Tetra protein targeted killer engagers (tetrakes) in bladder cancer treatment

Abstract

The invention provides a solution to deficiencies in innate immune cell targeting in solid tumors, including bladder cancer, through IL-18 or IL-15 Tetra protein targeted Killer Engagers (TetraKEs). These leverage CD16 and CD56 to enhance natural killer (NK) cell targeting of Nectin-4+ bladder tumor cells, offering a novel immunotherapeutic approach.

Description

TETRA PROTEIN TARGETED KILLER ENGAGERS (TETRAKES) IN BLADDER CANCER TREATMENT PRIORITY PARAGRAPH

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 684,157 filed August 16, 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 "UTSKP0572" which is 5 KB (as measured in Microsoft Windows®) and was created on August 18, 2025. BACKGROUND

[0004] Bladder cancer (BCa) is a prevalent malignancy in the U.S., with approximately 80,000 new cases and 16,000 deaths annually. Its high relapse rate necessitates lifelong monitoring and treatment, making BCa one of the costliest cancers, with an estimated annual healthcare expenditure of $3 billion. Over the past decade, immunotherapy, particularly immune checkpoint inhibitors, has transformed BCa treatment, with all five inhibitors receiving rapid FDA approval. However, approximately 80% of patients do not respond to these therapies due to poor immune infiltrate in immunotherapy-resistant “cold” bladder tumors. Research has focused on enhancing immune cell recruitment to address this challenge, primarily targeting adaptive immune cells like T cells. While T cells are critical, their activation and anti-tumor functions rely on collaboration with natural killer (NK) cells. Some BCa tumors evade CD8+ T cell-mediated killing by downregulating MHC molecule expression, underscoring the need for innovative immunotherapies. Cytotoxic NK cells, which kill independently of MHC, offer a promising alternative. Despite their prognostic significance in BCa, NK cell-targeted therapies remain limited, hindered by challenges such as inadequate recruitment and limited persistence in vivo.There is a critical need for improved compositions and methods to treat bladder cancer and other malignancies.

[0005] There remains a need for improved compositions and methods for treating various cancers, including bladder cancer. SUMMARY

[0006] A solution to the problem of deficient targeting of innate immune cells, such as natural killer (NK) cells, includes an immunotherapeutic approach using IL-18 or IL-15 Tetra protein targeted Killer Engagers (TetraKEs), which leverage the properties of CD16 and CD56 to enhance the targeting of Nectin4+ bladder tumor cells by NK cells. This platform can potentially also be used to target other specific antigens on bladder tumors and other solid tumors.

[0007] The invention encompasses a Natural Killer (NK) cell activator polypeptide comprising, from amino terminus to carboxy terminus: (a) a CD16 binding portion, preferably an anti-CD16 single-chain variable fragment (scFv); (b) an IL-18 portion or an IL-15 portion, with the IL-18 portion having an amino acid sequence at least 90% identical to amino acids 248 to 404 of SEQ ID NO:1 or the IL-15 portion at least 90% identical to amino acids 266 to 377 of SEQ ID NO:2; (c) a Nectin-4 binding portion, preferably an anti-Nectin-4 scFv with an amino acid sequence at least 90% identical to amino acids 413 to 654 of SEQ ID NO:1; and (d) a CD56 binding portion, preferably an anti-CD56 scFv. The polypeptide may include a linker between the CD16 binding portion and the IL-18 or IL-15 portion, and an amino acid hinge between the Nectin-4 and CD56 binding portions to ensure proper folding and functionality. The activator polypeptide may have an amino acid sequence at least 90% identical to SEQ ID NO:1 or SEQ ID NO:2. These TetraKEs leverage the properties of CD16 and CD56 to enhance NK cell recruitment, activation, and persistence, targeting Nectin-4 positive bladder tumor cells with high specificity. The invention also includes polynucleotides encoding the activator polypeptide, vectors (such as adenoviral, adeno-associated viral, retroviral, or lentiviral vectors) comprising these polynucleotides, host cells containing the polynucleotides or vectors, and pharmaceutical compositions comprising the activator polypeptide with a pharmaceutically acceptable carrier. Methods of treating bladder cancer, particularly Nectin-4 positive bladder cancer, are provided, involving administering a therapeutically effective amount of the activator polypeptide or pharmaceutical composition, either intratumorally or intravesically, optionally in combinationwith additional anti-cancer therapies such as Bacillus Calmette-Guérin (BCG) therapy or immune checkpoint inhibitors (e.g., anti-PD-1 or anti-PD-L1 antibodies). The invention further encompasses kits for treating bladder cancer, including the activator polypeptide or pharmaceutical composition, instructions for use, packaging, and optionally an additional anti-cancer drug.

[0008] Certain embodiments are directed to a Natural Killer cell activator comprising, from amino terminus to carboxy terminus, a CD16 binding portion, an IL-18 portion, a Nectin 4 binding portion, and a CD56 binding portion. In certain aspects, the activator further comprises a linker between the CD16 binding portion and the IL-18 portion. In certain aspects, the activator further comprises an amino acid hinge positioned between the Nectin 4 binding portion and the CD56 binding portion. In certain aspects, the CD16 binding portion is an anti-CD16 single-chain antibody, in certain aspects the anti-CD16 single-chain antibody is at least 90%, 95%, 98%, or 100% identical an anti-CD16 single-chain antibody as disclosed herein. The IL-18 portion can have an amino acid sequence that is at least 90%, 95%, 98%, or 100% identical to amino acids 248 to 404 of SEQ ID NO:1. The Nectin 4 binding portion can have an amino acid sequence that is at least 90%, 95%, 98%, or 100% identical to amino acids 413 to 654 of SEQ ID NO:1. The CD56 binding portion can be an anti-CD56 single-chain antibody. In certain aspects, the anti-CD56 single-chain antibody is at least 90%, 95%, 98%, or 100% identical an anti-CD16 single-chain antibody as disclosed herein.

[0009] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0010] 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.”

[0011] 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.

[0012] 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.”

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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 characteristic(s) of the claimed invention. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.

[0017] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detaileddescription 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

[0018] Bladder cancer (BCa): A malignancy originating in the bladder, with approximately 80,000 new cases and 16,000 deaths annually in the U.S., characterized by a high relapse rate and significant healthcare costs estimated at $3 billion annually.

[0019] Natural Killer (NK) Cells: A type of lymphocyte in the innate immune system capable of selectively killing stressed or tumor cells without harming healthy cells, functioning through the formation of an immunological synapse that triggers cytoskeletal reorganization and exocytosis of cytolytic granules such as perforin and granzymes.

[0020] CD16: An activating receptor (FcγRIII) expressed on NK cells that mediates antibody- dependent cell-mediated cytotoxicity (ADCC) by recognizing the Fc portion of IgG antibodies, inducing calcium flux and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs).

[0021] CD56: A surface marker expressed on NK cells, contributing to NK cell adhesion to tumor cells and activation of the Pyk2-mediated cytotoxic signaling cascade, with CD56bright and CD56dim subsets exhibiting distinct functional properties in bladder tumors.

[0022] Nectin4: A tumor antigen highly expressed in advanced or metastatic bladder cancer, associated with unfavorable prognosis, and targeted by the TetraKE to direct NK cell cytotoxicity specifically against Nectin4+ bladder tumor cells.

[0023] IL-18: An interleukin cytokine that promotes NK cell proliferation, activation, and increased expression of CD56, enhancing NK cell persistence and functionality in vivo.

[0024] IL-15: An interleukin cytokine that supports NK cell activation and survival, used in some TetraKE embodiments as an alternative to IL-18 to enhance NK cell functionality.

[0025] Tetra protein targeted Killer Engagers (TetraKEs): A novel immunotherapeutic platform comprising a fusion polypeptide with binding portions for CD16, IL-18 or IL-15, Nectin- 4, and CD56, designed to enhance NK cell-mediated cytotoxicity against bladder tumor cells by leveraging multiple activation pathways.

[0026] Immune Checkpoint Inhibitors / Blockers: Therapeutic agents that block inhibitory pathways (e.g., PD-1 / PD-L1) to enhance immune responses against cancer, with five such inhibitors approved by the FDA for bladder cancer treatment.

[0027] Immunological Synapse: A specialized interface formed between NK cells and target cells (e.g., tumor cells), facilitating the release of cytolytic granules and cytokines to mediate cell killing.

[0028] Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC): A mechanism by which NK cells eliminate target cells coated with antibodies, mediated through CD16 binding to the Fc portion of IgG antibodies.

[0029] Single-Chain Variable Fragment (scFv): A fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an antibody, connected by a short linker peptide, used in TetraKEs to target specific antigens or receptors.

[0030] Linker: A short amino acid sequence used to connect different functional domains (e.g., scFv, IL-18) in a fusion polypeptide like TetraKE, ensuring proper folding and functionality.

[0031] Hinge: An amino acid sequence, often derived from immunoglobulin regions, used in TetraKEs to provide flexibility between functional domains, such as between the Nectin4 and CD56 binding portions.

[0032] Polynucleotide, nucleic acid molecule or nucleic acid: Refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.

[0033] Polypeptide: Refers to any peptide or protein comprising amino acids joined by peptide bonds or modified peptide bonds.

[0034] Antibody: Refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric and polymeric forms of each isotype, unless otherwise specified.

[0035] Functional fragments: Refers to fragments or segments of antibodies that comprise portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. For example, functional fragments can comprise at least the CDRs of either the heavy chain or light chain variable region. Functional fragments can also comprise the heavy chain or light chain variable region, or sequences that are substantially similar to the heavy or light chain variable region. Further suitable functional fragments include, without limitation, antibodies with multiple epitope specificity, bispecific antibodies, diabodies, and single-chain molecules, as well as Fab,F(ab’)2, Fd, Fabc, and Fv molecules, single chain (Sc) antibodies (also called ScFv), individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like. All antibody isotypes can be used to produce functional fragments of the antibodies herein. Functional fragments can be recombinantly or synthetically produced, with natural or unnatural nucleic acid or amino acid molecules.

[0036] Treating or treatment: Refer to any success or indicia of success in the attenuation or amelioration of a pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms, diminishing of pathology, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival. The treatment or amelioration of symptoms can be based on objective or subjective parameters including the results of a physical examination, neurological examination, and / or psychiatric evaluations.

[0037] Effective amount and therapeutically effective amount: The terms are used interchangeably herein and refer to an amount of a polypeptide or biologic or functional fragment thereof, as described herein, effective to achieve a particular biological or therapeutic result such as, but not limited to, the biological or therapeutic results disclosed herein. A therapeutically effective amount of a biologic may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the biologic to elicit a desired response in the individual. Such results may include, but are not limited to, the treatment of cancer, as determined by any means suitable in the art.

[0038] Providing: Providing refers to supply or furnish for use. In some embodiments, the protein is provided directly by administering the protein, while in other embodiments, the protein is effectively provided by administering a nucleic acid that encodes the protein. In certain aspects the invention contemplates compositions comprising various combinations of nucleic acid, antigens, peptides, and / or epitopes. DESCRIPTION OF THE DRAWINGS

[0039] 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 betterunderstood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0040] FIG.1. Immune therapies in BCa: Dark-colored antibodies: currently approved T cell Immune checkpoint inhibitors; circled light-colored antibodies: in clinical trials. BCG, Bacillus Calmette-Guérin; CIS, carcinoma in situ. Modified from Zang et al.

[0041] FIG.2A-2C. CD56 expression on NK cells correlate with survival in BCa. (A) Human bladder tumor samples from 50 patients were processed into single cell suspensions and analyzed with flow cytometry. Plotted CD56dimNK cells as a percentage of intratumoral live CD45+ lymphocytes across pathologic tumor stage. Kaplan-Meier plots of cancer specific survival (CSS) of patients with BCa (n=50) according to intratumoral CD56bright(B) and CD56dimNK cells (C) P- values represent log-rank (Mantel-Cox) test.

[0042] FIG. 3A-3E. Deletion of CD56 in NK cells reduces its cytotoxicity against BCa. (A) CFSE cytotoxicity assays were performed using NK92 WT and CD56 KO NK92 as effectors against T24 BCa target cells (4 hour assay). Flow cytometry analysis of NKG2D (B) and granzyme B (C) in NK92 WT or CD56 KO NK92 cells upon 4 hour stimulation. (D) Pseudo-3D Atomic Force Microscopy images of live WT and CD56 KO NK cells attached to T24 cells acquired in an adhesion channel projected on the height channel. T24 cells are detected as flat features at the image floor. An NK cell is a round, slightly fuzzy object on the top. (E) Adhesion data comparing adhesion of NK cells to T24 are presented in nanonewtons (larger values = stronger adhesion).

[0043] FIG. 4. Construction and design of TetraKE: Construction of hybrid tetra protein targeted1618Nec56 NK cell engager. From left to right, the plasmid contains VH and VL regions of anti-CD16 spliced to a 20 amino acid linker, then IL-18, the VH and VL region of anti-Nectin4, mutated IgG / hinge, and then the VH and VL of anti-CD56 to form 1618Nec56 TetraKE.

[0044] FIG. 5. Expression of CD16 on patient derived NK cells. Expression level of CD16 (Mean Fluorescence Intensity, MFI) is measured by flow cytometry on patient derived CD56dim and CD16bright NK cells.

[0045] FIG.6. Hypothetical Model.1618Nec56 TetraKE recruiting and inducing activation of both CD56brightCD16lo and CD16hiCD56dim NK cells against BCa cells.

[0046] FIG. 7. IL-18 treatment increases expression of CD56 on NK cells. NK.92 cells are treated with human recombinant IL-18 (100ng / ml) for 12 hours and expression level of CD56 (Mean Fluorescence Intensity, MFI) is measured by flow cytometry.

[0047] FIG. 8. Establishment of UMUC3 BCa model with NK cell adoptive transfer. NSG mice were subcutaneously challenged with 1 x 106human UMUC3 BCa cells. Tumor growth is compared between control mice and mice which received NK cell adoptive transfer (3 x 106NK cells).

[0048] FIG. 9. Conceptual framework for Cellular Neighborhood mapping combining cell type as identified by label with the known composition and morphology of the tissue.

[0049] FIG.10. Pie chart listing NK cell markers.

[0050] FIG. 11A-11G. IL15 TetraKE increases activation of cell line NK cells. In a dose- escalation experiment, NK92 cells were co-incubated with Nectin4+ UMUC14 (E:T=5:1) for ~4.5 hours with IL15 TetraKE at specified concentrations or control (buffer) with Brefeldin A. Shown is the expression of CD25, CD107a, and Granzyme B as MFI and percentage of NK cells. Mean±SEM. * p<0.05, two-sided, unpaired t-test. (A) % CD25+ NK cells, (B) % CD107a+ NK cells, (C) % Granzyme B+ NK cells, (D) CD107a MFI, (E) IFNγ MFI in live NK cells, (F) Granzyme B MFI in NK cells, (G) % IFNγ+ NK cells.

[0051] FIG. 12A-12B. TetraKEs increase activation and cytotoxicity of NK cells against bladder cancer. Nectin4+ (HT1197 or UMUC14) or Nectin4- (T24 or UMUC3) BCa cell lines were pre-stained with CFSE. In a 96-well plate, NK92 and BCa cancer cells were plated at an E:T ratio of 5:1 for 4 hours in the presence of IL15 or IL18 TetraKE at specified concentrations or buffer as the vehicle control. Expression of NKG2D shown as the proportion of NK cells expressing NKG2D (A). In vitro cytotoxicity assay of WT NK92 cells, shown as the percentage of dead UMUC14 BCa cells from total UMUC14 BCa cells after 4-hour incubation (B). Mean±SEM. * p<0.05, two-sided, unpaired t-test.

[0052] FIG.13A-13H. TetraKEs increase activation of NK cells derived from bladder cancer patient samples. Patient-derived PBMCs were expanded using the GREX expansion column. In 8 mL of cR-10, ~10 x 106PBMCs were seeded with 400 U / mL IL-2 and 3 mM IPP. Starting day 4 and every 3 days thereafter, 4 mL of media was removed without disturbing immune cells and replenished with fresh cR-10 supplemented with 400 U / mL IL-2. On day ~30, PBMCs were collected and counted. Using the PE isolation kit, CD3+ cells were removed. Flow staining was used to confirm the NK cell population. Following depletion, CD3- cells were incubated for ~4 hours with Brefeldin A and specified concentrations of IL18 TetraKE. Data are from CD45+CD56+ cells. Mean±SEM. * p<0.05, two-sided, unpaired t-test. (A) % CD25+ NK cells,(B) % CD107a+ NK cells, (C) % Granzyme B+ NK cells, (D) % IFNγ+ NK cells, (E) % Perforin+ NK cells, (F) KI67 MFI in NK cells, (G) IFNγ MFI in NK cells, (H) Granzyme B MFI in NK cells.

[0053] FIG. 14. IL18 TetraKE reduces tumor burden in a humanized bladder cancer murine model. On day 0, male NSG mice were challenged subcutaneously with 0.5 x 106UMUC14 (control), a mix of 0.5 x 106UMUC14 and 1.5 x 106NK92.MI (E:T=3:1), or 0.5 x 106UMUC14 subcutaneously with ~3 x 106PBMCs intravenously. On day 8 and every other day (QOD) thereafter, mice were injected with 10 µg of IL18 TetraKE / tumor in 50 µL of PBS. Dosing was determined from other experiments with BiKEs / BiTEs and TriKEs as well as human clinical trials of BiTEs (Kennedy et al., Front. Immunol., 2023; Vallera et al., Clinical Cancer Therapy, 2016; Pawlowski et al., Cells, 2023; Suurs et al., Journal of Nuclear Medicine, 2020).

[0054] FIG.15A-15D. IL15 TetraKE reduces tumor burden without affecting body weight and shows no toxicity (no effect on body weight) in a humanized bladder cancer murine model. On day 0, male NSG mice were challenged subcutaneously with 0.5 x 106UMUC14 (control) or a mix of 0.5 x 106UMUC14 and 1.5 x 106NK92.MI (E:T=3:1). On day 12 and every other day (QOD) thereafter, mice were injected with 10 µg of IL-15 or IL-18 TetraKE / tumor in 50 µL of PBS. (A) Tumor volume in NSG mice treated with UMUC14 and IL-15 TetraKE, (B) Body weights of NSG mice treated with UMUC14 and IL-15 TetraKE, (C) Body weights of NSG mice treated with UMUC14 and IL-18 TetraKE, (D) Tumor volume in NSG mice treated with UMUC14 and IL-15 TetraKE. DESCRIPTION

[0055] 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.

[0056] 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 thetechniques 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 that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. I. Tetra protein targeted Killer Engagers (TetraKEs)

[0057] Research on immune cells in bladder cancer (BCa) has primarily focused on T cells, with limited emphasis on natural killer (NK) cells. Expanding NK cell-targeted therapies is expected to complement T cell immunotherapies and provide alternatives for BCa patients unresponsive to conventional treatments. The 1618Nec56 TetraKE, an NK cell-based biopharmaceutical, offers a streamlined approach to redirect NK cells in vivo without genetic modifications. This novel TetraKE targets two NK cell activation pathways, potentially transforming BCa immunotherapy. Using UMUC3 / NK92 and PDX257S / PBMCs adoptive transfer murine models, the therapeutic potential of TetraKEs can be further evaluated. Cutting- edge spatial phenotyping technology will enable the study of NK cell population diversity, functional states, and interactions within the tumor immune microenvironment (TME), unlike bulk measurements or other single-cell techniques. The 1618Nec56 TetraKE is expected to enhance immune specificity, tolerability, and reduce toxicity in vivo. Alone or combined with standard immunotherapies, it holds promise for revolutionizing BCa treatment, particularly for advanced cases resistant to conventional approaches. The FDA’s accelerated approval of enfortumab vedotin, a Nectin-4-targeting antibody-drug conjugate, in 2019 highlights the potential of Nectin- 4 as a BCa antigen. The TetraKE is the first NK cell-targeted therapy leveraging this antigen to recruit NK cells, offering an accessible, cost-effective, “off-the-shelf” intervention that avoids complex genetic modifications or ex vivo NK cell expansion protocols.

[0058] In certain aspects a 1618Nec56 TetraKE (CD16 / IL18 / Nec4 / CD56) fusion polypeptide can have for example an amino acid sequence that is at least 90, 95, 98, to 100% identical to the following amino acid sequence: MEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGG STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVT VSR1GGGGSGGGGSGGGGS2SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFG GGTKLTVL3PSGQAGAAASESLFVSNHAYFGKLESKLSVIRNLNDQVLFIDQGNRPLFED MTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKD TKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQ NED4EASGGPEA5LTQPSSVSANPGETVKITCSGGSSNYYGWYQQKSPGSAPVTLIYNNNK RPSDIPSRFSASKSGSTHTLTITGVRAEDEAVYFCGGWDKSAGIFGAGTTLTVLGQSSR6S SGGGGSSGGGG7SAVTLDESGGGLQTPGGGLSLVCKASGFTFSSNGMAWVRQAPGKGL EWVAGVNAAGSWTGYGAAVKGRATISRDNGQSTVRLQLNDLRAEDTGTYYCAKTADD WYGADDIDAWGHGTDVIVSS8EPKSSDKTHTSPPSPEVQLVQSGAEVKKPGSSVKVSCK ASGGTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAY MELSRLRSDDTAVYYCARDLSSGYSGYFDYWGQGTLVTV9SSGGGGSGGGGSGGGG10S DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNFLDWYLQKPGQSPQLLIYLGSNRA SGVPDRFSGSGSGTDFTLKISRVEADDVGVYYCMQSLQTPWTFGHGTKVEIKRTVAAA ALE11(SEQ ID NO:1) 1 = amino acids 1-121 is an Anti-CD16 Single-Chain Variable Fragment (scFv) Variable Heavy (VH) Chain (CDRs in bold italics), 2 = amino acids 122-136 is a spacer / linker, 3 = amino acids 137-247 is Anti-CD16 scFv Variable Light (VL) Chain (CDRs in bold italics), 4 = amino acids 248-404 IL-18, 5 = amino acids 405-412 spacer / linker, 6 = 413- 518 Anti-Nectin-4 scFv Variable Light (VL) Chain (CDRs in bold italics), 7 = amino acids 519-530 spacer / linker, 8 = amino acids 531-654 Anti-Nectin-4 scFv Variable Heavy (VH) Chain (CDRs in bold italics), 9 = amino acids 655-775 is an Anti-CD56 scFv Variable Heavy (VH) Chain, 10 = amino acids 776-790 is a spacer / linker, and 11 = amino acids 791-938 is Anti-CD56 scFv Variable Light (VL) Chain (CDRs in bold italics).

[0059] In certain aspects a 1615Nec56 TetraKE (CD16 / IL15 / Nec4 / CD56) fusion polypeptide can have and amino acid sequence that is at least 90, 95, 98, to 100% identical to the following amino acid sequence: MEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGG STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVT VSR1GGGGSGGGGSGGGGS2SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPG QAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFG GGTKLTVL3PSGQAGAAASESLFVSNHAY4NWVNVISDLKKIEDLIQSMHIDATLYTESD VHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS5EASGGPEALTQPSSVSANPGETVKITCSGGSSNYYGW YQQKSPGSAPVTLIYNNNKRPSDIPSRFSASKSGSTHTLTITGVRAEDEAVYFCGGWDKS AGIFGAGTTLTVL6GQSSRSSGGGGSSGGGGS7AVTLDESGGGLQTPGGGLSLVCKASGF TFSSNGMAWVRQAPGKGLEWVAGVNAAGSWTGYGAAVKGRATISRDNGQSTVRLQLN DLRAEDTGTYYCAKTADDWYGADDIDAWGHGTDVIVSS8EPKSSDKTHTSPPSP9EVQLV QSGAEVKKPGSSVKVSCKASGGTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLSSGYSGYFDYWGQGTLVTV SS10GGGGSGGGGSGGGG11SDVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNFLDW YLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEADDVGVYYCMQSLQT PWTFGHGTKVEIKRTVAAAALE12(SEQ ID NO:2) 1 = amino acids 1-120 is an Anti-CD16 Single-Chain Variable Fragment (scFv) Variable Heavy (VH) Chain (CDRs in bold italics), 2 = amino acids 121-135 is a spacer / linker, 3 = amino acids 136-246 is Anti-CD16 scFv Variable Light (VL) Chain (CDRs in bold italics), 4 = amino acids 247-265 spacer / linker, 5 = amino acids 266- 377 IL-15, 6 = amino acids 378-487 is an Anti-Nectin-4 scFv Variable Light (VL) Chain (CDRs in bold italics), 7 = amino acids 488-504 spacer / linker, 8 = amino acids 505-632 Anti-Nectin-4 scFv Variable Heavy (VH) Chain (CDRs in bold italics), 9 = amino acids 633-647 is a spacer / linker, 10 = amino acids 648-767 is an Anti-CD56 scFv Variable Heavy (VH) Chain, 11 = amino acids 768-782 is a spacer / linker, and and 12 = amino acids 783-896 is Anti-CD56 scFv Variable Light (VL) Chain (CDRs in bold italics). II. Natural Killer (NK) Cells

[0060] Natural killer (NK) cells selectively kill stressed or tumor cells without harming healthy cells. Unlike T cells, which rely on antigen-specific receptors, NK cells integrate activating and inhibitory signals from regulatory receptors to distinguish abnormal cells. Upon adhering to a stressed or tumor cell, NK cells form an immunological synapse, triggering cytoskeletal reorganization and exocytosis of cytolytic granules (e.g., perforin, granzymes) to mediate cell killing. NK cells also produce cytokines like IFN-γ to stimulate CD4+ and CD8+ T cell activation. Additionally, NK cells mediate antibody-dependent cell-mediated cytotoxicity (ADCC) via CD16 (FcγRIII), which recognizes the Fc portion of IgG and triggers robust, independent cytotoxicity. Immunophenotyping of intratumoral lymphocytes from BCa patients using multi-parametric flow cytometry revealed NK cells as a dominant population (~25–30% of CD45+ lymphocytes). NKcells are divided into CD56bright and CD56dim subsets, with CD56bright NK cells in tissues showing higher cytotoxicity and activation marker expression compared to CD56dim counterparts. In bladder tumors, CD56bright NK cells are associated with improved cancer-specific survival (CSS) (FIG. 2B), while CD56dim NK cells accumulate in higher-stage BCa and are linked to dysfunction (FIG. 2A, 2C). Deletion of CD56 in NK cells reduced cytotoxicity, NKG2D, and granzyme B expression, as well as adhesion to BCa cells (FIG. 3A–E), establishing CD56 as a novel mediator of NK cell cytotoxicity independent of CD16. While NK cell transfer shows promise in BCa, challenges include exhaustion of peripheral blood-derived NK cells from high- grade BCa patients and non-specific tumor targeting due to MHC downregulation in some tumors. The proposed TetraKE strategy addresses these challenges by enhancing NK cell specificity and persistence, potentially becoming the first FDA-approved NK cell-targeted therapy for BCa.

[0061] Cracking the Biomedical Puzzle: Importance of developing NK cell killer engagers. The concept of bispecific antibodies originated from T cells in the late 1980s. In 1985, two pivotal publications laid the groundwork for a novel hybrid antibody capable of precisely directing T cells toward cancer cells (23), bypassing the MHC and costimulatory pathways for T cell activation (24). Blinatumomab emerged as the first FDA-approved bispecific antibody, featuring an antibody targeting CD3 on T cells and CD19 on blast cells. Nonetheless, for years, the exploration of bispecific antibodies remained predominantly confined to T cells and hematologic malignancies.

[0062] Development of tetra protein targeted 1618Nec56 NK cell killer engager (TetraKE). Recent technological breakthroughs have propelled the bispecific antibody platform into an exciting frontier, offering a fresh approach to drive NK cell cytotoxicity against tumor cells. Pioneered by Dr. Vallera, bi-specific NK cell engagers (BiKES) are small molecules designed to simultaneously interact with both an NK cell-associated activation receptor and a tumor-specific antigen (25). Further, the engagement of CD3 in bispecific T cell antibodies triggers the release of interleukin (IL)-2, which contributes to the development of cytokine toxicity which is not expected with BiKES. Yet, despite these advancements, the application of this technology has not yet found a foothold in solid tumors, with no investigations conducted in BCa. Described herein is a project that uses this platform to develop a cutting-edge novel tetra protein targeted 1618Nec56 NK cell killer engager (TetraKE).

[0063] CD16 expressed on NK cells plays a pivotal role in antibody-directed cellular cytotoxicity (ADCC) by inducing calcium flux and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs). This activation triggers the release of lytic molecules and cytokines like IFN-γ and TNF-α. In contrast, CD56 expressed on bladder intratumoral NK cells contributes to cytotoxicity by influencing NK cell adhesion to tumor cells and downstream activation of the Pyk2-mediated cytotoxic signaling cascade. Here, development of a TetraKE by incorporating anti-CD16 single-chain variable fragments (scFv) and anti-CD56 scFV with scFv against Nectin4, a bladder tumor antigen is described (FIG.4). scFVs consist of a heavy and light chain linked by a linker sequence. Once bound to either CD16 or CD56, the relevant NK cell receptors are stimulated and direct a cytotoxic response to the bound Nectin 4+ tumor cell. Nectin- 4 was found highly expressed in advanced or metastatic BCa and was also linked to unfavorable prognosis in BCa with variant histology (BCVH), a subtype that is in urgent need of novel therapeutic agents (26). This specific binding to Nectin4 reduces off-target cytotoxicity, as the NK cells mediate their cytotoxicity specifically against their target cells. The TetraKE is expected to mediate the formation of an immunological synapse between tumor cells and NK cells, resulting in enhanced NK cell-mediated cytotoxicity against Netin4+ BCa cells. Notably, as the expression level of CD16 is significantly higher on low CD56 expressing cells than CD56bright NK cells (FIG. 5), this TetraKE strategy will effectively target both CD56dim and CD56bright NK cell subsets within the bladder (FIG.6). Furthermore, by incorporating an IL-18-crosslinker, an increase in NK cell in vivo persistence and functionality is anticipated, particularly of CD56dim NK cells, based on promising observations of recombinant human IL-18 increasing CD56 expression (FIG. 7) and NK cell functionality and expansion (27). After harvesting and refolding of the tetraKE (expressed as recombinant proteins in E. coli bacteria, then refolding and purification), fast-flow sepharose will be used to confirm adequate peaks and the expected size of TetraKE.

[0064] TetraKE BCa treatment. To test the specificity of the 1618Nec56 TetraKE, flow cytometry is used to determine the binding of fluorochrome-labeled TetraKE with Nectin 4+ BCa cell line (HT-1376, HT-1197) and Nectin 4- cell lines (UMUC-3, TCCSUP). Blocking with saturating concentrations of unlabeled anti-Nectin4 scFv will also be used to confirm Nectin4- dependent binding. It is expect that TetraKE will not only result in the recruitment of CD16loCD56bright NK cells but also CD16highCD56dim, however, it was previously found that a significant percentage of CD56dim NK cells were defective in cytokine production and mediating cytotoxicity (7, 8). IL-18 has been shown to induce proliferation and development of NK cells. Further, preliminary data shows that IL-18 increases CD56 expression on these CD56dim NK cells,potentially increasing their cytotoxicity. NK.92 cell line and NK cells enriched from human bladder tumors derived from patients (established using a previously published technique (7, 8)), and PBMCs from patients are exposed to anti-CD16 scFv, anti-CD56 scFv, TetraKE, and IL-18 to determine whether the TetraKE induces expansion and activation of the NK cells similar to the IL- 18 group. To test the capacity of the TetraKE to enhance survival of NK cells, purified NK cells can be cultured with TetraKE and viability assessed after 7-14 days. Carboxyfluorescein succinimidyl ester (CFSE) cytotoxicity assays can be used to evaluate the ability of 1618Nec56 to mediate NK cell cytotoxicity against Nectin4+ BCa cells. Anti-CD16 scFv, anti-CD56 scFv, anti- Nectin4 scFv, and IL-18 alone will be used as controls. Degranulation and IFNγ production assays will be done alongside cytotoxicity assays. A biorepository of PBMCs and tumor samples from ~300 patients with BCa has been established. Pharmacokinetic and toxicity studies can be carried out including in vitro absorption, distribution, metabolism, and excretion (ADME) assays, aqueous stability (bioavailability), plasma stability (rate of decomposition in plasma), PAMPA assay (absorbance in GI tract), HepG2 assay (hepatoxicity risks) hERG and NaV1.5 assays (cardiovascular risks).

[0065] In addition to in vitro and ex vivo studies, two novel humanized NK cell adoptive transfer models have been established that can be used to test the efficacy of 1618Nec56 TetraKE in vivo. TetraKE will be injected intratumorally in both PDX257S (previously published model)(28) and UMUC3 subcutaneous tumors where NK cells are adoptively transferred (FIG.8). Further, the therapeutic efficacy of TetraKE will also be tested in PDX257S and UMUC3 orthotopic BCa models where NK cells will be given intravesically before the intravesical instillation of the TetraKE. Detailed immunophenotyping of NK cells can be carried out using multi-parametric flow cytometry, CyTOF, and RNA sequencing (all techniques previously published and established in the lab). A comprehensive NK cell panel encompassing the activation and inhibitory receptors, migration, residency, and maturation markers can be used. A list of NK cell potential markers is provided. In addition to standard analyses, spatial analysis can be used to visualize and quantitate immune protein markers expression across the entire tissue landscape using whole-slide imaging at single-cell resolution. After phenotyping, the percentage of each phenotype within each sample is calculated and plotted as a pie chart. CytoMap can generate cellular neighborhoods (CN) that display patterns of the organization across the entire sample(FIG. 9). After cell segmentation, the data can contain spatial information on each cell as X / Y coordinates, allowing the nearest-neighbor analysis.

[0066] The TetraKE treatment is expected to enhance NK cell cytotoxicity and to activate and restore the functionality of dysfunctional low CD56 NK cells within the bladder tumors. Overall, our 1618Nec56 TetraKE is anticipated to emerge as a novel and viable treatment avenue for BCa by endowing antigen specificity (Nectin4), enhancing two dimensions of NK cell functionality - CD16-mediated ADCC and innovative CD56-mediated NK cell cytotoxicity - and by augmenting both the expansion and activation of NK cells through IL-18 modulation.

[0067] Synergy between TetraKES BCG and checkpoint inhibitors. Combination studies of TetraKE with standard BCa treatment strategies - BCG and αPD-L1. The combination strategies were chosen based on preliminary findings showing the involvement of NK cells in BCG response (29) and mediating sensitivity to αPD-L1 immune therapy (30). It is contemplated that the TetraKE co-treatment will improve the tumor suppressive effects of both BCG and anti-PD-L1 treatment in BCa. As previously published, for αPD-L1 treatment, 100 μg αPD-L1 will be given intraperitoneally on days 7, 12, and 17 after tumor challenge. Finally, for BCG treatment, BCG (1× 106CFU) can be instilled directly into the tumors or in the bladder weekly after the tumor challenge. To determine if 1618Nec56 TetraKE can potentiate the effects of existing BCa therapy, TetraKE will be given intratumorally or intravesically ± BCG / αPD-L1, and test survival in in vivo humanized murine models as discussed previously. A second cohort will be sacrificed for the measurement of bladder weights and immune analysis. Pharmacokinetic-pharmacodynamic (PK- PD) modeling will be employed to ascertain the ideal combination of drugs using the combination index method for in vivo synergism detection. This approach will aid in identifying dosage combinations that exhibit enhanced synergy in terms of potency and efficacy, thereby facilitating the clinical advancement of combination treatment strategies.

[0068] Clinical Significance. Bladder tumors carry one of the highest known cancer mutational loads, which correlates with the best immunotherapy responses (31). Thus, the rationale for immunotherapy to treat BCa is strong, and BCa has shown responsiveness to both old and new- generation immune therapies (BCG Therapy, αPD-L1, αPD-1). Unfortunately, not all bladder tumors respond to immunotherapy and the lack of response correlates with the poor immune infiltrate in these immunotherapy-resistant-resistant “cold” bladder tumors (4, 32). Certain bladder tumors may also evade CD8+ T cell-mediated killing by losing their expression of MHC molecules(6). Thus, targeting the function of intratumoral NK cells, which are not dependent on MHC for cell killing, holds significance as alternative immunotherapeutic strategies. The TetraKE described herein presents a cost-effective alternative by activating endogenous NK cells, potentially removing the requirement for complex NK cell adoptive transfers in patients and paving the way for a new age of immunotherapy.

[0069] Mechanistic Significance. NK cells, known for their potent cytotoxic capabilities, present a promising opportunity for new successful immunotherapeutic treatment options. However, the effectiveness of NK cell therapies against tumors has been hindered by challenges such as the absence of precise antigen targeting and the limited persistence of NK cells within the body. To overcome these roadblocks, a novel TetraKE is designed which is expected to mediate potent and specific NK cell-mediated cytotoxicity against Nectin4+ bladder tumor cells. Based on the finding that CD56 is responsible for activating NK cell signaling and cytotoxicity instead of just spatially modulating other integrins as an adhesion molecule, we designed the TetraKE to activate both CD56 and CD16-induced NK cell pathways. Finally, increasing CD56 via IL-18 to activate intratumoral NK cells and improve their functional efficacy is a new idea that holds significance even beyond the BCa field. III. Polypeptide compositions

[0070] Modifications and / or changes may be made in the amino acid composition of polypeptides or biologics, and thus the present invention contemplates variation in sequences of the polypeptides, and nucleic acids coding therefor, where they are nonetheless able retain substantial activity with respect to the therapeutic, preventative, and curative aspects of the present invention.

[0071] The biological functional equivalent may comprise a polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to encode the “wild-type” or recombinant polypeptide. This can be accomplished through the degeneracy of the genetic code which encode for the same amino acids.

[0072] In another example, a polynucleotide may encode a biological functional equivalent with more significant changes. Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors,and such like. So-called “conservative” changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges on the protein's ability to carry out its designed function. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.

[0073] In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a “biologically functional equivalent” protein and / or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalents are thus defined herein as those proteins (and polynucleotides) in selected amino acids (or nucleotides) may be substituted. In certain aspects, a polypeptide is or is at least 80, 85, 90, 92, 94, 96, 98, or 100% identical to the therapeutic polypeptide. In certain aspects, polypeptide(s) 80, 85, 90, 92, 94, 96, 98, or 100% identical to SEQ ID NO:1 or 2 are used or nucleic acids encoding the same.

[0074] In general, the shorter the length of the molecule, the fewer changes that can be made within the molecule while retaining function. Longer domains may have an intermediate number of changes. The full-length protein will have the most tolerance for a larger number of changes. However, it must be appreciated that certain molecules or domains that are highly dependent upon their structure may tolerate little or no modification. Function of a polypeptide can be determined by using various assays know to detect the activity of the polypeptide of interest.

[0075] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and / or the like. An analysis of the size, shape and / or type of the amino acid side-chain substituents reveals that arginine, lysine, and / or histidine are all positively charged residues; that alanine, glycine, and / or serine are all a similar size; and / or that phenylalanine, tryptophan, and / or tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine, and / or histidine; alanine, glycine, and / or serine; and / or phenylalanine, tryptophan, and / or tyrosine are defined herein as biologically functional equivalents.

[0076] To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and / or charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and / or arginine (−4.5).

[0077] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index and / or score and / or still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those that are within ±1 are particularly preferred, and / or those within ±0.5 are even more particularly preferred.

[0078] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. As detailed in U.S. Patent 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). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and / or those within ±0.5 are even more particularly preferred.

[0079] Delivery Vectors. In certain aspects, components are provided to a subject or organ or tissue by using nucleic acids that encode or express such components. Viral and non-viral delivery vectors can be used in the methods described herein (e.g., Syner-III).

[0080] The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to express virally encoded genes have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Viruses may thus be utilized that encode and express agents of the invention. Non-limiting examples of virus vectors that may be used to deliver nucleic acids are described below.

[0081] Adenoviral Vectors. A particular method for delivery of nucleic acid involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of genetransfer afforded by these vectors. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein.

[0082] AAV Vectors. A nucleic acid may be introduced into the cell using adenovirus-assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus-coupled systems. Adeno-associated virus (AAV) has a low frequency of integration and it can infect non-dividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture or in vivo. AAV has a broad host range for infectivity.

[0083] Retroviral Vectors. Retroviruses have the ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, U.S. Patents 6,013,516 and 5,994,136, each of which is incorporated herein by reference). Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.

[0084] Lipid-Mediated Transfection. In a further embodiment, a nucleic acid may be entrapped in a lipid particle such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Wu et al. (Eds.), Marcel Dekker, NY, 87-104, 1991). Also contemplated is a nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).IV. Pharmaceutical Compositions

[0085] 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 acceptable carrier. 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.

[0086] 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. In certain aspects compositions of the invention can be instilled an organ such as the bladder or the lung. The routes of administration described herein are merely an example and in no way limiting.

[0087] 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.

[0088] 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.

[0089] The amount of the therapeutic composition must be effective to achieve an enhanced therapeutic index. If multiple doses are employed, the frequency of administration will depend, for example, on the type of subject. One skilled in the art can ascertain upon routine experimentationthe appropriate route and frequency of administration in a given subject that are most effective in any particular case. Suitable doses and dosage regimens can be determined by conventionally known range-finding techniques. Generally, treatment is initiated with smaller dosages, which are less than the optimal dose. Thereafter, the dosage is increased by small increments until the optimal effect under the circumstances is obtained.

[0090] 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 chemical and physical considerations, such as solubility and reactivity with the therapeutic agent. In addition, the therapeutic composition may be formulated as polymeric compositions, inclusion complexes, such as cyclodextrin inclusion complexes, liposomes, microspheres, microcapsules, and the like, without limitation.

[0091] 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.

[0092] 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, and oral formulations such as, but not limited to, liquid solutions, including suspensions and emulsions, capsules, sachets, tablets, lozenges, and the like. Non-limiting formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, including non-active ingredients such as antioxidants, buffers, bacteriostats, solubilizers, thickening agents, stabilizers, preservatives, surfactants, and the like. The solutions can include oils, fatty acids, including detergents and the like, as well as other well known and common ingredients in such compositions, without limitation. V. Kits

[0093] In another aspect, the present invention provides kits for treatment of cancer, which kits are used to administer therapeutic compositions described herein. The kit can include acomposition including a therapeutically effective amount of a biologic as described herein and optionally a first anti-cancer drug for treating a cancer in the subject; instructions for use, and packaging. The kit can further include a second anti-cancer drug (e.g., checkpoint inhibitor, imatinib, standard chemotherapy agents such as cytarabine or doxorubicin, etc.). VI. References 1. Botteman et al., Pharmacoeconomics 21, 1315-1330, (2003). 2. Eble et al., The World Health Organization Classification of Tumours of the Urinary System and Male Genital System. Lyon, France: IARC Press, 209–211 (2004). 3. Roviello et al., Cancers (Basel) 13, (2021). 4. Darvin et al, Exp Mol Med 50, 1-11, (2018). 5. Schuster et al., Front Immunol 7, 235, (2016). 6. Cornel et al., Cancers (Basel) 12, (2020). 7. Mukherjee et al., Cancer Med 12, 8970-8980, (2023). 8. Mukherjee et al., Oncotarget 9, 36492-36502, (2018). 9. Zang et al., Front Oncol 11, 696716, (2021). 10. Yoon et al., Exp Mol Med 47, e141, (2015). 11. Pallmer and Oxenius, Front Immunol 7, 251, (2016). 12. Farag and Caligiuri, Blood Rev 20, 123-137, (2006). 13. Poli et al., Immunology 126, 458-465, (2009). 14. Nagler et al., Journal of immunology 143, 3183-3191 (1989). 15. Wagner et al., J Clin Invest 127, 4042-4058, (2017). 16. Michel et al., J Immunol 196, 2923-2931, (2016). 17. Gunesch et al., Elife 9, (2020). 18. Sancho et al., J Cell Biol 149, 1249-1262, (2000). 19. Ferreira-Teixeira et al., BMC Med 14, 163, (2016). 20. Rouanne et al., J Clin Invest 132, (2022). 21. Dhatchinamoorthy et al., Front Immunol 12, 636568, (2021). 22. Raulet, Semin Immunol 18, 145-150, (2006). 23. Staerz et al., Nature 314, 628-631, (1985). 24. Offner et al., Mol Immunol 43, 763-771, (2006).

Claims

CLAIMS 1. A Natural Killer (NK) cell activator polypeptide comprising, from amino terminus to carboxy terminus: (a) a CD16 binding portion; (b) an IL-18 portion or an IL-15 portion; (c) a Nectin-4 binding portion; and (d) a CD56 binding portion.

2. The activator polypeptide of claim 1, further comprising a linker between the CD16 binding portion and the IL-18 portion or IL-15 portion.

3. The activator polypeptide of claim 1 or 2, further comprising an amino acid hinge between the Nectin-4 binding portion and the CD56 binding portion.

4. The activator polypeptide of any one of claims 1-3, wherein the CD16 binding portion is an anti-CD16 single-chain variable fragment (scFv).

5. The activator polypeptide of claim 4, wherein the anti-CD16 scFv comprises: (a) a variable heavy chain (VH) comprising complementarity-determining regions (CDRs) having the amino acid sequences of DYGMS, GINWNGGSTGYADSVKG, and GRSLLFDY; and (b) a variable light chain (VL) comprising CDRs having the amino acid sequences of GDSLRSYYAS, GKNNRPS, and NSRDSSGNHVV.

6. The activator polypeptide of any one of claims 1-5, wherein the IL-18 portion comprises an amino acid sequence at least 90% identical to amino acids 248 to 404 of SEQ ID NO:

1.

7. The activator polypeptide of any one of claims 1-5, wherein the IL-15 portion comprises an amino acid sequence at least 90% identical to amino acids 266 to 377 of SEQ ID NO:

2.

8. The activator polypeptide of any one of claims 1-7, wherein the Nectin-4 binding portion is an anti-Nectin-4 scFv.

9. The activator polypeptide of claim 8, wherein the anti-Nectin-4 scFv comprises an amino acid sequence at least 90% identical to amino acids 413 to 654 of SEQ ID NO:

1.

10. The activator polypeptide of any one of claims 1-9, wherein the CD56 binding portion is an anti-CD56 scFv.

11. The activator polypeptide of claim 10, wherein the anti-CD56 scFv comprises: (a) a variable heavy chain (VH) comprising CDRs having the amino acid sequences of GYYMH, WINPNSGGTNYAQKFQG, and DLSSGYSGYFDY; and (b) a variable light chain (VL) comprising CDRs having the amino acid sequences of RSSQSLLHSNGYNFLD, LGSNRAS, and MQSLQTPWT.

12. The activator polypeptide of any one of claims 1-11, comprising an amino acid sequence at least 90% identical to SEQ ID NO:1 or SEQ ID NO:

2.

13. A polynucleotide encoding the activator polypeptide of any one of claims 1-12.

14. A vector comprising the polynucleotide of claim 13.

15. The vector of claim 14, wherein the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, or a lentiviral vector.

16. A host cell comprising the polynucleotide of claim 13 or the vector of claim 14 or 15.

17. A pharmaceutical composition comprising the activator polypeptide of any one of claims 1-12 and a pharmaceutically acceptable carrier.

18. A method of treating bladder cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the activator polypeptide of any one of claims 1-12 or the pharmaceutical composition of claim 17.

19. The method of claim 18, wherein the bladder cancer is Nectin-4 positive.

20. The method of claim 18 or 19, wherein the activator polypeptide is administered intratumorally or intravesically.

21. The method of any one of claims 18-20, further comprising administering to the subject an additional anti-cancer therapy.

22. The method of claim 21, wherein the additional anti-cancer therapy comprises Bacillus Calmette-Guérin (BCG) therapy, an immune checkpoint inhibitor, or a combination thereof.

23. The method of claim 22, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

24. A method of enhancing NK cell-mediated cytotoxicity against Nectin-4 positive bladder cancer cells in a subject, comprising administering to the subject a therapeutically effective amount of the activator polypeptide of any one of claims 1-12 or the pharmaceutical composition of claim 17.

25. A kit for treating bladder cancer, comprising: (a) the activator polypeptide of any one of claims 1-12 or the pharmaceutical composition of claim 17; (b) instructions for use; and (c) packaging.

26. The kit of claim 25, further comprising an additional anti-cancer drug selected from the group consisting of Bacillus Calmette-Guérin (BCG), an immune checkpoint inhibitor, and a chemotherapeutic agent.

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