGGTTTGGAGTCCAGCATTGAGACCCCAGACCAGCAGGAAAACAAAGAGAGGCAGCAGCAGCAGAAGAGAAGAGCCCGAGACAAGCAACAGAAATCGGTATCAGTCAGCACTGTACACTCTGGTGGCGGAGGCTCGGGCGGAGGTGGGTCGGGTGGCGGCGGATCAGAGCCCCACAGTCTTCGTTATAACCTCACGGTGCTGTCCTGGGATGGATCTGTGCAGTCAGGGTTTCTCACTGAGGTACATCTGGATGGTCAGCCCTTCCTGCGCTGTGACAGGCAGAAATGCAGGGCAAAGCCCCAGGGACAGTGGGCAGAAGATGTCCTGGGAAATAAGACATGGGACAGAGAGACCAGAGACTTGACAGGGAACGGAAAGGACCTCAGGATGACCCTGGCTCATATCAAGGACCAGAAAGAAGGCTTGCATTCCCTCCAGGAGATTAGGGTCTGTGAGATCCATGAAGACAACAGCACCAGGAGCTCCCAGCATTTCTACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTAAGGAATGGACAATGCCCCAGTCCTCCAGAGCTCAGACCTTGGCCATGAACGTCAGGAATTTCTTGAAGGAAGATGCCATGAAGACCAAGACACACTATCACGCTATGCATGCAGACTGCCTGCAGGAACTACGGCGATATCTAAAATCCGGCGTAGTCCTGAGGAGAACAGTGCCCCCCATGGTGAATGTCACCCGCAGCGAGGCCTCAGAGGGCAACATTACCGTGACATGCAGGGCTTCTGGCTTCTATCCCTGGAATATCACACTGAGCTGGCGTCAGGATGGGGTATCTTTGAGCCACGACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGGATGGAACCTTCCAGAAGTGGGTGGCCACCAGGATTTGCCAAGGAGAGGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACAGCACTCACCCTGTGCCCTCTGGGAAAGTGCTGGTGCTTCAGAGTCATTGGCAGACATTCCATGTTTCTGCTGTTGCTGCTGCTGCTATTTTTGTTATTATTATTTTCTATGTCCGTTGTTGTAAGAAGAAAACATCAGCTGCAGAGGGTCCAGAGCTCGTGAGCCTGCAGGTCCTGGATCAACACCCAGTTGGGACGAGTGACCACAGGGATGCCACACAGCTCGGATTTCAGCCTCTGATGTCAGATCTTGGGTCCACTGGCTCCACTGAGGGCGCCTCTGGTGGCGGAGGCTCG (SEQ ID NO: 1). The SFA nucleic acid sequence above encodes a protein having thefollowing amino acid sequence:MHLRLISCFFIILNFMEYIGSQNASRGRRQRRMHPNVSQGCQGGCATCSDYNGCLSCKP RLFFVLERIGMKQIGVCLSSCPSGYYGTRYPDINKCTKCKVDCDTCFNKNFCTKCKSGF YLHLGKCLDSCPEGLEANNHTMECVSIVHCEASEWSPWSPCMKKGKTCGFKRGTETRV RDILQHPS AKGNLCPPT SETRTCIVQRKKC SKGERGKKGRERKRKKLNKEERKETS S S SD SKGLESSIETPDQQENKERQQQQKRRARDKQQKSVSVSTVHSGGGGSGGGGSGGGGSE PHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKT WDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELF LSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKS GVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGD VLPDGDGTFQKWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVS AVAAAAIFVIIIFYVRCCKKKTSAAEGPELVSLQVLDQHPVGTSDHRDATQLGFQPLMSD LGSTGSTEGASGGGGS (SEQ ID NO:2). The R-spondin-3 (Rspo-3) segment includes amino acids 1 to 277 of SEQ ID NO:2(MHLRLISCFFIILNFMEYIGSQNASRGRRQRRMHPNVSQGCQGGCATCSDYNGCLSCKP RLFFVLERIGMKQIGVCLSSCPSGYYGTRYPDINKCTKCKVDCDTCFNKNFCTKCKSGF YLHLGKCLDSCPEGLEANNHTMECVSIVHCEASEWSPWSPCMKKGKTCGFKRGTETRV RDILQHPS AKGNLCPPT SETRTCIVQRKKC SKGERGKKGRERKRKKLNKEERKETS S S SD SKGLESSIETPDQQENKERQQQQKRRARDKQQKSVSVSTVH, SEQ ID NO:3). Amino acids 295 to 647 of SEQ ID NO:2 (EPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNK TWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGEL FLSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLK SGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWG DVLPDGDGTFQKWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFH VS AVAAAAIF VIIIF YVRC CKKKT S AAEGPELVSLQ VLDQHP VGT SDHRD ATQLGFQPLM SDLGSTGSTEGA, SEQ ID NO:4) is the MHC class I polypeptide-related sequence A isoform 1 (MICA) segment of one embodiment of a SFA. The segments can be linked using a linker, for example a peptide linker having the amino acid sequence SGGGGSGGGGSGGGGS (SEQ ID NO: 5) or a functionally similar peptide or chemical linker.

[0050] The disclosure provides isolated or encoded activator polypeptides. The polypeptide containing an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity(optionally at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, to 659 consecutive amino acids of SEQ ID NO:2. The Rspo-3 segment can comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (optionally at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to at least 50, 100, 150, 200, 250, to 277 consecutive amino acids of SEQ ID NO:3. The MICA segment can comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity(optionally at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to at least 50, 100, 150, 200, 250, 300, to 352 consecutive amino acids of SEQ ID NO:4.

[0051] The phrases “nucleic acid” or “nucleic acid sequence” as used herein refer to an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of synthetic origin which may be single-stranded or double-stranded.

[0052] A “coding sequence of’ or a “nucleotide sequence encoding” a particular polypeptide or protein, is a nucleic acid sequence which is transcribed and translated into a polypeptide or protein when placed under the control of appropriate regulatory sequences in the appropriate environment.

[0053] The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).

[0054] “Amino acid” or “amino acid sequence” as used herein refer to an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to synthetic molecules.

[0055] The term “polypeptide” as used herein refers to amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques ,including but not limited to acetylation, glycosylation, phosphorylation, or pegylation, which are well known in the art.

[0056] As used herein, the term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and / or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

[0057] As used herein, the term “recombinant nucleic acid” means that the nucleic acid is adjacent to a heterologous nucleic acid to which it is not adjacent in its natural environment. Backbone molecules according to the invention include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest.

[0058] “Recombinant polypeptides” refer to polypeptides or proteins produced by recombinant DNA techniques; i.e., produced from cells or systems transformed by an exogenous DNA construct encoding the desired polypeptide or protein.

[0059] A promoter sequence is a nucleic acid sequence “operably linked to” a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA.

[0060] “Plasmids” are designated by a lower case “p” preceded and / or followed by capital letters and / or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.

[0061] The phrase “substantially identical” in the context of two nucleic acids or polypeptides refers to two or more sequences that have at least 80%, 85%, 90%, 95%, or 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the known sequence comparison algorithms or by visual inspection. Such sequences may retain the essential functional properties of the reference sequence, such as NK cell activation for polypeptides or encoding capability for nucleic acids.

[0062] Additionally a “substantially identical” amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site or functional portion (e.g., receptor binding portion) of the molecule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine). One or more amino acids can be deleted or inserted resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal amino acids that are not required for biological activity can be removed. Modified polypeptide sequences of the invention can be assayed for biological activity by any number of methods.

[0063] “Fragments” or “segments” as used herein are a portion of a naturally occurring protein which can exist in at least two different conformations. Fragments can have the same or substantially the same amino acid sequence as the naturally occurring protein. “Substantially the same” means that an amino acid sequence is largely, but not entirely, the same, but retains at least one functional activity of the sequence to which it is related. In general, two amino acid sequences are “substantially the same” or “substantially homologous” if they are at least about 85% to 99% identical. Fragments which have different three-dimensional structures as the naturally occurring protein are also included. An example is a “pro-form” molecule, such as a low activity proprotein that can be modified by cleavage to produce a mature protein with higher activity.

[0064] The term “variant” refers to polynucleotides or polypeptides of the invention modified at one or more base pairs, codons, introns, exons, or amino acid residues (respectively) yet still retain a biological activity. Variants can be produced by any number of means included methods such as, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM and any combination thereof.

[0065] A further object of the present invention relates to a nucleic acid sequence encoding for a polypeptide or a fusion protein according to the invention. As used herein, a sequence “encoding” an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon. These nucleic acid sequences can be obtained by conventional methods well known to those skilled in the art. Typically, said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0066] A further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule encoding for a polypeptide or a fusion protein of the invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted. These recombinant vectors may, for example, be cloning vectors, or expression vectors.

[0067] As used herein, the terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Any expression vector for an animal cell can be used. Examples of suitable vectors include pAGE107 (Miyaji et al., 1990), pAGE103 (Mizukami and Itoh, 1987), pHSG274 (Brady et al., 1984), pKCR (O'Hare et al., 1981), pSGl beta d2-4 (Miyaji et al., 1990) and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art.

[0068] A further aspect of the invention relates to a host cell comprising a nucleic acid molecule encoding for a polypeptide or a fusion protein according to the invention or a vector according to the invention. In particular, a subject of the present invention is a prokaryotic or eukaryotic host cell genetically transformed with at least one nucleic acid molecule or vector according to the invention.

[0069] The term “transformation” means the introduction of a “foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been “transformed”.

[0070] Administration of a therapeutic T cell refers to a therapy involving a process comprising (i) extracting T cells from a patient's bloodstream through a process called leukapheresis; (ii) genetically modifying the extracted cells to express chimeric antigen receptors (CARs) and / or other recombinant proteins (e.g., SFA) on their surface, which enables them to recognize and target specific proteins found on cancer cells or in tumors; (iii) amplifying the engineered CAR-T cells or cells; and (iv) infusing or injecting the amplified cells back into the patient's bloodstream or into a tumor. Once inside the body, these modified cells seek out and destroy cancer cells, effectively reprogramming the immune system to recognize and attack the tumor.

[0071] A further aspect of the invention relates to a method for producing a polypeptide or a fusion protein of the invention comprising the step consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said polypeptide or fusion protein; and (ii) recovering the expressed polypeptide or fusion protein or using the cell expressing the polypeptide or fusion protein for therapeutic purposes.I. Polypeptide Compositions

[0072] Certain embodiments are directed to a fusion polypeptide that comprises a first peptide or polypeptide (e.g., Rspo-3 segment) coupled or fused to a second peptide or polypeptide (e.g., MICA segment). The first polypeptide can be linked at the N- or C-terminus to the second polypeptide. In other embodiments, the fusion polypeptide comprises a linker interposed between the first polypeptide and the second polypeptide. Furthermore, the fusion polypeptides set forth herein may comprise a third amino acid sequence of any number of additional amino acid residues at either the N-terminus or C-terminus of the fusion polypeptide amino acid sequence.

[0073] The fusion polypeptide may include an epitope or other tag to facilitate identification, targeting, and / or purification of the polypeptide. The use of 6><His and GST (glutathione Stransferase) as tags is well known. Inclusion of a cleavage site at or near the epitope or tag junction will facilitate removal of the extraneous third polypeptide after purification or other manipulation. Other amino acid sequences that may be included in the fusion polypeptide include functional domains, such as active sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals or transmembrane regions. The fusion polypeptide may further include one or more additional tissue-targeting moi eties.

[0074] The fusion polypeptides may possess deletions and / or substitutions of amino acids relative to the native sequences. Sequences with amino acid substitutions are contemplated, as are sequences with a deletion, and sequences with a deletion and a substitution. In some embodiments, these polypeptides may further include insertions or added amino acids.

[0075] Substitutional or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly to increase its efficacy or specificity. Substitutions of this kind may or may not be conservative substitutions. Conservative substitution is when one amino acid is replaced with one of similar shape and charge. Conservative substitutions include, for example, alanine to serine, arginine to lysine, or aspartate to glutamate. Changes other than those discussed immediately above are generally considered not to be conservative (non-conservative) substitutions. It is specifically contemplated that one or more of the conservative substitutions above may be included as embodiments. In some embodiments, such substitutions are specifically excluded. Furthermore, in additional embodiments, non-conservative substitutions can be employed in variants.

[0076] The following is a discussion based upon changing of the amino acids of a polypeptide to create a library of molecules or a second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a polypeptide without appreciable loss of function, such as ability to interact with a target peptide sequence. Since it is the interactive capacity and nature of a polypeptide that defines that polypeptide's functional activity, certain amino acid substitutions can be made in a polypeptide sequence and nevertheless produce a polypeptide with like properties.

[0077] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0078] It also is understood that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (—0.4); proline (-0.5+1); alanine (_0.5); histidine (-0.5); cysteine (-1.0); methionine (—1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (—2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0079] It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0080] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. However, in some aspects a non-conservative substitution is contemplated. In certain aspects a random substitution is also contemplated. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0081] Amino acid sequence variants of other polypeptides of these compositions can be substitutional, insertional, or deletion variants. A modification in a polypeptide may affect 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more non-contiguous or contiguous amino acids of a fusion polypeptide, as compared to the original fusion polypeptide.

[0082] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' nucleic acid sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0083] The polypeptides described herein may be fused, conjugated, or operatively linked to a label. As used herein, the term “label” intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein to generate a “labeled” composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and / or other property. In luminescence or fluoresecence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0084] II. Polynucleotides

[0085] Aspects of the disclosure relate to polypeptides and polynucleotides encoding such polypeptides (e.g., super fusion activator). The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three- dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single- stranded molecules. Unless otherwise specified or required, any embodiment of the disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single- stranded forms known or predicted to make up the double-stranded form.

[0086] The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3'-TCGA-5' is 100% complementary to the nucleotide sequence 5'-AGCT- 3'. Further, the nucleotide sequence 3'-TCGA- is 100% complementary to a region of the nucleotide sequence 5'-TTAGCTGG-3'. It will be recognized by one of skill in the art that two complementary nucleotide sequences include a sense strand and an antisense strand.

[0087] Polypeptides may be encoded by a nucleic acid molecule in the composition. In certain embodiments, the nucleic acid molecule can be in the form of a nucleic acid vector. The term “vector” is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated andexpressed. A nucleic acid sequence can be “heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced or to the nucleic acid in which is incorporated, which includes a sequence homologous to a sequence in the cell or nucleic acid but in a position within the host cell or nucleic acid where it is ordinarily not found. Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for example Sambrook et al., 2001; Ausubel et al., 1996, both incorporated herein by reference). Vectors may be used in a host cell to produce an antibody.

[0088] The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed or stably integrate into a host cell's genome and subsequently be transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described herein. It is contemplated that expression vectors that express a marker may be useful in the invention. In other embodiments, the marker is encoded on an mRNA and not in an expression vector.

[0089] A “promoter” is a control sequence. The promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and / or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0090] The particular promoter that is employed to control the expression of a peptide or protein encoding polynucleotide is not believed to be critical, so long as it is capable ofexpressing the polynucleotide in a targeted cell, preferably a bacterial cell. Where a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a bacterial, human or viral promoter. In some embodiments, the host cell is an eukaryotic cell. In some embodiments, using eukaryotic cells is beneficial, as it provides for secondary modifications that may not be present in certain prokaryotic systems.

[0091] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.

[0092] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and / or acceptor splicing sites to ensure proper processing of the transcript for protein expression. (See Chandler et al., 1997, incorporated herein by reference.)

[0093] The vectors or constructs will generally comprise at least one termination signal. A “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels. In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (poly A) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message.

[0094] In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper poly adenylation of the transcript.

[0095] In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed “ori”), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

[0096] Some vectors may employ control sequences that allow it to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0097] The polynucleotides and polypeptides of the disclosure may be transfected of transformed into host cells or expressed in host cells. As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include both freshly isolated cells and ex vivo cultured, activated or expanded cells. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. Common host cells include bacteria (such as E. coli, B. subtilis, S. viofoceoruber), yeast (such as S. cerevisiae, P. pastoris), fungi (such as A. oryzae) or eukaryotic cells.III. Pharmaceutical Compositions

[0098] In light of the current specification, the determination of an appropriate treatment regimen (e.g., dosage, frequency of administration, systemic vs. local, etc.) is within the skill of the art. For administration, the components described herein will be formulated in a unit dosage form (solution, suspension, emulsion, etc.) in association with a pharmaceutically acceptablecarrier. Such vehicles are usually nontoxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and Hank's solution. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. A preferred vehicle is 5% (w / w) human albumin in saline. The vehicle may contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0099] The therapeutic compositions described herein, as well as their biological equivalents, can be administered independently or in combination by any suitable route. Examples of parenteral administration include intravenous, intraarterial, intramuscular, intraperitoneal, and the like. The routes of administration described herein are merely an example and in no way limiting.

[0100] The dose of the therapeutic compositions administered to an animal, particularly in a human, in accordance with embodiments of the invention, should be sufficient to result in a desired response in the subject over a reasonable time frame. It is known that the dosage of therapeutic compositions depends upon a variety of factors, including the strength of the particular therapeutic composition employed, the age, species, condition or disease state, and the body weight of the animal.

[0101] Moreover, dose and dosage regimen, will depend mainly on the type of biological damage to the host, the type of subject, the history of the subject, and the type of therapeutic composition being administered. The size of the dose will be determined by the route, timing and frequency of administration as well as the existence, nature and extent of any adverse side effects that might accompany the administration of a particular therapeutic composition and the desired physiological effect. It is also known that various conditions or disease states, in particular, chronic conditions or disease states, may require prolonged treatment involving multiple administrations.

[0102] The therapeutic compositions for use in embodiments of the invention generally include carriers. These carriers may be any of those conventionally used and are limited only by the route of administration and other considerations. The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers, or diluents, are well known and readily available. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert with respect to the therapeutic composition and one that has no detrimental side effects or toxicity under the conditions of use.

[0103] The choice of excipient will be determined, in part, by the particular therapeutic composition, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the pharmaceutical composition used in the embodiments of the invention. For example, the non-limiting formulations can be injectable formulations such as, but not limited to, those for intravenous, subcutaneous, intramuscular, intraperitoneal injection, and the like.A. Combination of SFA Components

[0104] Certain embodiments of the invention are directed to compositions comprising combinations of super fusion activator (SFA) components and other therapeutics to enhance the treatment of solid tumors and hematologic malignancies. In addition to the RSPO3-MICA fusion polypeptide, SFAs may include other polypeptide segments such as IL-15, IL-21, or anti-PD-Ll to further potentiate NK cell activation and overcome immunosuppression in the tumor immune microenvironment (TIME). For example, an SFA may comprise an IL-15 polypeptide segment to enhance NK cell proliferation and survival by upregulating NKG2D receptor expression, operatively linked to the RSPO3-MICA fusion via a peptide linker (e.g., SEQ ID NO:5). Alternatively, an anti-PD-Ll segment can be included to block inhibitory checkpoint signals, such as PD-1 / PD-L1 interactions, thereby enhancing NK cell cytotoxicity in the TIME. These combination SFAs can be expressed in host cells, such as NK cells or CAR-T cells engineered using viral vectors or CRISPR-based methods, to synergistically improve anti-tumor activity against solid tumors, as demonstrated in experimental models (see Examples). In certain embodiments, the SFA combination includes a targeting moiety, such as an antibody fragment (e.g., single-chain variable fragment (scFv) or Fab) specific to tumor-associated antigens (e.g., EGFR , HER2, or GPC3), to enhance tumor-specific delivery and retention within solid tumors by improving penetration into the tumor mass.

[0105] In another embodiment, the SFA components are co-administered as separate polypeptides or expressed from distinct polynucleotides within the same host cell to achieve a combinatorial effect. For instance, a host cell may express an RSPO3-MICA SFA alongside a separate IL- 15 polypeptide to promote both NK cell maturation and proliferation, or an anti-PD- Ll polypeptide to block inhibitory signals and enhance cytotoxicity. These combination strategies are designed to address the immunosuppressive barriers in solid tumors, such as poor immune cell infiltration and downregulation of NK cell activity, by simultaneously targetingmultiple pathways in the TIME , as supported by experimental data showing enhanced NK cell function and tumor suppression (see Examples).B. Methods for Treating Solid Tumors

[0106] Certain embodiments are directed to methods for treating solid tumors by administering SFAs or SFA-expressing cells to a patient in a manner that optimizes delivery to the tumor site. One method involves intratumoral injection of SFA polypeptides or SFA- expressing cells (e.g., CAR-T or NK cells) to ensure high local concentrations within the TME, minimizing systemic exposure and reducing side effects such as cytokine release syndrome. In certain embodiments, the SFA is delivered using nanoparticle-based carriers or viral vectors (e.g., adeno-associated virus or lentivirus) engineered to target tumor-specific markers, enhancing penetration into solid tumor masses. For example, nanoparticles conjugated with anti- GPC3 antibodies can deliver SFA-encoding polynucleotides to hepatocellular carcinoma cells, promoting localized NK cell activation.

[0107] Another embodiment includes a combination therapy where SFA administration is paired with immune checkpoint inhibitors (e.g., anti-PD-1 or anti-CTLA-4 antibodies) to further enhance the anti-tumor immune response. In such methods, the SFA activates NK cells within the TME, while the checkpoint inhibitor mitigates T-cell exhaustion, resulting in a synergistic effect against solid tumors. In certain embodiments, the method involves sequential administration, where SFA-expressing CAR-T cells targeting GPC3 are infused first, followed by systemic administration of an anti-PD-Ll antibody to sustain immune activation. These methods are particularly effective for solid tumors such as hepatocellular carcinoma, pancreatic cancer, or lung cancer, where the TME poses significant barriers to immune cell function.

[0108] The term “solid tumor” refers to a malignant neoplasm characterized by a mass of abnormal tissue, including but not limited to carcinomas, sarcomas, and melanomas, that typically lacks a fluid-filled cavity. Examples include hepatocellular carcinoma, pancreatic cancer, lung cancer, breast cancer, and colorectal cancer, which are characterized by a complex tumor microenvironment that suppresses immune cell function.

[0109] The term “nanoparticle-based carrier” refers to a nanoscale delivery system, typically ranging from 1 to 1000 nanometers in size, designed to encapsulate or conjugate therapeutic agents, such as SFA polypeptides or polynucleotides, for targeted delivery to tumor cells. Thesecarriers may include liposomes, polymeric nanoparticles, or metallic nanoparticles, and can be functionalized with targeting moieties to improve tumor penetration and retention.

[0110] The term “viral vector” as used herein refers to a genetically engineered virus, such as an adeno-associated virus (AAV) or lentivirus, used to deliver a nucleic acid sequence, such as an SFA-encoding polynucleotide, into target cells. Viral vectors may be modified to express targeting ligands or antibodies to enhance specificity for tumor cells in the treatment of solid tumors.

[0111] The term “intratumoral injection” refers to the direct administration of a therapeutic agent, such as an SFA polypeptide, SFA-expressing host cell, or delivery vehicle (e.g., nanoparticle or viral vector), into the tumor mass. This method aims to achieve high local concentrations of the therapeutic agent within the tumor microenvironment while minimizing systemic exposure and associated side effects.

[0112] The term “immune checkpoint inhibitor” refers to a therapeutic agent, typically an antibody or antibody fragment, that blocks inhibitory pathways in the immune system, such as programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), or cytotoxic T- lymphocyte-associated protein 4 (CTLA-4), to enhance immune cell activity against tumors. These inhibitors are used in combination with SFAs to augment anti-tumor responses in solid tumors.IV. Kits

[0113] In another aspect, the present invention provides kits for immunotherapy, which kits are used to administer a composition (e.g., SFA expression vector) or therapy (e.g., SFA expressing cell) described herein. In one embodiment, the kit comprises reagents and composition(s)(e.g., SFA expression vectors) for making and administering the immunotherapy described herein. In a further embodiment, such a kit can comprise instructions for suitable operational parameters in the form of a label or separate insert. In yet another embodiment, the kit can comprise one or more containers with reagents or components to be used in preparation or administration of the immunotherapy described.V. Examples

[0114] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMPLE 1BOOSTING NATURAL KILLER CELLS ANTITUMOR IMMUNITY IN THE TUMOR I UNE MICROENVIRONMENT

[0115] Immunotherapy efficiently utilizes patients’ immune systems to fight cancer with fewer side effects (Myers and Miller, Nat Rev Clin Oncol., 2021, 18:85-100). Clinically, immunotherapy is the last line of therapy for cancer patients whose immune systems have already been severely damaged at this point. In addition, the hostile tumor immune microenvironment (TIME) continues to impede immune cell activity and reduce therapeutic efficacy (Dean et al., Nat Comm., 2024, 15:683). Natural Killer (NK) cells are the front-line responders in immunosurveillance, which can kill cancer cells and interact with other immune cells (Balatsoukas et al., Trends Mol Med, 2022, 28(3): 194-209). It is well-documented that NK cell functions are highly suppressed in the TIME, leading to patients' poor responses to conventional T-cell therapy (Fang et al. Front Med, 2018, 12:440-450). A novel and robust NK activator strategy is needed. We hypothesize that the engineered super fusion activator (SFA) RSPO3-MICA activates NK cells in the TIME and suppresses tumor growth.

[0116] NK terminal maturation - Matured NK cells have higher cytotoxicity compared to immature NK cells. (Bi and Wang, Front Immunol, 2020, 11 : 1945) The membrane-bound protein Rspo3 regulates NK cells’ maturation, reduces tumor progression, & correlates with better prognosis in multiple cancers. (Tang et al., Cancer Discov, 2021, 11(12):3142-3157)

[0117] NK cytotoxicity activation - mbRspo3 was fused with an NK-activating ligand MHC class I chain-related protein A (MICA). The MICA-NKG2D axis activates NK cell’scytotoxicity. (Xu et al., Cancer Jmmunol Immunotherapy 2019, 68: 1429-1441). MICA membrane-bound expression increases NK cell recognition, antitumor activity, and tumor suppression (Fuertes et al., Front Immunol, 2021, 12:713158).

[0118] FIG. 1 illustrates the design of the super fusion activator.

[0119] FIG. 2 illustrates a summary of the methods used to test the function of the SFA.

[0120] FIG. 3 A shows a flow plot characterizing NK development stages with the CD27 and CDl lb surface markers. FIG. 3B shows a bar chart summarizing the % of Q3: CD27highCDl Ibhigh matured NK and Q4: CD271ow CDl lbhigh terminally-matured NK cells in the TIME. Fig. 3C shows a flow plot indicating % of NK cells population at each developmental stage. These populations are gated from the CD3-CD45+NK1.1+DX5+ tumorinfiltrating NK cells. Error bars = SEM. n= 6-8 mice / group. *p < 0.05; **p <0.01; ***p < 0.001.

[0121] FIG. 4 shows bar charts summarizing the % of cytolytic granules with FIG. 4A showing Granzyme B, FIG. 4B showing Perforin, and FIG. 4C showing cytokine IFN-y released by CD3-CD45+NK1.1+DX5+ tumor-infiltrating NK cells in the TIME. n= 6-8 mice / group. Error bars = SEM. *p < 0.05; **p <0.01; ***p < 0.001.

[0122] FIG. 5 shows growth curves (left) and representative pictures (right) of tumors dissected 20 days after inoculation of B16F10-EV, B16F10-mbMICA, B16F10-mbRpo3, and B16F10-SFA in FIG. 5A showing immunocompetent C57BL / 6 mice and FIG. 5B showing immunodeficient NRG mice, n = 10 mice / group. Error bars = SEM. *p < 0.05; **p <0.01; ***p < 0.001.

[0123] FIG. 6 shows a line curve summarizing the % NK cells cytotoxicity at various ratios. YAC-1 lacks the MHC-1 receptor and is an optimal target cell line for this assay. n= 3-5 mice / group. Error bars = SEM. *p < 0.05; **p <0.01; ***p < 0.001.

[0124] These results show that (i) NK cell maturation stage and activation signals are crucial for NK cell function, (ii) mbRspo3 terminally differentiates NK cells and increases their cytotoxicity, (iii) mbMICA enhances NK activation signal and increases their cytotoxicity, (iv) The SFA (Rspo3 -mbMICA) terminally differentiates and activates NK cells, and (v) The SFA enhances the TIME anti-tumor immunity and reduces tumor progression.

Claims

CLAIMS1. A super fusion activator (SFA) comprising polypeptide having an amino terminal Rspo3 polypeptide segment operatively coupled a carboxy terminal MICA polypeptide segment.

2. The super fusion activator of claim 1, wherein the amino terminal polypeptide is linked to the carboxy terminal polypeptide by a peptide linker.

3. The super fusion activator of claim 1, wherein the super fusion activator has the amino acid sequence of SEQ ID NO:2.

4. The super fusion activator of claim 3, wherein the super fusion activator is encoded by the nucleic acid sequence of SEQ ID NO: 1.

5. The activator of claim 1, wherein the amino terminal Rspo3 polypeptide segment has the amino acid sequence of SEQ ID NO:3.

6. The activator of claim 3, wherein the carboxy terminal MICA polypeptide segment is has the amino acid sequence of SEQ ID NO:4.

7. A host cell expressing the SFA of claim 1.

8. The host cell of claim 7, wherein the host cell is a T cell.

9. The host cell of claim 8, wherein the T cell is a chimeric antigen receptor expressing T cell (CAR-T).

10. A chimeric antigen receptor T (CAR-T) cell expressing a chimeric antigen receptor and the activator of claim 1.

11. The CAR-T of claim 10, wherein the chimeric antigen receptor is GPC3.

12. A polynucleotide encoding the SFA of claim 1 .

13. A method of treating cancer comprising administering the SFA of claim 1 to a patient having a tumor.

14. A composition comprising a super fusion activator (SFA) polypeptide and at least one additional polypeptide segment selected from the group consisting of IL-15, IL-21, and anti-PD- Ll, wherein the additional polypeptide segment is operatively linked to the SFA polypeptide or expressed separately in a host cell to enhance NK cell activity against solid tumors.

15. The composition of claim 14, further comprising a targeting moiety specific to a tumor- associated antigen, wherein the targeting moiety is selected from the group consisting of an anti- EGFR antibody fragment and an anti-HER2 antibody fragment.

16. A method of treating a solid tumor in a patient, comprising administering to the patient a super fusion activator (SFA) or a host cell expressing the SFA of claim 1, wherein the administration is via intratumoral injection to enhance localized NK cell activation within the tumor microenvironment.

17. The method of claim 16, wherein the SFA is delivered using a nanoparticle-based carrier or a viral vector targeting a tumor-specific marker to enhance penetration into the solid tumor.

18. A method of treating a solid tumor in a patient, comprising administering to the patient a combination therapy comprising: (a) the SFA of claim 1 or a host cell expressing the SFA, and (b) an immune checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody and an anti-CTLA-4 antibody, wherein the combination therapy enhances anti-tumor immune responses in the tumor microenvironment.

19. The method of claim 18, wherein the SFA or SFA-expressing host cell is administered first, followed by the immune checkpoint inhibitor to sustain immune activation against the solid tumor.

20. The method of claim 16 or 18, wherein the solid tumor is selected from the group consisting of hepatocellular carcinoma, pancreatic cancer, and lung cancer.

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