Poly-antigen cytokine-receptor complexes (PACCS), compositions, and methods of use

Abstract

A protein complex includes a first protein and a second protein. The first protein includes an IL- 15 domain operably linked to both a first functional domain and a second functional domain. The second protein includes an IL-15Rα domain operably linked to at least one functional domain. In one or more embodiments, at least one of the functional domains is a targeting domain. In one or more embodiments, at least one functional domain includes albumin. In one or more embodiments, the IL15Rα domain is operably linked to two or more functional domains.

Description

[0001] PCT Application Attorney Docket No. 0110.000730W001

[0002] POLY-ANTIGEN CYTOKINE-RECEPTOR COMPLEXES (PACCs), COMPOSITIONS, AND METHODS OF USE

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 611,290, filed December 18, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0005] SEQUENCE LISTING

[0006] This application contains a Sequence Listing electronically submitted via Patent Center to the United States Patent and Trademark Office as an xml file entitled “0110000730W001.xml” having a size of 181 ,295 bytes and created on December 12, 2024. The information contained in the Sequence Listing is incorporated by reference herein.

[0007] SUMMARY

[0008] This disclosure describes, in one aspect, a multispecific compound that generally includes a complex of two proteins. The first protein includes an IL- 15 domain operably linked to both a first functional domain and a second functional domain. The second protein includes an IL-15Ra domain operably linked to at least one functional domain.

[0009] In one or more embodiments, at least one of the functional domains is a targeting domain.

[0010] In one or more embodiments, at least one functional domain includes albumin.

[0011] In one or more embodiments, the IL15Ra domain is operably linked to two or more functional domains.

[0012] In one or more embodiments, the IL-15 domain is complexed to the IL-15Ra domains via non-covalent affinity.

[0013] In one or more embodiments, each functional domain independently includes an amino acid sequence having at least 70% identity with any one of SEQ ID NOs: 19-31 and 73-78.

[0014] In another aspect, this disclosure describes an isolated nucleic acid encoding the amino acid sequence of any PACC complex component protein. In another aspect, this disclosure describes a host cell that includes a nucleic acid that encodes the amino acid sequence of any PACC complex component protein.

[0015] In another aspect, this disclosure describes a pharmaceutical composition that includes any embodiment of a PACC complex and a pharmaceutically acceptable carrier.

[0016] In another aspect, this disclosure describes a method that includes administering to a subject any embodiment of a PACC complex in an amount effective to induce natural killer (NK)-mediated killing of a cell.

[0017] In another aspect, this disclosure describes a method for stimulating expansion of natural killer (NK) cells in vivo. Generally, the method includes administering to a subject any embodiment of a PACC complex in an amount effective to stimulated expansion of NK cells in the subject.

[0018] In another aspect, this disclosure describes a method of treating a subject having, or at risk of having cancer. Generally, the method includes administering to the subject any embodiment of a PACC complex that targets cells of the tumor in an amount effective to ameliorate at least one symptom or clinical sign of cancer or decrease the likelihood that the subject develops cancer compared to an untreated individual.

[0019] In one or more embodiments, the cancer can include prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematopoietic cancer.

[0020] In one or more embodiments, the PACC complex is administered prior to, simultaneously with, or following chemotherapy, surgical resection of a tumor, or radiation therapy.

[0021] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0022] BRIEF DESCRIPTION OF THE FIGURES FIG 1 . Schematic representations of compounds consistent with one or more embodiments described herein, such as Poly-Antigen Cytokine-receptor Complexes (PACCs). (A) Schematic representation of a compound including two domains attached to IL- 15 and two domains attached to IL-15Ra. FIG. (B) Schematic representation of a compound including two moi eties attached to IL- 15 and one domain attached to IL-15Ra. (C) Schematic representation of a subclass of compounds represented by FIG. 1A wherein each of the attached moieties is a targeting domain such as an scFv or a sdAb. (D) Schematic representation of a subclass of compounds represented by FIG. 1A wherein the two moieties attached to IL- 15 are targeting domains and one of the moieties attached to IL-15Ra is a targeting domain and the other domain attached to IL-15Ra is another protein, such as human serum albumin (HSA).

[0023] FIG. 2 shows two schematics of custom ELIS As used to detect camB7H3 and camCD16 on the TriKE portion of the molecule while the PACC portion of the molecule, in the B7H3 / HSA TriKE-PACC, adheres to the plate via an anti-HSA antibody. The graph shows binding of biotinylated CD16 extracellular domain (ECD) or biotinylated B7H3 by a TriKE-PACC as measured by chemiluminescent ELISA at various concentrations of TriKE-PACC and ECD.

[0024] FIG. 3 shows an SDS-PAGE gel interrogating migration of non-reduced and reduced B7H3 TriKE and B7H3 / HSA TriKE-PACC molecules. Within the TriKE-PACC molecules, the complex migrates slower while the faster migrating band represents TriKE molecule alone. Left lane: Molecular weight standards; Lane 1 : B7H3 / HSA TriKE-PACC co-transfection (nonreduced); Lane 2: B7H3 TriKE (non-reduced); Lane 3: B7H3 / HSA TriKE-PACC co-transfection (reduced); Lane 4: B7H3 TriKE (reduced); Right lane: Molecular weight standards.

[0025] FIG. 4 shows a denaturing SDS-PAGE gel used to assay several (noted) TriKE-PACC compounds. The complex migrates slower while the faster migrating band represents TriKE molecule alone. Left lane: molecular weight standards; Lane 1: CD33 / HSA TriKE-PACC; Lane 2: B7H3 / HSA TriKE-PACC; Lane 3: B7H3 / HSA TriKE-PACC.

[0026] FIG. 5. NK cell response to treatment with B7H3 TriKE or TriKE-PACC. FIG. (A) Response ofNK-92 cells to increasing concentrations of either B7H3 TriKE alone or complexed with HSA-PACC to form a TriKE-PACC (B7H3 / HSA TriKE-PACC). This assay focuses on the potency of the IL-15 or IL-15 / IL-15a potency of the molecules. (B) Expression of CD107a, a marker of NK cell degranulation, in CD56+CD3- NK cells treated in a mixture of PBMCs incubated for five hours with B7H3+ 22RV1 cells (3 nM; N=3). (C) Expression of IFNy in CD56+CD3- NK cells treated in a mixture of PBMCs incubated for five hours with B7H3+ 22RV1 cells (3 nM; N=3). These assays measure the cytolytic potential of the molecule, mediated by the anti-CD16 and anti-B7H3 arms of the TriKE portion. The results show that the PACC arm does not interfere with function in a TriKE-PACC compared with a TriKE.

[0027] FIG. 6 Schematic illustration of TriKE-PACC compounds investigated in Example 4. (A) A schematic illustration of an anti-CD16 and anti-B7H3 TriKE. (B) A schematic illustration of an anti -CD 16 and anti-B7H3 / HSA TriKE- PACC. (C) A schematic illustration of an anti- B7H3 / mesothelin TriKE-PACC.

[0028] FIG 7. Response of NK cells, within PBMCs, to no treatment (PBMC alone) or prostate cancer tumor targets (22RVls) following incubation with different B7H3 TriKEs (Batch 10, TFN 338.1, TFN 325.3, TFN 359) and B7H3 / HSA TriKE-PACC (TFN 362). (A) and (B) show NK cell degranulation (CD 107a) while (C) and (D) show NK cell intracellular cytokine production (Interferon gamma=IFNg). This figure again shows that the PACC portion of the TriKE-PACC does not interfere with activity.

[0029] FIG 8. Schematic representations of linear (TetraKE) or molecular complex (TriKE- PACC) constructs investigated in Example 5. (A) Molecule (linear) approach including an anti- CD16 sdAb (caml6), an IL-15, an anti-B7H3 sdAb (camB7H3), and an anti-ADAM17 scFv (MEDI scFv) arms expressed linearly. (B) A second linear (TetraKE) configuration of a construct including an anti-CD16 sdAb (caml6), an IL-15, an anti-ADAM17 scFv (MEDI scFv), and an anti-B7H3 sdAb (camB7H3) arms expressed linearly. (C) Configuration of a B7H3 / Medi TriKE-PACC where the core TriKE targets B7H3 and the PACC targets ADAMI 7 (Medi). (D) Configuration of a Medi / B7H3 TriKE-PACC, where the core TriKE portion targets ADAM17 (Medi) and the PACC portion targets B7H3.

[0030] FIG. 9 shows a schematic representation of the experimental protocol followed in the Examples to produce TriKE and TriKE-PACC proteins.

[0031] FIG 10. Protein gels used to measure the molecular weight and concentration of the B7H3 / Medi TriKE-PACC described in Example 5. (A) SDS-PAGE gel of a B7H3 / Medi TriKE- PACC used to estimate protein purity and demonstrate the presence of both the PACC arm and the TriKE arm after purification via the HIS tag. (B) Native gel of the B7H3 / Medi TriKE-PACC used to determine whether the complex stayed bound. Lane 1 : BSA; Lane 2: empty control; Lane 3: B7H3 / Medi TriKE-PACC. FIG. 11 shows flow cytometry results demonstrating that B7H3 ligands on HSNCC cells bound the B7H3 sdAb of the B7H3 TriKE, caml6-IL-15-B7H3-MEDI TetraKE, and caml6-IL- 15-MEDI-B7H3 TetraKE.

[0032] FIG. 12 shows flow cytometry results demonstrating that B7H3 ligands on HNSCC cells bind to B7H3 TriKE or as a B7H3 / Medi TriKE-PACC. 10x HIS tag, present in the TriKE portion of the molecule, was leveraged in this assay using a fluorochrome labeled anti-HIS antibody to identify binding of molecules at noted concentrations to B7H3 -expressing HNSCC cells.

[0033] FIG 13. Results from interrogation molecule binding, through anti-B7H3 sdAb portion, to HNSCC cells using an anti-HIS fluorochrome labeled antibody. (A) Flow cytometry results from cells bound to the B7H3 / Medi TriKE-PACC, the two B7H3-Medi TetraKEs (TetraKE 1 and TetraKE 2), the B7H3 TriKE, an isotype control (IC), or unstained cells. (B) Quantification of flow cytometry fluorescence intensity measurements from cells treated with the noted molecules at three concentrations.

[0034] FIG. 14. Flow cytometry results from labeling from tags present on the TriKE side (His- tag) or the PACC side (Strep-2-tag) of the B7H3 / Medi TriKE PACC molecule, at noted concentrations, when incubated with B7H3 -expressing HNSCC cells. This figure essentially demonstrates that the full molecule is present (including the PACC side) when the binding occurs through the TriKE portion of the molecule.

[0035] FIG. 15. Evaluation of NK cell activation against HNSCC tumor targets using anti-B7H3 and Medi TriKE, TetraKE, or TriKE-PACC compounds. PBMCs were cultured with Cal27 and Cal33 tumor targets and noted treatments at 3 nM concentration and NK cell degranulation was assessed after five hours of co-culture using CD107a staining by flow cytometry. The purpose of these assays was to assess how well the TriKE-PACC functions in comparison to the TriKEs alone or the TetraKE molecules.

[0036] FIG. 16. Evaluation of NK cell CD16 and CD62L, typically clipped off the surface of activated NK cells, when PBMCs are incubated with TriKE, TetraKE or TriKE-PACC compounds post strong activation with PMA and lonomycin. Surface expression of CD 16 (left) and CD62L (right) was assessed by flow cytometry on CD56+CD3- NK cells without stimulation (black bar) and with PMA / Ionomycin stimulation (gray bar). This Figure shows that the B7H3 / Medi TriKE-PACC is better at retaining surface CD16 and CD62L than the TetraKE molecules, and similar to Medi antibody alone, Medi TriKE, and B7H3 TriKE + Medi antibody.

[0037] FIG. 17. Quantification of cytotoxicity in Cal27 and Cal33 cells following treatment with TriKE, TetraKE, or TriKE-PACC compounds. NuclightRed labeled Cal27 or Cal33 targets were plated and allowed to attach overnight. NK cells and noted treatments (3 nM) were then added and disappearance of (red) tumor targets was measured every hour for 50 hours. This Figure shows that the TriKE-PACCs provide similar cytotoxic activity to TriKEs alone and are superior than the TetraKE format for B7H3 / Medi targeting.

[0038] FIG. 18. Schematic representations of targeting domains used to prepare TriKE-PACC compounds having anti-CD133 activity tested in Examples 6 and 7.

[0039] FIG. 19. Flow cytometric phenotyping of cells to determine expression of B7H3 and CD133.

[0040] FIG. 20. Flow cytometric phenotyping of cells to determine expression of B7H3 and CD133.

[0041] FIG. 21. Activation ofNK cells against BT16-B7H3 KO (B7H3- / CD133- / PSMA-), C4-2 (B7H3+ / CD133- / PSMA+), HT29 (B7H3+ / CD133+ / PSMA-), and MA 148 (B7H3+ / CD133+ / PSMA-) cells following incubation with TriKE and TriKE-PACC constructs as measured by CD 107a (degranulation) expression. This figure shows that in several contexts both the TriKE and the PACC components within the TriKE-PACC are functional.

[0042] FIG. 22. Activation ofNK cells against BT16-B7H3 KO (B7H3- / CD133- / PSMA-), C4-2 (B7H3+ / CD133- / PSMA+), HT29 (B7H3+ / CD133+ / PSMA-), and MA148 (B7H3+ / CD133+ / PSMA-) cells following incubation with TriKE and TriKE-PACC constructs as measured by IFN-y (intracellular inflammatory cytokine) expression. This figure shows that in several contexts both the TriKE and the PACC components within the TriKE-PACC are functional.

[0043] FIG. 23. Activation ofNK cells against BT16-B7H3 KO (B7H3- / CD133- / PSMA-), C4-2 (B7H3+ / CD133- / PSMA+), HT29 (B7H3+ / CD133+ / PSMA-), and MA 148 (B7H3+ / CD133+ / PSMA-) cells following incubation with TriKE and TriKE-PACC constructs as measured by TNF-a (intracellular inflammatory cytokine) expression. This figure shows that in several contexts both the TriKE and the PACC components within the TriKE-PACC are functional. FIG. 24 shows the gating strategy used to determine proliferating and high proliferating PBMCs when utilizing CellTrace Dye dilution to evaluate proliferation. Briefly, PMBCs are thawed, rested overnight, and labeled with CellTrace dye. Cells are then incubated with different conditions to induce proliferation and assessed for CellTrace Dye dilution seven days after by flow cytometry.

[0044] FIG. 25. Proliferation of cryopreserved PBMCs following incubation with TriKE and TriKE-PACC compounds. Briefly, PMBCs are thawed, rested overnight, and labeled with CellTrace dye. Cells are then incubated with different conditions to induce proliferation and assessed for CellTrace Dye dilution seven days after by flow cytometry.

[0045] FIG. 26. Proliferation of freshly isolated NK cells following incubation with TriKE and TriKE-PACC compounds. Briefly, PMBCs are thawed, rested overnight, and labeled with CellTrace dye. Cells are then incubated with different conditions to induce proliferation and assessed for CellTrace Dye dilution seven days after by flow cytometry.

[0046] FIG. 27. ELISA showing selectivity of antibodies that specifically bind human IL 15- IL15Ra complexes. (A) Schematic illustration of assay. In this exemplary schematic illustration, the capture antibody is a mouse anti -human IL15 / IL15Ra complex antibody and the detection antibody is a biotinylated goat anti-human IL15 / IL15Ra antibody. (B) ELISA binding to capture antibodies specific to human IL15-IL15Ra complexes. IL15 / IL15Ra complexes, such as the TriKE-PACC molecules, are captured by an anti-human IL15-IL15Ra complex antibody immobilized to a plastic plate, whereas molecules containing only IL15, e g. TriKE molecules, do not bind. The bound molecules are detected via a biotinylated anti-IL15 / IL15Ra complex antibody and Streptavidin-HRP.

[0047] FIG. 28. Dual ELISA. (A) Schematic of a Dual ELISA in which the IL15 / IL15Ra complex of the TriKE-PACC binds to the capture antibody immobilized to the plate, and detection is through binding of the natural ligand of the TriKE PACC conjugated to HRP. (B) An example of Dual ELISA data using recombinant human B7H3 conjugated to HRP as the detection molecule.

[0048] FIG. 29 shows denaturing SDS-PAGE used to assay expression and purity of various TriKE-PACC constructs. (A) Coomassie Blue stain. (B) HIS stain (detects epitope tag present on the TriKE arm). Left lane: Molecular weight standards; Lane 1: caml6-IL15-B7H3 / IL15RotSU- CD83; Lane 2: caml6-IL15-B7H3 / IL15RaECD-CD83; Lane 3: caml6-IL15-CD83 / IL15RaSU- B7H3; Lane 4: cam!6-IL15-CD83 / IL15RaECD-B7H3; Lane 5: cam!6-ILl 5-CD83 TriKE; Lane 6: caml6-IL15-B7H3 TriKE; Lane 7: caml6-IL15-B7H3 / IL15RaSU-Nkp30; Lane 8: caml6- IL15-B7H3 / IL15RaECD-Nkp30; Lane 9: caml6-IL15-CD83 / IL15RaECD-B7H3; Lane 10: caml6-IL15-SSl / IL15RaSU-B7H3; Lane 11 : 16-15-SS1 TriKE.

[0049] FIG. 30. Western Blot of a denaturing SDS-PAGE gel probed with a Streptactin-HRP conjugated antibody used to detect the presence of the Strep tag that is expressed only on the PACC arm of the TriKE-PACC complexes. Left lane: Molecular weight standards; Lane 1 : caml6-IL15-B7H3 / IL15RaSU-CD83 (405); Lane 2: caml6-IL15-B7H3 / IL15RaECD-CD83 (406); Lane 3: caml64L15-CD83 / IL15RotSU-B7H3 (419); Lane 4: caml6-IL15- CD83 / IL15RaECD-B7H3 (420); Lane 5: cam 16-IL15-CD83 TriKE (255); Lane 6: caml6-IL15- B7H3 TriKE (385); Lane 7: caml6-IL15-SS l TriKE (124); Lane 8: caml6-IL15- SSl / IL15RaECD B7H3 (427); Lane 9: caml6-IL15-SSl / IL15RaSU B7H3 (428); Lane 10: caml64L15-B7H3 / IL15RaSU-Nkp30 (438); Lane 11 : caml6-IL15-B7H3 / IL15RaECD-Nkp30 (439).

[0050] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0051] Targeting domains, such as antibodies that harness the immune system provide methods of treating conditions such as infection and cancer that specifically target diseased tissue with relatively minimal off-target effects. In particular, therapies that recruit natural killer (NK) cells to promote anti-cancer and anti -infection activities are potentially powerful, targeted tools. While targeting a single antigen is powerful, targeting combinations of antigens can improve specificity to diseased cells. Compounds of the present disclosure target multiple antigens, improving specificity and efficacy of treatment, such as NK cell recruitment.

[0052] This disclosure describes multi-specific compounds that include IL- 15 and at least a portion of IL-15Ra, and methods of producing and using such compounds. Typically, compounds of the present disclosure include a first protein and a second protein. The first protein and the second protein are complexed via the interaction of IL-15 and an IL-15 receptor, such as IL-15Ra or the sushi domain of IL-15Ra, described in greater detail herein. Compounds of the present disclosure typically include one or more targeting domains such as antibodies. In one or more embodiments, a compound includes a first protein including IL-15, a first functional domain, and a second functional domain, and a second protein including IL-15Ra and a third functional domain. In one or more embodiments, at least two of the first functional domain, the second functional domain, and the third functional domain are independently a first targeting domain and a second targeting domain. The remaining functional domain may be a third targeting domain or it may confer a different activity or function to the compound.

[0053] Multi specific compounds

[0054] In one aspect, therefore, this disclosure describes the design, construction, and use of a multispecific molecule that includes two domains capable of driving NK-cell-mediated killing of a cell and an intramolecular NK activating domain capable of generating an NK cell self- sustaining signal. The multispecific molecule can drive NK cell proliferation and / or enhance NK-cell-driven cytotoxicity against, for example, cancer cells or infected cells. In one or more embodiments, the compounds disclosed here are referred to as “multi specific killer engager molecules.” Multispecific killer engagers can refer to Tri-specific Killer Engagers (TriKEs) or other IL- 15 expressing molecules. In one or more embodiments, the compound disclosed herein are referred to as “Poly-Antigen Cytokine-receptor Complexes” (PACCs).

[0055] The PACCs described herein are characterized as one component of a two-molecule complex, typically proteins, that are associated via the IL-15 / IL-15Ra interaction. The IL-15Ra- containing molecule is sometimes referred to herein as the “PACC arm.” In one or more embodiments, the IL- 15 -containing molecule includes two targeting domains. In some such embodiments, the IL-15-containing molecule is referred to as a trispecific killer engager (TriKE); this would be termed a TriKE-PACC. In one or more embodiments, the IL-15- containing molecule includes three targeting domains. In some such embodiments, the IL- 15- containing molecule is referred to as a tetraspecific killer engager (TetraKE); this would be termed a TetraKE-PACC. In some embodiments the IL-15 molecule could have a single targeting domain. In other embodiments the IL- 15 molecule would be monomeric; this would be termed IL15-PACC.

[0056] The PACC arms described herein include at least two functional domains that confer additional functionality to the complex between the IL-15 expressing protein and the PACC. Different types of functional PACC domains are described in greater detail herein.

[0057] FIG. 1 shows several schematic representations of exemplary IL-15-containing proteins coupled with PACCs. Two exemplary classes of PACCs coupled to IL- 15 -containing proteins are disclosed here. A first class is illustrated in FIG. 1 A and includes two functional domains operably linked to IL15Ra. This PACC is shown complexed with an IL- 15 -containing protein in which two functional domains are operably linked to the IL- 15 domain. Another example of a complex that includes a PACC arm is illustrated in FIG. 1C. This exemplary complex includes a PACC that includes a first targeting domain (depicted in gray as an scFv) and a second targeting domain (depicted in white as a single-domain antibody (sdAb). The PACC is complexed with an IL- 15 -containing protein that itself has two functional domains operably linked to the IL- 15 domain. As illustrated, the IL- 15 -containing protein has a first targeting domain (depicted in white), such as an scFv, and a second targeting domain (depicted in gray).

[0058] A second class of complex that contains a PACC arm includes a first protein including a first functional domain and a second functional domain operably linked to IL- 15 and a third functional domain operably linked to IL-15Ra in the PACC (FIG. IB). Another exemplary embodiment of a PACC complex in this class is illustrated schematically in FIG. ID. In this example complex, the IL-15-containing protein includes a first targeting domain (depicted in white), such as an scFv, operably linked to IL-15, and a second targeting domain (depicted in gray), such as an sdAb, also operably linked to IL-15. The PACC arm of the complex includes a functional domain (depicted in white) operably linked to IL-15Ra.

[0059] The terms “multispecific compound” and “multispecific protein” refer to a “fusion molecule” or “fusion protein” and refer to a biologically active polypeptide, including two or more binding domains, with or without a further effector molecule, covalently linked (e.g., fused) by recombinant, chemical, or other suitable method. For example, a binding domain can be linked to another binding domain through a peptide linker sequence. Alternatively, a peptide linker may be used to assist in constructing the fusion molecule.

[0060] As used herein, the term “operably linked” refers to a direct or indirect covalent linking between the domains of the multispecific compound. Thus, two domains that are operably linked may be directly covalently coupled to one another. Conversely, two operably linked domains may be connected by mutual covalent linking to an intervening domain (e.g., a flanking sequence or linker). Two domains may be considered operably linked if, for example, they are separated by the third domain, with or without one or more intervening flanking sequences.

[0061] Domains of a multispecific compound can be in assembled operable linkage with one another using one or more linkers. The term “linker” as used herein refers any bond, small molecule, peptide sequence, or other vehicle that physically links the domains. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage at conditions under which the compound or the antibody remains active. Linkers are classified upon their chemical motifs, well known in the art, including disulfide groups, hydrazine or peptides (cleavable), or thioester groups (non-cleavable). Linkers also include charged linkers, and hydrophilic forms thereof as known in the art.

[0062] Suitable linkers for linking domains of a multispecific compound can include natural linkers, empirical linkers, or a combination of natural and empirical linkers. Natural linkers are derived from multi-domain proteins, which are naturally present between protein domains. Properties of natural linkers such as, for example, length, hydrophobicity, amino acid residues, and / or secondary structure can be exploited to confer desirable properties to a multi-domain compound that includes natural linkers connecting functional domains.

[0063] The studies of linkers in natural multi-domain proteins have led to the generation of many empirical linkers with various sequences and conformations for the construction of recombinant fusion proteins. Empirical linkers can be classified in three types: flexible linkers, rigid linkers, and cleavable linkers. Flexible linkers can provide a certain degree of movement or interaction at the joined domains. Flexible linkers typically include small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, which provide flexibility, and allow for mobility of the connected functional domains. Rigid linkers can successfully keep a fixed distance between domains to maintain their independent functions, which can provide efficient separation of the protein domains and / or sufficiently reduce interference between functional domains. Cleavable linkers can allow one to control release of functional domains in vivo. By taking advantage of unique in vivo processes, cleavable linkers can be cleaved under specific conditions such as the presence of reducing reagents or proteases. This type of linker can reduce steric hindrance, improve bioactivity, and / or achieve independent actions / metabolism of individual domains of recombinant fusion proteins after linker cleavage. Exemplary linkers are reflected in the amino acid sequences of SEQ ID NOs:3-17.

[0064] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system capable of immune surveillance. Like T cells, NK cells deliver a store of membrane penetrating and apoptosis-inducing granzyme and perforin granules. Unlike T cells, NK cells do not require antigen priming and recognize targets by engaging activating receptors in the absence of MHC recognition.

[0065] NK cells express CD16, an activation receptor that binds to the Fc portion of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15, which can induce increased antigen-dependent cytotoxicity, lymphokine- activated killer activity, and / or mediate interferon (IFN) gamma, tumor-necrosis factor (TNF) alpha and / or granulocyte-macrophage colony-stimulating factor (GM-CSF) responses. IL-15 can also drive NK cell proliferation and survival, thus enhancing expansion and persistence of NK cells. All of these IL- 15 -activated functions contribute to improved cancer defense.

[0066] Therapeutically, adoptive transfer of NK cells can, for example, induce remission in subjects with refractory acute myeloid leukemia (AML) when combined with lymphodepl eting chemotherapy and IL-2 to stimulate survival and in vivo expansion of NK cells. This therapy can be limited by lack of antigen specificity and IL-2-mediated induction of regulatory T (Treg) cells that suppress NK cell proliferation and function. Generating a reagent that drives NK cell antigen specificity, expansion, and / or persistence, while bypassing the negative effects of Treg inhibition, can enhance NK-cell-based immunotherapies.

[0067] Complete PACC sequences

[0068] In one or more embodiments, a PACC includes a protein including the amino acid sequence of any one of SEQ ID NOs:34-63 and 73-78. In one or more embodiments, a PACC includes a protein including the amino acid sequence of any one of SEQ ID NOs:34-63 and 79- 90. In one or more embodiments, a PACC includes a protein including an amino acid sequence having at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to any one of SEQ ID NOs:34-63 and 73-78.

[0069] In one or more embodiments, the first protein of the complex, such as the PACC arm, includes a protein including the amino acid sequence of any one of SEQ ID NOs:34-59 and 79- 90. In one or more embodiments, the first protein of the complex, such as the PACC arm, includes a protein including an amino acid sequence having at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to any one of SEQ ID NOs:34-59 and 79-90.

[0070] In one or more embodiments, the second protein bound to a PACC, such as a TriKE, includes a protein including the amino acid sequence of any one of SEQ ID NOs: 60-63 and 73- 78. In one or more embodiments, the second protein of a PACC, such as a TriKE, includes a protein including an amino acid sequence having at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to any one of SEQ ID NOs:60-63 and 73-78.

[0071] In one or more embodiments, the PACC includes an STII domain and a short spacer. The sequence of the STII domain may be “WSHPQFEK,” as is included in SEQ ID NOs: 34-51, 56- 59, 62-65, 72, and 79-90. In one or more embodiments, the PACC includes a short linker adjacent to the STI domain. The sequence of the spacer may be “VDE” as is included in SEQ ID NOs: 34-51, 56-59, 62-65, 72, and 79-90. Alternatively, the PACC may not include an STII domain and / or a short spacer. While the STII domain and spacer may be included for research purposes, it may be desirable to exclude the STII domain and spacer for certain applications, such as certain clinical uses. In one or more embodiments, the PACC may include a sequence including the amino acid sequence of any one of SEQ ID NOs: 34-51, 56-59, 62-65, 72, and 79- 90 without the STII domain and spacer. In one or more embodiments, the PACC may include a sequence including the amino acid sequence of any one of SEQ ID NOs: 34-51, 56-59, 62-65, 72, and 79-90 without the last 11 amino acids.

[0072] IL-15 / IL-15Ra

[0073] Cytokines are a broad category of small proteins (~5 kDa-20 kDa) involved in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm, but are nevertheless involved in autocrine, paracrine, and endocrine signaling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally not hormones or growth factors (despite some overlap in the terminology). Cytokines are produced by a broad range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells, endothelial cells, fibroblasts, and various stromal cells. Cytokines modulate the balance between humoral and cellbased immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations.

[0074] For brevity in this description, reference to an NK activating domain by identifying the cytokine on which it is based includes both the full amino acid sequence of the cytokine, any suitable amino acid fragment of the cytokine, and or a modified version of the cytokine that includes one or more amino acid substitutions. Thus, reference to an “IL-15” NK activating domain includes an NK activating domain that includes the full amino acid sequence of IL- 15, an NK activating domain that includes a fragment of IL-15 (e.g., SEQ ID NO: 18), a functional variant thereof, or an NK activating domain that includes an amino acid substitution compared to the wild-type IL- 15 amino acid sequence. For example, an NK activating domain can include a fragment of IL-15 that includes an N-to-D or an N-to-A amino acid substitution at position 72 of SEQ ID NO: 18, denoted in bold. Reference to position 72 of SEQ ID NO: 18 merely refers to the location of the amino acid substitution regardless of the particular fragment of IL- 15 that may be used as the NK activating domain. Thus, the NK activating domain can include a fragment of IL- 15 other than the fragment reflected in SEQ ID NO: 18 and that fragment can have an N-to-D or an N-to-A amino acid substitution at the position of the alternative IL- 15 fragment that corresponds to position 72 of SEQ ID NO: 18.

[0075] Interleukin 15 (IL-15) is a cytokine that has been important to different cytolytic immune cell populations, particularly NK cells. IL-15 binds to a tripartite receptor including a, P, and y chains. The interaction between IL-15 and IL-15Ra is noncovalent but is very strong. When IL- 15 is bound to IL-15Ra, the complex can bind additional factors that promote NK cell activation. While IL-15 naturally binds to the fully assembled receptor on a cell membrane, it has been found that IL- 15 will bind to only the extracellular domain (ECD) of IL-15Ra.

[0076] As described herein, the compounds of the present disclosure typically include two proteins. A first protein may include IL-15. In one or more embodiments, the first protein is a TriKE. A second protein may include IL-15Ra, such as the IL-15Ra ECD or the IL5Ra sushi domain. In one or more embodiments, the second protein is a PACC arm. The first protein and the second protein may associate via the IL-15 / IL-15Ra interaction to form a compound. In one or more embodiments, a compound includes IL-15. In one or more embodiments, the IL-15 includes the amino acid sequence of SEQ ID NO: 18. In one or more embodiments, the IL-15 includes an amino acid sequence having at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to SEQ ID NO: 18. In one or more embodiments wherein the compound includes IL-15, the IL-15 includes a D8N or D8A amino acid substitution.

[0077] In one or more embodiments, the IL-15Ra ECD includes the amino acid sequence of SEQ ID NO:2. In one or more embodiments, the IL-15Ra ECD includes an amino acid sequence having at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to SEQ ID NO:2.

[0078] IL- 15 can also bind to a truncated “sushi domain” form of IL-15Ra. A sushi domain, also known as a short consensus repeat or a type 1 glycoprotein motif, is a common motif in proteinprotein interaction. Sushi domains have been identified on a number of protein-binding molecules, including complement components Clr, Cis, factor H, and C2m, as well as the nonimmunologic molecules factor XIII and b2- glycoprotein. A typical sushi domain has approximately 60 amino acid residues and contains four cysteines (Ranganathan, Pac. Symp Biocomput.2000: 155-67). The first cysteine can form a disulfide bond with the third cysteine, and the second cysteine can form a disulfide bridge with the fourth cysteine.

[0079] In one or more embodiments, a compound includes a sushi domain of IL-15Ra. In one or more embodiments, the sushi domain of IL-15Ra includes the amino acid sequence of SEQ ID NO: 1. In one or more embodiments, the sushi domain of IL-15Ra includes an amino acid sequence having at least at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to SEQ ID NO:1.

[0080] A “protein coding sequence” or a sequence that “encodes” a particular polypeptide is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.

[0081] Functional domains

[0082] In one or more embodiments, a complex containing a PACC includes at least three functional domains, such as four functional domains. One or more of the functional domains may be a targeting domain. In one or more embodiments, one or more of the functional domains of a PACC provide a function other than targeting.

[0083] For example, in one or more embodiments, one or more functional domains can include an immunologically active protein or immunologically active fragment (i.e., a peptide) thereof. Attaching a molecule recognized by the immune system and naturally kept in circulation may improve bioactivity of a complex containing a PACC. For example, when human serum albumin (HSA) was attached to the PACC arm of Example 3, the biological effects of the complex were amplified. While exemplified herein in the context of an exemplary embodiment in which HSA provides prolonged bioavailability of a PACC-containing complex, the compositions and methods described herein can involve the use of other molecules associated with extended bioavailability to prolong bioavailability of a PACC-containing complex.

[0084] In one or more embodiments, a PACC arm includes albumin. Different sources of albumin may be used depending on the type of cell to be treated with the complex containing a PACC. For example, human serum albumin may be used in a PACC arm intended for use in humans. In one or more embodiments, a PACC arm includes human serum albumin, bovine serum albumin, murine serum albumin, or serum albumin from any primate.

[0085] In one or more embodiments wherein the PACC arm includes human serum albumin, the PACC includes the amino acid sequence of SEQ ID NO:22.

[0086] Targeting domains In one or more embodiments, a complex containing a PACC includes at least one targeting domain. In one or more embodiments, a targeting domain includes an antibody. In one or more embodiments, a targeting domain includes a ligand, such as a ligand of a receptor. In one or more embodiments, a targeting domain includes an extracellular domain (ECD) of a membrane-associated molecule.

[0087] The term “antibody” refers to a molecule that contains at least one antigen binding site that immunospecifically binds to a particular antigen target of interest. The term “antibody” thus includes but is not limited to a full length antibody and / or its variants, a fragment thereof, peptibodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, and antibody mimetics that mimic the structure and / or function of an antibody or a specified fragment or portion thereof, including single chain antibodies and fragments thereof. Thus, as used herein, the term “antibody” encompasses antibody fragments capable of binding to a biological molecule (such as an antigen or receptor) or a portion thereof, including but not limited to Fab, Fab' and F(ab')2, pFc', Fd, a single domain antibody (sdAb), a variable fragment (Fv), a single-chain variable fragment (scFv) or a disulfide-linked Fv (sdFv); a PACC or a bivalent PACC; a linear antibody; a single-chain antibody molecule; and a multispecific antibody (e.g., a tribody) formed from antibody fragments. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or subclass.

[0088] While the principles and compound architectures described herein are anticipated to apply to many combinations of targeting domains, several specific combinations are exemplified in the embodiments.

[0089] The PACC arm may include a targeting domain that selectively targets a molecule associated with a cancer, such as a tumor. In one or more embodiments, the PACC arm includes a targeting domain that selectively targets B7H3 (sometimes referred to as CD276), mesothelin, prostate-specific membrane antigen (PSMA), A Disintegrin and Metalloprotease 17 (ADAMI 7), PD-L1, PD-1, TGFP receptor 1 (RI), TGFP receptor 2 (RII), CTLA-4, CXCL10, or CX3CL1. In one or more embodiments, the PACC includes a TGFP RI ECD, a TGFP RII ECD, a CTLA-4 ECD, or a PD-1 ECD. The PACC arm may additionally include a targeting domain that selectively targets an NK cell engager domain (e.g., CD16, CD16+CD2, CD16+DNAM, NKp46, NKp30, CD16+NKp46, NKG2D, NKG2C), and an NK activating domains (e.g., IL-15, IL-12, IL-18, IL- 21, or other NK cell enhancing cytokine, chemokine, and / or activating molecule), with each domain operably linked to the other domains. As used herein, the terms “selectively targets” and “selectively binds” refer to the ability to differentiate between two or more alternatives such as, for example, having differential affinity, to any degree, for a particular target.

[0090] In one or more embodiments, a compound includes a targeting domain including the amino acid sequence of any one of SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31. In one or more embodiments, a compound includes a targeting domain that is a functional variant of any one of SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO 21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.

[0091] As used herein, a protein is a “functional variant” to a reference protein if the amino acid sequence of the protein possesses a specified amount of identity compared to the reference protein and retains the activity of the reference protein. Structural similarity of two proteins can be determined by aligning the residues of the two proteins (for example, a candidate protein and the protein of, for example, SEQ ID NO:1) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate protein is the protein being compared to the reference protein (e.g., SEQ ID NO: 1). A candidate protein can be isolated, for example, from an animal, or can be produced using recombinant techniques, or chemically or enzymatically synthesized.

[0092] A pair-wise comparison analysis of amino acid sequences can be carried out using, for example, the BESTFIT algorithm in the GCG package (version 10.2, Madison WI). Alternatively, proteins may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website. The default values for all BLAST 2 search parameters may be used, including matrix = BLOSUM62; open gap penalty = 11 , extension gap penalty = 1, gap x_dropoff = 50, expect = 10, wordsize = 3, and filter on.

[0093] In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions. A conservative substitution for an amino acid in a protein may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free -OH is maintained; and Gin for Asn to maintain a free -NH2. Likewise, biologically active analogs of a protein containing deletions or additions of one or more contiguous or noncontiguous amino acids that do not eliminate a functional activity of the protein are also contemplated.

[0094] Generally, portions of antibodies such as SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO 23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31 other than the CDRs are more amenable to variation while maintaining functionality — i.e., specifically bind to the intended antigen.

[0095] Variants of the disclosed sequences also include proteins, or full-length protein, that contain substitutions, deletions, or insertions into the protein backbone, that would still leave at least about 70% homology to the original protein over the corresponding portion. A yet greater degree of departure from homology is allowed if like-amino acids, i.e., conservative amino acid substitutions, do not count as a change in the sequence. Examples of conservative substitutions involve amino acids that have the same or similar properties. Illustrative amino acid conservative substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine to leucine.

[0096] In one or more aspects, a compound can include additional sequences, such as, for example, amino acids appended to the C-terminal or N-terminal of the functional domain, IL-15, or IL-15Ra. Such modifications can, for example, facilitate purification by trapping on columns, the use of antibodies, or facilitate recovery when expressed recombinantly in a microbe. Such tags include, for example, a histidine-rich tag (e.g., SEQ ID NO:63) that allows purification of proteins on nickel columns and / or one or more linkers between domains of a protein. Such gene modification techniques and suitable additional sequences are well known in the molecular biology arts. In one or more embodiments, the C-terminal and / or N-terminal modification may be cleaved from the protein before being incorporated into, for example, a pharmaceutical composition. In other embodiments, retaining a C-terminal or N-terminal modification may be desired for a given application — i.e., to facilitate immobilization to a substrate.

[0097] The targeting domain can include any moiety that selectively binds to a target such as, for example, a tumor cell, a target in the cancer stroma, or an immobilized cell. Thus, a targeting domain can include, for example, an antibody. In one or more embodiments, an antibody can include a polypeptide as described in detail herein. In one exemplary embodiment, the complex containing the PACC can include one or more of the complementarity-determining regions (CDRs) of any one of SEQ ID NOs: 19, 21, 23, 24, 26, 29-31, 34, 35, 37-43, 48-51, 54-57, 60- 62, 64, 65, 67, 68, 70, 71, or 73-90. In one or more embodiments, the complex containing the PACC can include two or more of the CDRs of any one of SEQ ID NOs: 19, 21, 23, 24, 26, 29- 31, 34, 35, 37-43, 48-51, 54-57, 60-62, 64, 65, 67, 68, 70, 71, or 73-90. In one or more embodiments, the antibody can include any one of SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO 24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID N0:31. In one or more embodiments, the PACC can include any of the CLE 2a variants of any one of SEQ ID NOs:73-78. Suitable alternative variants are described herein.

[0098] In one or more embodiments, the PACC or the IL- 15 -containing protein can include an NK engaging domain. The NK engaging domain can include any moiety that binds to and / or activates an NK cell and / or any moiety that blocks inhibition of an NK cell. In one or more embodiments, the NK engaging domain can include an antibody that selectively binds to a component of the surface of an NK cell. In other embodiments, the NK engaging domain can include a ligand or small molecule that selectively binds to a component of the surface of an NK cell. Thus, for brevity, reference to an antibody that selectively binds to a component of the surface of an NK cell includes any antibody fragment (as defined above) that exhibits the described binding character. Similarly, reference to a ligand that selectively binds to a component of the surface of an NK cell includes any fragment of the ligand that exhibits the described binding character.

[0099] In one or more embodiments, the NK engaging domain can selectively bind to a receptor at least partially located at the surface of an NK cell. In certain embodiments, the NK engaging domain can serve a function of binding an NK cell and thereby bring the NK cell into spatial proximity with a target to which the targeting domain selectively binds. In certain embodiments, however, the NK engaging domain can selectively bind to a receptor that activates the NK cell and, therefore, also possess an activating function. As described above, activating the CD 16 receptor can elicit antibody-dependent cell-mediated cytotoxicity. Thus, in certain embodiments, the NK engaging domain can include at least a portion of an anti-CD16 receptor antibody effective to selectively bind to the CD16 receptor (e.g., SEQ ID NO: 19 or SEQ ID NO:29). Another example of this in NKp30L (SEQ ID NO:20), which engages the activating receptor NKp30 on NK cells. In other embodiments, the NK engager cell domain may interrupt mechanisms that inhibit NK cells. In such embodiments, the NK engager domain can include, for example, anti-PDl / PDLl, the PD1 ECD (SEQ ID NO:25), anti-NKG2A, anti-TIGIT, anti-killer- immunoglobulin receptor (KIR), the TGFPRII (SEQ ID NO:32), and / or any other inhibition blocking domain.

[0100] One can design the NK engaging domain to possess a desired degree of NK selectivity and, therefore, a desired immune engaging character. For example, CD 16 has been identified as Fc receptors FcyRIIIa (CD16a) and FcyRIIIb (CD16b). These receptors bind to the Fc portion of IgG antibodies that then activates the NK cell for antibody-dependent cell-mediated cytotoxicity. Anti-CD16 antibodies selectively bind to NK cells, but also can bind to neutrophils. Anti-CD16a antibodies selectively bind to NK cells, but do not bind to neutrophils. A complex containing a PACC that includes an NK engaging domain that includes an anti-CD16a antibody can bind to NK cells but not bind to neutrophils. Thus, in circumstances where one may want to engage NK cells but not engage neutrophils, one can design the NK engaging domain of the complex containing the PACC to include an anti-CD16a antibody.

[0101] In one or more embodiments, the NK cell engaging domain can involve the use of a humanized CD 16 engager derived from an animal nanobody. While an scFv has a heavy variable chain component and a light variable chain component joined by a linker, a nanobody consists of a single monomeric variable chain — i.e., a variable heavy chin or a variable light chain — that is capable of specifically engaging a target. A single domain antibody (sdAb) may be derived from an antibody of any suitable animal such as, for example, a camelid (e.g., a llama or camel) or a cartilaginous fish. A single domain antibody can provide superior physical stability, an ability to bind deep grooves, and increased production yields compared to larger antibody fragments.

[0102] In one exemplary embodiment, an sdAb-based NK engager molecule can involve a humanized CD 16 nanobody derived from a llama nanobody (GenBank sequence EF561291 ; Behar et al., 2008. Protein Eng Des Sei. 21(1): 1-10), termed EF91. Upon confirming functionality of the molecule, the CDRs were cloned into a humanized camelid scaffold (Vincke et al., 2009. J Biol Chem. 284(5):3273 -3284) to humanize the CD16 engager (SEQ ID NO: 19). The use of a humanized camelid sdAb in the NK engaging domain of a complex containing a PACC can increase drug yield, increase stability, and / or increase NK-cell-mediated antibodydependent cellular cytotoxicity (ADCC) efficacy.

[0103] While described herein in the context of various embodiments in which the NK engaging domain includes an anti-CD16 sdAb or anti -CD 16 scFv, the NK engaging domain can include any antibody or other ligand that selectively binds to CD 16. Moreover, the NK engaging domain can include an antibody or ligand that selectively binds to any NK cell receptor such as, for example, the cell cytotoxicity receptor 2B4, low affinity Fc receptor CD 16, killer immunoglobulin like receptors (KIR), CD2, NKG2A, TIGIT, NKG2C, LIR-1, and / or DNAM-1.

[0104] In one aspect, the immune cell is a T cell or a natural killer (NK) cell. In another aspect, the immune cell is an NK cell; and the immune cell engaging domain includes a ligand or antibody that specifically binds to CD 16. In one or more aspects, the antibody that specifically binds to CD16 includes an scFv, an F(ab)2, a Fab, or a single domain antibody.

[0105] As explained in more detail above, “antibody” refers generally to an immunoglobulin or a fragment thereof and thus encompasses a monoclonal antibody, a fragment thereof (e.g., scFv, Fab, F(ab’)2, Fv, sdAb, or other modified form of an antibody, including a humanized form of an antibody or fragment thereof). Thus, for brevity, reference to an antibody that selectively binds to a target includes any antibody or antibody fragment that exhibits the described binding character. Similarly, reference to an antibody that selectively binds to CD 16 (or any other NK cell receptor) includes any antibody or antibody fragment that exhibits the described binding character. In one or more aspects, the immune cell engaging domain includes a ligand or antibody that specifically binds to CD 16 such as, for example, an antibody fragment having the amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO:29.

[0106] In one or more embodiments, the NK engaging domain and the targeting domain can be linked using any one of the linkers reflected in SEQ ID NOs:3-17.

[0107] In another aspect, the complex containing the PACC further includes an additional immune cell activating domain. In one or more embodiments, the immune cell can be an NK cell and the immune cell activating domain includes a NK activating cytokine or a functional portion thereof.

[0108] In one or more embodiments, the PACC includes a CLEC12 binding single domain antibody (sdAb). The CLEC12 sdAb may include the amino acid sequence of any one of SEQ ID NOs:73-78. A CLEC12 binding PACCs may provide a dual targeting approach. In one or more embodiments, a PACC including a CLEC12 binding domain may be used to treat a myeloid malignancy. In one or more embodiments, a PACC including a CLEC12 sdAb is be used in combination with TriKEs targeting CD33.

[0109] The NK activating domain can include a “immune cell activating domain”, e.g., an amino acid sequence that activates NK cells, promotes sustaining NK cells, or otherwise promotes NK cell activity. For example, NK cells are responsive to a variety of cytokines including, but not limited to, IL-2, which is involved in NK cell homeostasis, proliferation, survival, activation, and / or development. IL- 15 and IL-2 share several signaling components, including the IL-2 / IL- 15R0 (CD 122) and the common gamma chain (CD 132). Unlike IL-2, IL- 15 does not stimulate Tregs, allowing for NK cell activation while bypassing Treg inhibition of the immune response. Besides promoting NK cell homeostasis and proliferation, IL-15 can rescue NK cell functional defects that can occur in the post-transplant setting. IL-15 also can stimulate CD8+T cell function, further enhancing its immunotherapeutic potential. In addition, based on pre-clinical studies, toxicity profdes of IL-15 may be more favorable than IL-2 at low doses.

[0110] Therefore, the NK activating domain can be, or can be derived from, one or more cytokines that can activate and / or sustain NK cells. As used herein, the term “derived from” refers to an amino acid fragment of a cytokine (e.g., IL-2) that is sufficient to provide NK cell activating and / or sustaining activity. In embodiments that include more than one NK activating domain, the NK activating domains may be provided in series or in any other combination. Additionally, each cytokine-based NK activating domain can include either the full amino acid sequence of the cytokine or may be an amino acid fragment, independent of the nature of other NK activating domains included in the complex bearing the PACC. Exemplary cytokines on which an NK activating domain may be based include, for example, IL-15, IL-18, IL-12, and IL- 21. Thus, in one or more embodiments, the IL- 15 -containing compound may have one IL-15- containing domain that complexes with the IL-15Ra of the PACC and a second IL- 15 domain that includes a sufficient portion of IL-15 (or a variant thereof) sufficient to activate NK cells.

[0111] In one or more embodiments, PACCs include domains linked by one or more of the linkers reflected in SEQ ID NOs:3-17 or a combination of the linkers reflected in SEQ ID NOs:3-17.

[0112] In another aspect, this disclosure describes an isolated nucleic acid sequence that encodes any embodiment of a PACC arm or complexed IL-15-containing protein described herein. Given the amino acid sequence of any PACC arm or IL-15-containng protein, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods.

[0113] As used herein, the term “nucleic acid” or “oligonucleotide” refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, nucleic acids conjugates, and oligonucleotides. A nucleic acid may be single-stranded, double-stranded, linear, or covalently circularly closed molecule. A nucleic acid can be isolated. The term “isolated nucleic acid” means, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, (iv) was synthesized, for example, by chemical synthesis, or (vi) extracted from a sample. A nucleic acid might be introduced — i.e., transfected — into cells. When RNA is used to transfect cells, the RNA may be modified by stabilizing modifications, capping, or polyadenylation.

[0114] As used herein “amplified DNA” or “PCR product” refers to an amplified fragment of DNA of defined size. Various techniques are available and well known in the art to detect PCR products. PCR product detection methods include, but are not restricted to, gel electrophoresis using agarose or polyacrylamide gel and adding ethidium bromide staining (a DNA intercalant), labeled probes (radioactive or non-radioactive labels, southern blotting), labeled deoxyribonucleotides (for the direct incorporation of radioactive or non-radioactive labels) or silver staining for the direct visualization of the amplified PCR products; restriction endonuclease digestion, that relies agarose or polyacrylamide gel or high-performance liquid chromatography (HPLC); dot blots, using the hybridization of the amplified DNA on specific labeled probes (radioactive or non-radioactive labels); high-pressure liquid chromatography using ultraviolet detection; electro-chemiluminescence coupled with voltage-initiated chemical reaction / photon detection; and direct sequencing using radioactive or fluorescently labeled deoxyribonucleotides for the determination of the precise order of nucleotides with a DNA fragment of interest, oligo ligation assay (OLA), PCR, qPCR, DNA sequencing, fluorescence, gel electrophoresis, magnetic beads, allele specific primer extension (ASPE) and / or direct hybridization.

[0115] Generally, nucleic acid can be extracted, isolated, amplified, or analyzed by a variety of techniques such as those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 pages (2012); or as described in U.S. Pat. 7,957,913; U.S. Pat. 7,776,616; U.S. Pat. 5,234,809; U.S. Pub. 2010 / 0285578; and U.S. Pub. 2002 / 0190663. Examples of nucleic acid analysis include, but are not limited to, sequencing and DNA-protein interaction. Sequencing may be by any method known in the art. DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, and next generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina / Solexa sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing. Separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes.

[0116] Methods of making multispecific compounds

[0117] In another aspect, the present disclosure describes methods of making multispecific compounds such as complexes containing PACCs. Typically, a complex containing a PACC includes two molecules, wherein the molecules are non-covalently attached. In one or more embodiments, the complex includes a first molecule including IL-15 and a second molecule including IL-15Ra, termed the PACC arm, wherein the first molecule is associated with the second molecule via the IL-15 / IL-15Ra interaction to form a complex.

[0118] Accordingly, in one or more embodiments, a method of making a complex containing a PACC includes associating an IL-15 domain with an IL-15Ra domain.

[0119] In one or more embodiments, a method of making a complex containing a PACC includes providing a first protein including IL-15, providing a second protein including IL-15Ra (the PACC), and contacting the first protein with the second protein to form a complex via the IL-15 / IL-15Ra interaction. In one or more embodiments, providing a first protein can include expressing (e.g., transcribing and translating) a nucleic acid sequence encoding the first protein. In one or more embodiments, providing the second protein can include expressing (e.g., transcribing and translating) a nucleic acid sequence encoding the second protein. In one or more embodiments, a single cell may be co-transfected to express both the first protein and he second protein.

[0120] In one or more embodiments, contacting the first protein with the second protein involves allowing the components of the PACC complex that have been expressed and secreted by transfected cells to form PACC complexes (e.g., self-assembly) in the culture supernatant. In one or more alternative embodiments, the PACC complex components may self-assemble within the cell prior to being released from the cell. In one or more embodiments, contacting the first protein with the second protein occurs inside of a cell. For example, a cell may be transfected with a first nucleic acid encoding the first protein and a second nucleic acid encoding the second protein, each of the first and second nucleic acids may be expressed to provide the first and second proteins, and the first protein may contact the second protein within the cytoplasm to form a complex containing at PACC. The complex is secreted and isolated from the supernatant via an epitope tag, size exclusion chromatography, or other forms of affinity chromatography.

[0121] Nucleic acids

[0122] In another aspect, this disclosure describes isolated nucleic acid sequences encoding any of the proteins described herein.

[0123] In another aspect, this disclosure describes a host cell including any of the isolated nucleic acid sequences and / or proteins described herein.

[0124] The nucleic acid constructs of the present invention may be introduced into a host cell to be altered, thus allowing expression of the chimeric protein within the cell, thereby generating a genetically engineered cell. A variety of methods are known in the art and suitable for introducing a nucleic acid into a cell, including viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Other methods of transfection include proprietary transfection reagents such as EXPIFECTAMINE, LIPOFECT AMINE (Thermo Fisher Scientific, Inc., Waltham, MA), HILYMAX (Dojindo Molecular Technologies, Inc., Rockville, MD), FUGENE (Promega Corp., Madison, WI), FETPEI (Polyplus Transfection, Illkirch, France), EFFECTENE (Qiagen, Hilden, Germany) and DreamFect (OZ Biosciences, Inc USA, San Diego, CA).

[0125] The nucleic acid constructs described herein may be introduced into a host cell to be altered, thus allowing expression within the cell of the protein encoded by the nucleic acid. A variety of host cells are known in the art and suitable for protein expression. Examples of typical cell used for transfection and protein expression include, but are not limited to, a bacterial cell, a eukaryotic cell, a yeast cell, an insect cell, or a plant cell such as, for example, E. coli, Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e.g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F.

[0126] In one or more aspects, the host cell is a T cell, an NK cell, or a macrophage. Pharmaceutical compositions and methods

[0127] In a further aspect, this disclosure describes a pharmaceutical composition that includes any one of the PACCs and complexes containing PACCs described herein and a pharmaceutically acceptable carrier.

[0128] A multispecific compound, such as a complex containing a PACC as described herein may be formulated with a pharmaceutically acceptable carrier. As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial, and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and / or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with a complex containing a PACC without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0129] A multispecific compound, such as a complex containing a PACC may therefore be formulated into a pharmaceutical composition. The pharmaceutical composition may be formulated in a variety of forms adapted to a preferred route of administration. Thus, a composition can be administered via known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.). A pharmaceutical composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). A composition also can be administered via a sustained or delayed release. Thus, a multispecific compound, such as a complex containing a PACC may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The composition may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. The formulation may further include one or more additives including such as, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like.

[0130] A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing a multispecific compound or a complex containing a PACC into association with a carrier that constitutes one or more accessory ingredients. In general, a formulation may be prepared by uniformly and / or intimately bringing the active molecule into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

[0131] The amount of multispecific compound or a complex containing a PACC administered can vary depending on various factors including, but not limited to, the specific complex containing a PACC being used, the weight, physical condition, and / or age of the subject, and / or the route of administration. Thus, the absolute weight of the complex containing a PACC included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight, and physical condition of the subject, and / or the method of administration. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of multispecific compound or a complex containing a PACC effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.

[0132] In one or more embodiments, the method can include administering sufficient multispecific compound or a complex containing a PACC to provide a dose of, for example, from about 100 ng / kg to about 50 mg / kg to the subject, although in some embodiments the methods may be performed by administering the multispecific compound or a complex containing a PACC in a dose outside this range. In one or more of these embodiments, the method includes administering sufficient the multispecific compound or a complex containing a PACC to provide a dose of from about 10 pg / kg to about 5 mg / kg to the subject, for example, a dose of from about 100 ug / kg to about 1 mg / kg.

[0133] In another aspect, this disclosure describes a method including administering to a subject a multispecific compound in an amount effective to induce NK-mediated killing of a cell, the multi specific compound including a targeting domain including one of the complexes containing a PACC described herein.

[0134] In another aspect, this disclosure describes a method for stimulating expansion of NK cells in vivo including administering to a subject an effective amount of multispecific compound including a targeting domain including one of the complexes containing a PACC described herein.

[0135] In another aspect, this disclosure describes methods of killing a target cell in a subject. Generally, the method includes administering to the subject a complex containing a PACC in an amount effective to induce NK-mediated killing of the target cells. “Treat” or variations thereof refer to reducing, limiting progression, ameliorating, or resolving, to any extent, the symptoms or signs related to a condition. As used herein, “ameliorate” refers to any reduction in the extent, severity, frequency, and / or likelihood of a symptom or clinical sign characteristic of a particular condition; “symptom” refers to any subjective evidence of disease or of a subject’s condition; and “sign” or “clinical sign” refers to an objective physical finding relating to a particular condition capable of being found by one other than the subject.

[0136] A “treatment” may be therapeutic or prophylactic. “Therapeutic” and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition. “Prophylactic” and variations thereof refer to a treatment that limits, to any extent, the development and / or appearance of a symptom or clinical sign of a condition. Generally, a “therapeutic” treatment is initiated after the condition manifests in a subject, while “prophylactic” treatment is initiated before a condition manifests in a subject. Thus, in certain embodiments, the method can involve prophylactic treatment of a subject at risk of developing a condition. “At risk” refers to a subject that may or may not actually possess the described risk. Thus, for example, a subject “at risk” for developing a specified condition is a subject that possesses one or more indicia of increased risk of having, or developing, the specified condition compared to individuals who lack the one or more indicia, regardless of the whether the subject manifests any symptom or clinical sign of having or developing the condition. Exemplary indicia of a condition can include, for example, genetic predisposition, ancestry, age, sex, geographical location, lifestyle, or medical history. Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0137] In one or more cases, the treatment can involve administering a complex containing a PACC to a subject so that the complex can stimulate endogenous NK cells in vivo. Using a complex containing a PACC as a part of an in vivo protocol can make NK cells antigen specific with simultaneous co-stimulation, enhancement of survival, and expansion, which may be antigen specific. In other cases, the complex containing a PACC can be used in vitro as an adjuvant to NK cell adoptive transfer therapy. The terms “administration of’ and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical, or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intrasternal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration.

[0138] Accordingly, a complex containing a PACC may be administered before, during, or after the subject first exhibits a symptom or clinical sign of the condition. Treatment initiated before the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the likelihood that the subject experiences clinical evidence of the condition compared to a subject to which a complex containing a PACC is not administered, decreasing the severity of symptoms and / or clinical signs of the condition, and / or completely resolving the condition. Treatment initiated after the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the severity of symptoms and / or clinical signs of the condition compared to a subject to which the composition is not administered, and / or completely resolving the condition.

[0139] The complex containing a PACC used can be any embodiment of the complexes containing PACC arm described herein having a targeting domain that selectively binds to an appropriate target cell population. In one or more cases, the target cell can include a tumor cell so that the method can involve treating cancer associated with the tumor cells. Thus, in one or more embodiments, the method can include ameliorating at least one symptom or clinical sign of the tumor.

[0140] In embodiments in which the target cell includes a tumor cell, the method can further include surgically resecting the tumor and / or reducing the size of the tumor through chemical (e.g., chemotherapeutic) and / or radiation therapy. Exemplary tumors that may be treated include tumors associated with prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer and / or hematopoietic cancer.

[0141] Thus, in one or more embodiments, treating a subject includes a subject having, or at risk of having cancer. Generally, the method includes administering to the subject an effective amount of multispecific compound including a targeting domain including one of the complexes containing PACC proteins described herein. As used herein, the term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to other sites (secondary sites, metastases) which differentiates cancer (malignant tumor) from benign tumor. Virtually any organ can be affected, meaning more than 100 types of cancer can affect humans. Cancers can result from many causes including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to an environmental pollutant, tobacco and / or alcohol use, obesity, poor diet, lack of physical activity, or any combination thereof. As used herein, “neoplasm” or “tumor” (and grammatical variations thereof) means new and abnormal growth of tissue, which may be benign or cancerous. In a related aspect, the neoplasm is indicative of a neoplastic disease or disorder, including but not limited, to various cancers. For example, such cancers can include prostate, pancreatic, biliary, colon, rectal, liver, kidney, lung, testicular, breast, ovarian, brain, and head and neck cancers, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

[0142] Exemplary cancers described by the national cancer institute include: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS- Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma / Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma / Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas / Carcinoids, Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma / Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood;

[0143] Ewing’s Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin’ s Lymphoma, Adult; Hodgkin’s Lymphoma, Childhood; Hodgkin’s Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi’s Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS — Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin’s, Adult; Lymphoma, Hodgkin’s; Childhood; Lymphoma, Hodgkin’s During Pregnancy; Lymphoma, Non-Hodgkin’s, Adult; Lymphoma, Non-Hodgkin’s, Childhood; Lymphoma, Non-Hodgkin’s During Pregnancy;

[0144] Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom’s; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin’s Lymphoma, Adult; Non-Hodgkin’s Lymphoma, Childhood; Non-Hodgkin’s Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer;

[0145] Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor;

[0146] Pancreatic Cancer; Pancreatic Cancer, Childhood, Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm / Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer;

[0147] Pregnancy and Hodgkin’ s Lymphoma; Pregnancy and Non-Hodgkin’s Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma;

[0148] Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland’s Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi’s; Sarcoma (Osteosarcoma) Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant;

[0149] Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood;

[0150] Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom’s Macro globulinemia; and Wilms’ Tumor.

[0151] In one or more embodiments, the cancer can include or involve prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, and / or hematopoietic cancer.

[0152] In one aspect, the multispecific compound is administered prior to, simultaneously with, or following chemotherapy, surgical resection of a tumor, or radiation therapy.

[0153] In one or more embodiments, a multispecific compound or complex containing a PACC may be administered, for example, from a single dose to multiple doses per week, although in one or more embodiments the method can be performed by administering a complex containing a PACC at a frequency outside this range. In certain embodiments, a multispecific compound or complex containing a PACC may be administered from about once per month to about five times per week.

[0154] In one or more embodiments, the method further includes administering one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered before, after, and / or coincident to the administration of a multispecific compound or complex containing a PACC. A multispecific compound or complex containing a PACC and the additional therapeutic agents may be co-administered. As used herein, “co-administered” refers to two or more components of a combination administered so that the therapeutic or prophylactic effects of the combination can be greater than the therapeutic or prophylactic effects of either component administered alone. Two components may be co-administered simultaneously or sequentially. Simultaneously co-administered components may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes cases in which the components are administered so that each component can be present at the treatment site at the same time. Alternatively, sequential co-administration of two components can include cases in which at least one component has been cleared from a treatment site, but at least one cellular effect of administering the component (e.g., cytokine production, activation of a certain cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, a co-administered combination can, in certain circumstances, include components that never exist in a chemical mixture with one another. In other embodiments, the multispecific compound or complex containing a PACC and the additional therapeutic agent may be administered as part of a mixture or cocktail. In one or more aspects, the administration of the multispecific compound or complex containing a PACC may allow for the effectiveness of a lower dosage of other therapeutic modalities when compared to the administration of the other therapeutic agent or agents alone, thereby decreasing the likelihood, severity, and / or extent of the toxicity observed when a higher dose of the other therapeutic agent or agents is administered.

[0155] The term “chemotherapeutic agent” as used herein refers to any therapeutic agent used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, tioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, panitumamab, Erbitux™ (cetuximab), matuzumab, IMC-IIF 8, TheraCIM hR3, denosumab, Avastin™ (bevacizumab), Humira™ (adalimumab), Herceptin™ (trastuzumab), Remicade™ (infliximab), rituximab, Synagis™ (palivizumab), Mylotarg™ (gemtuzumab oxogamicin), Raptiva™ (efalizumab), Tysabri™ (natalizumab), Zenapax™ (dacliximab), NeutroSpec™ (Technetium (99mTc) fanolesomab), tocilizumab, ProstaScint™ (Indium-Ill labeled Capromab Pendetide), Bexxar™ (tositumomab), Zevalin™ (ibritumomab tiuxetan (IDEC-Y2B8) conjugated to yttrium 90), Xolair™ (omalizumab), MabThera™ (Rituximab), ReoPro™ (abciximab), MabCampath™ (alemtuzumab), Simulect™ (basiliximab), LeukoScan™ (sulesomab), CEA-Scan™ (arcitumomab), Verluma™ (nofetumomab), Panorex™ (Edrecolomab), alemtuzumab, CDP 870, natalizumab Gilotrif™ (afatinib), Lynparza™ (olaparib), Peijeta™ (pertuzumab), Otdivo™ (nivolumab), Bosulif™ (bosutinib), Cabometyx™ (cabozantinib), Ogivri™ (trastuzumab-dkst), Sutent™ (sunitinib malate), Adcetris™ (brentuximab vedotin), Alecensa™ (alectinib), Calquence™ (acalabrutinib), Yescarta™ (ciloleucel), Verzenio™ (abemaciclib), Keytruda™ (pembrolizumab), Aliqopa™ (copanlisib), Nerlynx™ (neratinib), Imfinzi™ (durvalumab), Darzalex™ (daratumumab), Tecentriq™ (atezolizumab), and Tarceva™ (erlotinib). Examples of immunotherapeutic agent include, but are not limited to, interleukins (IL-2, IL-7, IL- 12), cytokines (Interferons, G-CSF, imiquimod), chemokines (CCL3, CC126, CXCL7), immunomodulatory imide drugs (thalidomide and its analogues).

[0156] In one or more aspects, the chemotherapy is selected from the group consisting of altretamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphane, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamaide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, tioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, and vinorelbine.

[0157] In one or more embodiments, the method can include administering sufficient multispecific compound or a complex containing a PACC as described herein and administering the at least one additional therapeutic agent demonstrates therapeutic synergy. In one or more aspects of the methods of the present invention, a measurement of response to treatment observed after administering both a multispecific compound or a complex containing a PACC as described herein and the additional therapeutic agent is improved over the same measurement of response to treatment observed after administering either the multispecific compound or the complex containing a PACC or the additional therapeutic agent alone.

[0158] The term “subject” as used herein refers to any individual or subject to which the subject methods are performed. In many embodiments, the subject is human, although the subject may be any non-human animal. Suitable non-human animals include, but are limited to, a vertebrate such as a rodent (including a mouse, a rat, a hamster, or a guinea pig), a cat, a dog, a rabbit, a farm animal (including a cow, a horse, a goat, a sheep, a pig, a chicken, etc ), or a primate (including a monkey, a chimpanzee, an orangutan, or a gorilla).

[0159] In one or more embodiments of this aspect, the complex containing a PACC can include an immune cell activating domain that includes IL-15 or a functional portion thereof, operably linked to the NK engaging domain. In one or more embodiments, the PACC arm can include one or more of the amino acid sequences as set forth in any one of SEQ ID NOs:34-65.

[0160] In another aspect, this disclosure describes a targeted therapeutic compound that includes a targeting domain and a therapeutic domain linked to the targeting domain. The targeting domain includes any embodiment of the complex containing a PACC and PACC arms described herein. In one or more embodiments, the targeted therapeutic compound can provide immunotherapy and, therefore, be a targeted immunotherapeutic compound. In one or more embodiments, the therapeutic domain can include a drug, a therapeutic radioisotope, a toxin, a cytokine, or a chemokine.

[0161] As used herein, the term “drug” refers to any chemical substance, which, when administered to a living organism, produces a biological effect. A pharmaceutical drug is a chemical substance used to treat, cure, prevent, or diagnose a disease or to promote well-being. Drugs can be obtained through extraction from medicinal plants, or by organic synthesis. Pharmaceutical drugs may be used for a limited duration, or on a regular basis for chronic disorders.

[0162] A “radioisotope” or “radionuclide” is an atom that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferred to one of its electrons to release it as a conversion electron; or used to create and emit a new particle (alpha particle or beta particle) from the nucleus. During those processes, the radionuclide is said to undergo radioactive decay. These emissions are considered ionizing radiation because they are powerful enough to liberate an electron from another atom. The radioactive decay can produce a stable nuclide or will sometimes produce a new unstable radionuclide that may undergo further decay.

[0163] As used herein, the term “toxin” refers to a substance harmful to cells. Toxins can be small molecules, peptides, or proteins that are capable of causing disease or cell death on contact with, or absorption by, body tissues. Toxins vary greatly in their toxicity. Toxins are largely secondary metabolites, which are organic compounds that are not directly involved in an organism’s growth, development, or reproduction, instead often aiding the organism in matters of defense. In one or more applications, a toxin may be used therapeutically by targeting the effect of the toxin toward an undesirable cell or cells (e.g., tumor cells).

[0164] Imaging Compound

[0165] In a further aspect, this disclosure describes a targeted imaging compound that includes a targeting domain and an imaging domain linked to the targeting domain. The targeting domain includes any embodiment of one of the complex containing a PACCs described herein. The imaging domain can include any moiety that can produce a detectable signal. Exemplary imaging moieties include, but are not limited to a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzymatic label.

[0166] Capture Assay Device

[0167] In yet another aspect, this disclosure describes a capture assay device including any embodiment of one of the PACCs described herein immobilized to a substrate. For example, a PACC described herein can be incorporated into cell and / or ligand capture technology such as, for example, an ELISA-based assay. A substrate to immobilize the PACCs can include, for example, a cell culture plate or dish, a glass slide, or any other support than can be used to perform an assay requiring an immobilized PACC.

[0168] In the preceding description and following claims, the term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended — i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc ).

[0169] As used herein, “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” or the like are used in their open-ended inclusive sense, and generally mean “include, but not limited to,” “includes, but not limited to,” or “including, but not limited to.” Further, wherever embodiments are described herein with the language “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” and the like, otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. The term “consisting of’ means including, and limited to, that which follows the phrase “consisting of.” That is, “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of’ indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0170] As used herein, the word “exemplary” means to serve as an illustrative example and should not be construed as preferred or advantageous over other embodiments.

[0171] As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0172] In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.

[0173] In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously.

[0174] EXAMPLES

[0175] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0176] Assembly of plasmids encoding PACCs

[0177] A mammalian expression plasmid was prepared using Gibson assembly of the PACC arm sequences into the pMC.EFla-MCS-SV40polyA parental minicircle cloning vector (System Biosciences LLC, Palo Alto, CA). Each PACC arm sequence included an N-terminal signal peptide for export, the IL-15Ra extracellular domain or the IL15Ra Sushi binding domain, a linker between the fusion protein moieties, a tumor engager sequence, or other protein as described in the PACC sequence list, and a C-terminal Strep tag II.

[0178] Briefly, double-stranded DNA fragments of the expression cassette components were cloned into the plasmid backbone via the Gibson cloning method. Kanamycin-resistant DH5-a transformants were selected, confirmed by Sanger sequencing, expanded, and purified.

[0179] Protein expression and purification

[0180] The PACC arm-encoding plasmid was co-transfected with the TriKE-10*His-encoding plasmid by cationic lipid-mediated transfection into mammalian Expi293 cells. Following a four- day production period at 37°C, 8% CO2, 125 RPM shake speed, the supernatant was harvested via centrifugation and filtration and the His-tagged protein was isolated via immobilized metal affinity chromatography (IMAC) either manually on cobalt resin or on HISTRAP Excel Ni-NTA columns (Cytiva Bioprocess R&D, Uppsala, Sweden) using the AKTA chromatography system (Cytiva Bioprocess R&D, Uppsala, Sweden). Protein purity and size was determined by polyacrylamide gel electrophoresis, his tag in-gel staining and Coomassie staining. A schematic of the preparation and assembly of constructs is shown in FIG. 9. All proteins were isolated via the C-terminal His-tag present only on the IL15-inclusive TriKE. Therefore, any detection of the PACC-arm components after purification indicates that it is bound to the protein with IL-15. As described herein, IL-15Ra and IL-15 form a stable, high- affinity association. Complex formation was investigated using biolayer interferometry (BLiTz), Native PAGE, Western Blot, and ELISA.

[0181] Example 1 : Characterization of a TriKE-PACC including an anti-CD33 scFv, an anti-CLEC12A scFv, and two NK cell engaging domains.

[0182] In this Example, the PACC arm-encoding plasmid encoded an anti-CLEC12A scFv, IL15Ra, and NKp30L to engage NKp30 on NK cells. The plasmid encoding the TriKE encoded an anti-CD33 scFv, IL15, and caml6 to engage CD16 on NK cells.

[0183] Example 2: Characterization of a TriKE-PACC including anti-B7H3, anti-CD16, and HSA domains.

[0184] A plasmid encoding a PACC arm including IL15Ra and HSA was prepared as described in Example 1. A plasmid encoding a TriKE including a camB7H3, IL15, and caml6 was prepared, and each of the PACC arm and the TriKE were expressed recombinantly and purified as described in “Cloning and Protein Production Methods” above.

[0185] To determine functional binding of the entire complex containing a PACC, B7H3 / HSA TriKE-PACC, we carry out a compound ELISA assay in which the complex is immobilized to the plate through binding of the component on the PACC (HSA in FIG. 2) and detected through recombinant proteins of the antigens targeted by the arms on the TriKE. A first ELISA was conducted to detect the anti-B7H3 antibody (camB7H3). A second ELISA was conducted to detect the anti-CD16 antibody (camCD16). Luminescence was quantified for each ELISA at multiple concentrations of PACC using a 1 :3 dilution series. As the concentration increased, the signal for both CD16 and B7H3 increased, indicating that both domains were present and functional on the assembled B7H3 / HSA TriKE-PACC.

[0186] A protein gel was run with reduced and non-reduced samples of the TriKE alone and the TriKE-PACC (FIG. 3). Within the TriKE-PACC molecules, the complex migrates slower while the faster migrating band represents TriKE molecule alone. Example 3: Interrogation of HSA PACC effect on potency of the B7H3 / HSA PACC complex.

[0187] NK-92 cells, an immortalized NK cell line, was treated with either the TriKE produced in Example 2 alone or as a PACC produced in Example 2. The response IL-15 cytokine signaling level was measured for cells treated with increasing doses of either the TriKE alone or the TriKE-PACC. It was observed that cells treated with the complex showed an increased response, particularly when treated with the compound in a range of 1-100 nMol (FIG. 5 A).

[0188] PBMCs were incubated with 22RV1 cells and IL- 15 alone, the TriKE of Example 2, or TriKE-PACC of Example 2. Following incubation, NK cells were analyzed for expression of CD 107a and IFN-y (FIG. 5B). This data shows that addition of the PACC onto the TriKE molecule, by formation of the TriKE-PACC, does not impair the NK cytolytic and inflammatory potential of the molecul.

[0189] Example 4: Characterization of a TriKE-PACC including anti-CD16, anti-B7H3, and HSA domains.

[0190] A plasmid encoding a TriKE including an anti -CD 16 domain and an anti-B7H3 domain was prepared as described above. A plasmid encoding the PACC arm including IL15Ra and HSA from Example 2 was prepared. A plasmid encoding a PACC arm including IL15Ra and an anti-mesothelin domain was prepared (FIG. 6).

[0191] Each protein was recombinantly expressed and purified. A first TriKE-PACC was formed by combining the HSA PACC arm with the TriKE. A second TriKE-PACC was formed by combining the anti-mesothelin PACC arm with the TriKE. Data (FIG. 7) shows that TriKE- PACCs retain the same functional potential as that seen on the TriKEs, while now incorporating more functional moieties meant to impact PK and targeting diversity.

[0192] Example 5: Characterization of TriKE-PACC including anti-CD16, anti-B7H3, and anti-AD AM- 17 (MEDI3622) domains.

[0193] A plasmid encoding a first TetraKE (TetraKEl) and a plasmid encoding a second TetraKE (TetraKE2) were prepared as described in Example 1. Each TetraKE was recombinantly expressed and purified (FIG. 8 A, 8B).

[0194] A plasmid encoding a first TriKE including caml6, IL-15, and camB7H3 was prepared. A plasmid encoding a PACC arm including fL15Ra and an anti-AD AMI 7 antibody called “MEDT scFv” was prepared. Both constructs were recombinantly expressed and purified. A first TriKE-PACC consistent with FIG. 9C was prepared by combining the two proteins.

[0195] A plasmid encoding a second TriKE including cam 16, IL- 15, and MEDI scFv was prepared. A plasmid encoding a PACC arm including IL15Ra and camB7H3 was prepared. Both constructs were recombinantly expressed in mammalian cells, where they assembled intracellularly, and were then purified (FIG. 8D). A denaturing protein gel showing components of the TriKE-PACC of FIG. 8C is shown in FIG. 10A. A native protein gel showing the intact TriKE-PACC complex depicted in FIG. 8C is shown in FIG. 10B. An ELISA that demonstrated complex formation by specifically binding proteins containing the intact IL15-IL15Ra complex of the TriKE-PACC of FIG 8C is shown in FIG. 27B, with the schematic representation of the experimental setup shown in FIG. 27A.

[0196] HNSCC cells were incubated with the TetraKE or the PACC depicted in FIG. 8 at different concentrations. Cells were analyzed using flow cytometry to determine binding. Binding data shows that the TetraKE was able to bind cells (FIG. 11), but the TriKE-PACC (FIG. 12) induced more efficient binding (higher florescence) when compared to the TetraKE (FIG. 13). Moreover, both portions of the TriKE-PAC molecule were detectable by flow cytometry by evaluating the His-tag presence on the TriKE and the Strep2-tag presence on the PACC (FIG. 14). These surprising findings indicate that the TriKE-PACC complex modality improves the binding of antigens when compared to the linear TetraKE approach.

[0197] To determine whether the TetraKE and TriKE-PACCs exhibited similar antitumor activity, NK cells were incubated with each TetraKE or TriKE-PACC with NK cells and either Cal27 cells or Cal33 cells. Expression of CD 107a on NK cells was measured to determine N cell degranulation (FIG. 15). Once again, surprisingly, the TriKE-PACC modality induced better cytolytic activity (measured by degranulation) of NK cells when compared to the TetraKE approach.

[0198] The ADAMI 7-binding antibody interferes with the proteolytic activity of the ADAMI 7 metalloproteinase, which mediates rapid cleavage of receptors on the surface of cells upon cellular activation. On NK cells CD 16 and CD62L are both cleaved rapidly upon activation, limiting the immunotherapeutic potential of the cell. Cells were treated with a TetraKE, B7H3 / MEDI TriKE-PACC, or component thereof, optionally in combination with PMA / lo stimulation, which robustly activates NK cells and ADAM 17 activity, or nothing to display optimal expression of CD16 and CD62L. After a four-hour incubation, CD16 and CD62L expression were measured by flow cytometry (FIG. 16). The surprising findings show that the B7H3 / Medi TriKE-PACC is better at retaining surface CD 16 and CD62L than the TetraKE molecules, and similar to Medi antibody alone, Medi TriKE, and B7H3 TriKE + Medi antibody.

[0199] To determine the effect of the TetraKE and TriKE-PACC constructs on NK cell-mediated cytotoxicity, NuclightRed labeled Cal27 or Cal33 cells were incubated with NK cells treated with IL-15, the anti-B7H3 TriKE, two forms of the B7H3 / MEDI TetraKE, or the B7H3 / MEDI TriKE-PACC. Tumor cell viability was measured over 50 hours (FIG. 17). Cells treated with the TriKE or TriKE-PACC exhibited the strongest response, while cells treated with IL- 15 or either of the TetraKEs exhibited a weaker response. This surprising finding again demonstrates the benefits of utilizing at TriKE-PACC complex vs. a linear TetraKE approach.

[0200] Example 6: Characterization of TriKE-PACCs including anti-CD16, anti-PSMA or anti-B7H3, and anti-CD133 domains.

[0201] A construct encoding a TriKE including an anti-PSMA domain, IL-15, and an anti-CD16 domain was prepared. A construct encoding a TriKE including an anti-CD16 domain, IL- 15, and an anti-B7H3 domain was prepared. A construct encoding a PACC arm including IL-15a and an anti-CD133 domain was prepared. Each construct was recombinantly expressed, TriKE and PACC constructs were co-transfected into mammalian cells, then the supernatant was harvested and TriKE-PACCs were purified as described above.

[0202] From the expressed proteins, two TriKE-PACCs were purified from the transfection supernatant. A first PSMA / CD133 TriKE-PACC included the anti-PSMA TriKE and the anti- CD133 PACC arm (FIG. 18). A second B7H3 / CD133 TriKE-PACC included the anti-B7H3 TriKE and the anti-CD133 PACC arm (FIG. 18). In addition, several TriKE-PACCs were prepared to investigate the function of the IL-15Ra full extracellular domain (ECD) and the truncated IL-15Ra sushi domain (SU) in PACC constructs.

[0203] To confirm the formation of the TriKE-PACC complex and its ability to bind the B7-H3 receptor on target cells, a dual ELISA was conducted (schematic shown in FIG. 28A). This assay selectively captured only those molecules with the intact IL15 / IL15Ra complex, utilizing a complex-specific capture antibody immobilized on the plate (FIG. 28B). Detection was via HRP- labeled recombinant human B7-H3, resulting in observable signals exclusively in samples containing the intact TriKE-PACC complex that successfully bound to B7-H3.

[0204] To determine an appropriate cell type to test with an anti-B7H3 / CD133 TriKE-PACC, expression of B7H3 and CD133 on multiple cell lines was characterized (FIG. 19, FIG. 20). Most cell lines expressed very low levels of CD133 except MA148 cells. Accordingly, MA148 cells were used for subsequent testing of B7H3 / CD133 constructs.

[0205] NK cells were incubated with IL-15 alone, each of the TriKEs, or each of the TriKE- PACCs of FIG. 18. In addition, the NK cells were incubated alone or with BT16-B7H3 KO cells, C4-2 cells, HT29 cells, or MA148 cells. Expression of CD107a, ZFN-y, and TNF-a on the NK cells was measured to determine response to the treatment (FIGs. 21-23). The data illustrates that the dual targeting approach can bypass inefficient activation seen with single targeting approaches. As an example, cells that do not express PSMA but do express CD133 (for instance MA148s) do not induce NK cell activation in the presence of a single anti-PSMA TriKE, but the PSMA / CD133 TriKE-PACC induces NK cell activation similarly to that seen with an antiCD 133 TriKE. Thus, this surprising approach to generate dual targeting through a TriKE-PACC complex can be used to deal with tumor heterogeneity and antigen escape.

[0206] Example 7: Proliferation of PBMCs after treatment with multiple TriKE-PACCs.

[0207] Each construct depicted in FIG. 18 was evaluated for its ability to enhance proliferation of NK cells in frozen PBMCs (FIG. 25) and proliferation of freshly isolated NK cells (FIG. 26) to determine if addition of the PACC to the IL- 15 bearing molecule, a TriKE in this example, deters the ability of the IL-15 moiety to drive NK cell proliferation. The gating strategy used to count cells that were proliferating or highly proliferating is shown in FIG. 24. All treatments except the B7H3 TriKE-PACC increased proliferation and high proliferation of NK cells from frozen PBMCs and freshly isolated NK cells, thus indicating that the TriKE-PACC molecule induces similar IL-15 signaling as TriKE molecules alone.

[0208] Example 8: Confirmation of expression and purification of multiple TriKE-PACC proteins.

[0209] Several TriKE-PACC molecules, including those containing anti-CD16, anti-B7H3, anti- CD83, anti-SSl(mesothelin) and NKp30L domains, co-transfected into Expi293 cells and purified by IMAC via the HIS tag present on the TriKE arm of the protein complex. Expression levels and purity were confirmed by denaturing SDS-PAGE followed by Coomassie stain to visualize total protein present in the purified samples (FIG. 29A), and a HIS stain to detect the HIS epitope tag (FIG. 29B).

[0210] Example 9: Verification of TriKE-PACC complex formation across various protein configurations following IMAC purification.

[0211] Several TriKE-PACC molecules, including configurations containing anti-CD16, anti- B7H3, anti-CD83, anti-SSl(mesothelin) and NKp30L domains, were prepared and isolated via the His-tag on the TriKE arm of the molecules by IMAC. Denaturing SDS-PAGE was followed by a Western blot probed with a Streptactin-HRP conjugate for detection of the Strep tag II on the PACC arm (FIG. 30). The Western blot confirmed the presence of the IL15 / IL15Ra interaction post-purification, evidenced by the Strep tag’s presence on the TriKE-PACC proteins isolated via the HIS tag on the TriKE arm. Furthermore, the robust IL15 and IL15Ra interaction was highlighted by the persistence of intact TriKE-PACC proteins, as indicated by their migration size even after the proteins underwent boiling for the denaturing gel.

[0212] Example 10: PACCs including complexes other than TriKEs.

[0213] In some formats, the PACC could be used on IL- 15 expressing complexes other than TriKEs. One such example of this includes combining a monomeric recombinant human IL-15 (rhIL-15) with an HSA-PACC to extend its half-life, or an antigen targeting PACC, such as the B7H3 PACC, to direct the IL-15 to the site of B7H3 tumor expressing cells.

[0214] A plasmid encoding the rhIL-15 sequence is co-transfected with one of the HSA- containing PACCs of the present disclosure into mammalian production cells (such as CHOs, expi-CHOs, or expi-293 cells) or bacterial cells (such as E. coli). Protein complexes are then isolated from the supernatant, intracellular compartment, or inclusion bodies. The protein complexes are purified using affinity chromatography, size exclusion chromatography, or a similar methodology.

[0215] The rhIL-15 / HSA-PACC is tested by evaluating complex formation using a modified ELISA, where capture occurs with an anti-HSA antibody and detection occurs through an IL- 15 detection antibody. The rhIL-15 / B7H3-PACC is tested by incubating rhIL-15 / B7H3-PACC, or rhIL-15 alone as a control, with B7H3+ C4-2 cells or B7H3 CRISPR KO C4-2 cells and evaluating surface expression of IL-15 on these cells; should be detected on B7H3+ C4-2 cells when incubated with the rhIL-15 / B7H3-PACC but not rhIL-15 alone. These assays are used to confirm that the complex retains its ability to bind the desired molecules.

[0216] Evaluation of natural cytotoxicity of NK cells against B7H3+ C4-2 cells is assessed using an xCELLigence (impedance-based) cytolytic analysis platform. Cells are pre-incubated with rhIL-15 / B7H3-PACC or rhIL-15 and washed. Cells pre-incubated with rhIL-15 / B7H3-PACC are killed more than the rhIL-15 cells.

[0217] Example 11 : PACC application to NK cells

[0218] In some formats, the PACC could be a Natural Killer (NK) cellular product that expresses soluble IL-15. One example of this is the combination of soluble monomeric recombinant human IL- 15 (srhIL-15) with an HSA-PACC to extend its half-life, or an antigen targeting PACC, such as the B7H3 PACC, to direct the srhIL-15 to the site of B7H3 tumor expressing cells.

[0219] A non-viral knock-in (KI) transfection technique, such as CRISPR-KI, is used to express srhIL-15 and the PACC (HSA or B7H3) into cytokine-primed enriched primary NK cells. Cells co-express a fluorescent molecule linked to each transfected component to identify transfected cells. NK cells are expanded with feeder cells (K562 cells expressing membrane-bound 4-BB1L and IL-21). Prior to use in validation assays, cells can be flow sorted for positive clones if necessary. Protein complexes are isolated from the supernatant of cells for ELISA evaluation.

[0220] The srhIL-15 / HSA-PACC is tested by evaluating complex formation using a modified ELISA, where capture occurs with an anti -HSA antibody and detection occurs through an IL- 15 detection antibody. For the srhIL-15 / B7H3-PACC, transfected cells are co-incubated with B7H3+ C4-2 cells or B7H3 CRISPR KO C4-2 cells and subsequently evaluated for surface expression of IL- 15 by flow cytometry. Control transfected or rhIL-15 transfected NK cells can be used as controls. Surface IL- 15 is detected on B7H3+ C4-2 cells when incubated with the srhIL-15 / B7H3-PACC, but not detected on cells incubated with srhIL-15 alone. In the co-culture system, evaluation of natural cytotoxicity of transfected (control, srhIL-15, or srhIL-15 / B7H3- PACC) NK cells against B7H3+ C4-2 cells is assessed using an xCELLigence (impedancebased) cytolytic analysis platform. The cells transfected to express srhIL-15 / B7H3-PACC transfected cells drive more killing of B7H3+ C4-2 cells. The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0221] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0222] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0223] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0224] Sequence Listing Free Text

[0225] I. Amino acid Sequences of IL-15Ra domains

[0226] SEQ ID NO: 1 - IL-15Ra sushi domain I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA

[0227] TNIAHWTTPS LKCIR

[0228] SEQ ID N0:2 - IL-15Ra ECD

[0229] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTT

[0230] II. Amino acid sequences of possible linkers to be used between IL-15Ra and engager or protein:

[0231] SEQ ID NOG - LinkerSeql6

[0232] SGGGGSGGGG SGGGGSGGGG SG

[0233] SEQ ID NO:4 - whitlow

[0234] GSTSGSGKPG SGEGSTKG

[0235] SEQ ID NO:5 - (G4S)2

[0236] GGGGSGGGGS

[0237] SEQ ID NO:6 - (G4S)3

[0238] GGGGSGGGGS GGGGS

[0239] SEQ ID NO:7 - (G4S)4

[0240] GGGGSGGGGS GGGGSGGGGS

[0241] SEQ ID NO:8 - (G4S)7

[0242] GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS GGGGS

[0243] SEQ ID NO: 9 - Gly6

[0244] GGGGGG

[0245] SEQ ID NO: 10 -RL-1

[0246] AEAAKEAAKE AAKEAAKALE AEAAKEAAKE AAKEAAKA

[0247] SEQ ID NO: 11 - EAAAK

[0248] EAAAK

[0249] SEQ ID NO: 12 - (EAAAK)2

[0250] EAAAKEAAAK

[0251] SEQ ID NO: 13 - (EAAAK)3

[0252] EAAAKEAAAK EAAAK

[0253] SEQ ID NO: 14 - PAPAP PAPAP

[0254] SEQ ID NO: 15 - RL-2

[0255] AEAAKEAAKA

[0256] SEQ ID NO: 16 - RL-3

[0257] APAPAPAPAP

[0258] SEQ ID NO: 17 -RL-4

[0259] APAPAPAPAP APAPAPAPAP

[0260] SEQ ID NO: 18 - IL- 15 protein

[0261] NWVNVI SDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI

[0262] SLESGDAS IH DTVENLI ILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL

[0263] QS FVHIVQMF INTS

[0264] III. Engagers and proteins incorporated into PACC arm

[0265] SEQ ID NO: 19 - caml6

[0266] QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS I TWSGRDTFY ADSVKGRFT I SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SS

[0267] CDR1 : 26-32

[0268] CDR2: 52-57

[0269] CDR3: 99-111

[0270] SEQ ID NO: 20 - NKp30L

[0271] DLKVEMMAGG TQI TPLNDNV T I FCNI FYSQ PLNITSMGI T WFWKSLTFDK EVKVFEFFGD HQEAFRPGAI VSPWRLKSGD ASLRLPGIQL EEAGEYRCEV WTPLKAQGT VQLEWASPA SRLLLDQVGM KENEDKYMCE SSGFYPEAIN I TWEKQTQKF PHPIEI SEDV I TGPTIKNMD GTFNVTSCLK LNSSQEDPGT VYQCWRHAS LHTPLRSNFT LTAARHSLSE TEKTDNFS

[0272] SEQ ID NO:21 - B7-H3

[0273] QVQLVESGGG LVQPGGSLRL SCAASGFTFS SYWMYWVRQT PGKGLEWVST INRDGSATWY ADSVKGRFT I SRDNAKNTGY LQMNSLKPDD TAVYYCVSDP DNYSSDEMVP YWGQGTQVTV SS

[0274] CDR1 : 26-32

[0275] CDR2: 52-57

[0276] CDR3: 99-111

[0277] SEQ ID NO: 22 - HSA protein

[0278] RGVFRRDAHK SEVAHRFKDL GEENFKALVL IAFAQYLQQC PFEDHVKLVN

[0279] EVTEFAKTCV ADESAENCDK SLHTLFGDKL CTVATLRETY GEMADCCAKQ

[0280] EPERNECFLQ HKDDNPNLPR LVRPEVDVMC TAFHDNEETF LKKYLYE IAR RHPYFYAPEL LFFAKRYKAA FTECCQAADK AACLLPKLDE LRDEGKASSA KQRLKCASLQ KFGERAFKAW AVARLSQRFP KAEFAEVSKL VTDLTKVHTE CCHGDLLECA DDRADLAKYI CENQDS ISSK LKECCEKPLL EKSHCIAEVE NDEMPADLPS LAADFVESKD VCKNYAEAKD VFLGMFLYEY ARRHPDYSW LLLRLAKTYE TTLEKCCAAA DPHECYAKVF DEFKPLVEEP QNLIKQNCEL FEQLGEYKFQ NALLVRYTKK VPQVSTPTLV EVSRNLGKVG SKCCKHPEAK

[0281] RMPCAEDYLS WLNQLCVLH EKTPVSDRVT KCCTESLVNR RPCFSALEVD ETYVPKEFNA ETFTFHADIC TLSEKERQIK KQTALVELVK HKPKATKEQL KAVMDDFAAF VEKCCKADDK ETCFAEEGKK LVAASQAALG L

[0282] SEQ ID NO: 23 - CD83

[0283] AQVQLVESGG GLVQAGGSLT LSCAAFGDTS S I FLMGWFRQ APGKQRDMVA LINYDGTTNY KDTVKGRFAI SRDNAKNTVS LQMNSLSPED TAVYFCNARS I FGNSWGQGT QVTVSS

[0284] CDR1 : 27-33

[0285] CDR2: 53-57

[0286] CDR3: 99-105

[0287] SEQ ID NO:24 - CD133 scFv

[0288] ELDIVLSQSP AIMSASPGEK VTISCSASSS VSYMYWYQQK PGSSPKPWIY RTSNLASGVP ARFSGSGSGT SYSLTISSME AEDAATYYCQ QYHSYPPTFG AGTKLELKSS GGGGSGGGGG GSSRSSLEVK LVESGPELKK PGETVKISCK ASGYTFTDYS MHWVNQAPGK GLKWMGWINT ETGEPSYADD FKGRFAFSLE TSASTAYLQI NNLKNEDTAT YFCATDYGDY FDYWGQGTTL TVSSAKTTPP SVTS

[0289] VL: 1-108

[0290] Linker: 109-127

[0291] VH: 128-254

[0292] CDR1 : 26-35

[0293] CDR2: 51-57

[0294] CDR3: 90-98

[0295] CDR4: 153-159

[0296] CDR5: 179-184

[0297] CDR6: 226-233

[0298] SEQ ID NO:25 - PD-1 ECD

[0299] FLDSPDRPWN PPTFSPALLV VTEGDNATFT CSFSNTSESF VLNWYRMSPS NQTDKLAAFP EDRSQPGQDC RFRVTQLPNG RDFHMSWRA RRNDSGTYLC GAISLAPKAQ IKESLRAELR VTERRAEVPT AHPSPSPRPA GQFQTLV

[0300] SEQ ID NO:26 - J591 scFv

[0301] EVQLVQSGAE VKKPGASVKI SCKTSGYTFT EYTIHWVKQA SGKGLEWIGN

[0302] INPNNGGTTY NQKFEDRATL TVDKSTSTAY MELSSLRSED TAVYYCAAGW

[0303] NFDYWGQGTT VTVSSGSTSG GGSGGGSGGG GSSDIVMTQS PSSLSASVGD RVTITCKASQ DVGTAVDWYQ QKPGKAPKLL IYWASTRHTG VPDRFTGSGS GTDFTLTISS LQPEDFADYF CQQYNSYPLT FGGGTKLEIK

[0304] VL: 1-119

[0305] Linker: 120-133

[0306] VH: 134-240

[0307] CDR1 : 26-32

[0308] CDR2: 52-57

[0309] CDR3: 98-104

[0310] CDR4: 157-167

[0311] CDR5: 183-189

[0312] CDR6: 222-230

[0313] SEQ ID NO: 27 - MEDI scFv

[0314] EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYPMNWVRQA PGKGLEWVSY ISPFGGMTDY ATSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDA MRGAEVDYWG QGTLVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCRSS QS IPSYLNWY QQKPGKAPKL LIYAASRLQS GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCQQSYSTPL TFGGGTKVEI K

[0315] VL: 1-119

[0316] Linker: 120-133

[0317] VH: 134-241

[0318] CDR1 : 26-32

[0319] CDR2: 52-57

[0320] CDR3: 99-108

[0321] CDR4: 158-168

[0322] CDR5: 184-190

[0323] CDR6: 223-231

[0324] SEQ ID NO:28 - CD27 ECD

[0325] ATPAPKSCPE RHYWAQGKLC CQMCEPGTFL VKDCDQHRKA AQCDPCIPGV SFSPDHHTRP HCESCRHCNS GLLVRNCTIT ANAECACRNG WQCRDKECTE CDPLPNPSLT ARSSQALSPH PQPTHLPYVS EMLEARTAGH MQTLADFRQL PARTLSTHWP PQRSLCSSDF IR

[0326] SEQ ID NO:29 - CD16a clone F8

[0327] QVQLVESGGG SVQPGGSLRL SCDVSALNLD RYALGWFRQV PGKEREGIAC

[0328] LSSRGDGTDY ADSVKDRFAI SRGPDGNTFT LLMNTLKPED SGIYYCAADL

[0329] SLARSCDVRS SEYTYWGQGI QVTVSSGPGG

[0330] CDR1 : 26-32

[0331] CDR2: 52-57

[0332] CDR3: 99-115

[0333] SEQ ID NO:30 - HER2 scFv MDLQLTQSPA ILSASPGEKV TMTCRATPSV SYMHWYQQKP GSSPKPWIYT

[0334] TSNLASGVPA RFSGGGSGTS YSLTVSRVEA EDAATYYCQQ WSRSPPTFGG

[0335] GSKLEIKGST SGSGKSSEGK GVQLQESGPE WKPGGSMKI SCKTSGYSFT

[0336] GHTMNWVKQS HGKNLEWIGL INPYNGDTNY NQKFKGKATF TVDKSSSTAY MELLSLTSED SAVYYCARRV TDWYFDVWGA GTTVTVS

[0337] VL: 1-107

[0338] Linker: 108-121

[0339] VH: 122-237

[0340] CDRL 25-34

[0341] CDR2: 50-56

[0342] CDR3: 89-97

[0343] CDR4: 146-152

[0344] CDR5: 172-177

[0345] CDR6: 219-227

[0346] SEQ ID NO:31 - HER2 scFv (Trastuzumab-based)

[0347] EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS GSTSGSGKPG SGEGSTKGDI QMTQSPSSLS ASVGDRVTIT CRASQDVNTA VAWYQQKPGK APKLLIYSAS FLYSGVPSRF SGSRSGTDFT LTISSLQPED FATYYCQQHY TTPPTFGQGT KVEIKRTV

[0348] VL: 1-120

[0349] Linker: 121-138

[0350] VH: 139-248

[0351] CDR1 : 26-32

[0352] CDR2: 52-57

[0353] CDR3: 99-109

[0354] CDR4: 162-172

[0355] CDR5: 188-194

[0356] CDR6: 227-235

[0357] SEQ ID NO:32 - TGFP trap

[0358] TIPPHVQKSV NNDMIVTDNN GAVKFPQLCK FCDVRFSTCD NQKSCMSNCS ITS ICEKPQE VCVAVWRKND ENITLETVCH DPKLPYHDFI LEDAASPKCI MKEKKKPGET FFMCSCSSDE CNDNI I FSEE YNTSNPDLLL VI FQGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSGGGGST IPPHVQKSVN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCS I TS ICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNI I FSEEY NTSNPDLLLV I EQ

[0359] TGFprllb arm 1 : 1-144 Linker 145-179

[0360] TGFprllb arm 2: 180-323

[0361] SEQ ID NO:33 - strep tag II WSHPQFEK

[0362] V. Full sequence of PACC-arm recombinant proteins

[0363] General structure: IL-15Ra (Sushi domain or ECD)-(linker)-engager or protein-spacer-affinity tag

[0364] SEQ ID NO: 34 - IL-15RaSu-(linker)-caml6-STII

[0365] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRGGGGS GGGGSGGGGS QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS I TWSGRDTFY ADSVKGRFT I SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SSVDEWSHPQ FEK

[0366] IL-15RaSu: 1-65 Linker: 66-80 Cam 16: 67-202

[0367] Spacer: 203-205 STII:206-213 CDR1 : 106-112

[0368] CDR2: 132-137

[0369] CDR3: 179-191

[0370] SEQ ID NO:35 - IL-15RaECD-(linker)-caml6-STII

[0371] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS I TWSGRDTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT

[0372] YWGQGTLVTV SSVDEWSHPQ FEK

[0373] IL-15RaECD: 1-175

[0374] Linker: 176-190

[0375] Caml6: 191-312

[0376] Spacer: 313-315

[0377] STII:316-323

[0378] CDR1 : 216-221

[0379] CDR2: 242-247

[0380] CDR3: 289-301

[0381] SEQ ID NO:36 - IL-15RaECD-(linker)-CD27-STII

[0382] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA

[0383] TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAATPAPKS CPERHYWAQG KLCCQMCEPG TFLVKDCDQH

[0384] RKAAQCDPCI PGVSFSPDHH TRPHCESCRH CNSGLLVRNC T I TANAECAC RNGWQCRDKE CTECDPLPNP SLTARSSQAL SPHPQPTHLP YVSEMLEART AGHMQTLADF RQLPARTLST HWPPQRSLCS SDFIRVDEWS HPQFEK

[0385] IL-15RaECD: 1-175

[0386] Linker: 176-213

[0387] CD27: 214-385

[0388] Spacer: 383-388

[0389] STII: 389-396

[0390] SEQ ID NO:37 - IL-15RaECD-(linker)-CD16a clone F8-STII

[0391] ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA

[0392] TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA

[0393] ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTEISSHES SHGTPSQTTA

[0394] KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS QVQLVESGGG

[0395] SVQPGGSLRL SCDVSALNLD RYALGWFRQV PGKEREGIAC LSSRGDGTDY

[0396] ADSVKDRFAI SRGPDGNTFT LLMNTLKPED SGIYYCAADL SLARSCDVRS

[0397] SEYTYWGQGI QVTVSSGPGG QVDEWSHPQF EK

[0398] IL-15RaECD: 1-175

[0399] Linker: 176-190

[0400] CD18a clone F8: 191-321

[0401] Spacer: 322-324

[0402] STII: 325-332

[0403] CDR1 : 216-222

[0404] CDR2: 242-247

[0405] CDR3: 289-305

[0406] SEQ ID NO:38 - IL-15RaSu-(linker)-CD83 sd-STII

[0407] ITCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRGGGGS GGGGSGGGGS AQVQLVESGG GLVQAGGSLT LSCAAFGDTS S I FLMGWFRQ APGKQRDMVA LINYDGTTNY KDTVKGRFAI SRDNAKNTVS LQMNSLSPED TAVYFCNARS I FGNSWGQGT QVTVSSVDEW SHPQFEK

[0408] IL-15RaSu: 1-65 Linker: 66-80 CD83 sd: 81-196 Spacer: 197-199 STII: 200-207 CDR1 : 107-113 CDR2: 133-137 CDR3: 179-185

[0409] SEQ ID NO:39 - IL-15RaECD- (linker)-CD83 sd-STII

[0410] ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTEISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS AQVQLVESGG GLVQAGGSLT LSCAAFGDTS S I FLMGWFRQ APGKQRDMVA LINYDGTTNY KDTVKGRFAI SRDNAKNTVS LQMNSLSPED TAVYFCNARS I FGNSWGQGT QVTVSSVDEW SHPQFEK

[0411] IL-15RaECD: 1-175

[0412] Linker: 176-190

[0413] CD83 sd: 191-306

[0414] Spacer: 307-309

[0415] STII: 310-317

[0416] CDR1 : 217-223

[0417] CDR2: 243-247

[0418] CDR3: 289-295

[0419] SEQ ID NO:40 - IL-15RaSu-(linker)-B7H3 clone 2-STII

[0420] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLVES GGGLVQPGGS LRLSCAASGF TFSSYWMYWV RQTPGKGLEW VST INRDGSA TWYADSVKGR FT I SRDNAKN TGYLQMNSLK PDDTAVYYCV SDPDNYSSDE MVPYWGQGTQ VTVSSVDEWS HPQFEK

[0421] IL-15RaSu: 1-65

[0422] Linker: 66-103

[0423] B7H3 clone 2: 104-225

[0424] Spacer: 226-228

[0425] STII: 229-236

[0426] CDR1 : 129-135

[0427] CDR2: 155-160

[0428] CDR3: 202-214

[0429] SEQ ID NO:41 - IL-15RaECD-(linker)-B7H3 clone 2-STII

[0430] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLVES GGGLVQPGGS LRLSCAASGF TFSSYWMYWV

[0431] RQTPGKGLEW VST INRDGSA TWYADSVKGR FT I SRDNAKN TGYLQMNSLK PDDTAVYYCV SDPDNYSSDE MVPYWGQGTQ VTVSSVDEWS HPQFEK

[0432] IL-15RaECD: 1-175

[0433] Linker: 176-213

[0434] B7H3 clone 2: 214-335

[0435] Spacer: 336-338

[0436] STII: 339-346

[0437] CDR1 : 239-245

[0438] CDR2: 265-270

[0439] CDR3: 312-324 SEQ ID NO:42 - IL-15RaECD-(linker)-B7H3 clone 2-STII

[0440] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS QVQLVESGGG LVQPGGSLRL SCAASGFTFS SYWMYWVRQT PGKGLEWVST INRDGSATWY ADSVKGRFTI SRDNAKNTGY LQMNSLKPDD TAVYYCVSDP DNYSS DEMVP

[0441] YWGQGTQVTV SSVDEWSHPQ EEK

[0442] IL-15RaECD: 1-175

[0443] Linker: 176-190

[0444] B7H3 clone 2: 191-312

[0445] Spacer: 313-315

[0446] STII: 316-323

[0447] CDR1 : 216-222

[0448] CDR2: 242-247

[0449] CDR3: 289-301

[0450] SEQ ID NO:43 - IL-15RaSu-(linker)-B7H3 clone 2-STII

[0451] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRGGGGS GGGGSGGGGS QVQLVESGGG LVQPGGSLRL SCAASGFTFS SYWMYWVRQT PGKGLEWVST INRDGSATWY ADSVKGRFT I SRDNAKNTGY LQMNSLKPDD TAVYYCVSDP DNYSSDEMVP YWGQGTQVTV SSVDEWSHPQ FEK

[0452] IL-15RaSu: 1-65

[0453] Linker: 66-80

[0454] B7H3 clone 2: 81-202

[0455] Spacer: 203-205

[0456] STII: 206-213

[0457] CDR1 : 106-112

[0458] CDR2: 132-137

[0459] CDR3: 179-191

[0460] SEQ ID NO:44 - IL-15RaECD-(linker)-PD-l-STII

[0461] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS FLDSPDRPWN PPTFSPALLV VTEGDNATFT CS FSNTSES F VLNWYRMSPS NQTDKLAAFP

[0462] EDRSQPGQDC RFRVTQLPNG RDFHMSWRA RRNDSGTYLC GAI SLAPKAQ IKESLRAELR VTERRAEVPT AHPSPSPRPA GQFQTLWDE WSHPQFEK

[0463] IL-15RaECD: 1-175

[0464] Linker: 176-190

[0465] PD-1: 191-337 Spacer: 338-340

[0466] STII: 341-349

[0467] SEQ ID NO:45 - IL-15RaSu-(linker)-PD-l-STII

[0468] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRGGGGS GGGGSGGGGS FLDSPDRPWN PPTFSPALLV VTEGDNATFT CS FSNTSES F VLNWYRMSPS NQTDKLAAFP EDRSQPGQDC RFRVTQLPNG RDFHMSWRA RRNDSGTYLC GAI SLAPKAQ IKESLRAELR VTERRAEVPT AHPSPSPRPA GQFQTLWDE WSHPQFEK

[0469] IL-15RaSu: 1-65

[0470] Linker: 66-80

[0471] PD-1: 81-227 Spacer: 228-230 STII: 231-238

[0472] SEQ ID NO:46 - IL-15RaECD-(linker)-HSA-STII

[0473] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS RGVFRRDAHK SEVAHRFKDL GEENFKALVL IAFAQYLQQC PFEDHVKLVN EVTEFAKTCV ADESAENCDK SLHTLFGDKL CTVATLRETY GEMADCCAKQ EPERNECFLQ HKDDNPNLPR LVRPEVDVMC TAFHDNEETF LKKYLYE IAR RHPYFYAPEL LFFAKRYKAA FTECCQAADK AACLLPKLDE LRDEGKASSA KQRLKCASLQ KFGERAFKAW AVARLSQRFP KAEFAEVSKL VTDLTKVHTE CCHGDLLECA DDRADLAKYI CENQDS I SSK LKECCEKPLL EKSHCIAEVE NDEMPADLPS LAADFVESKD VCKNYAEAKD VFLGMFLYEY ARRHPDYSW LLLRLAKTYE TTLEKCCAAA DPHECYAKVF DEFKPLVEEP QNLIKQNCEL FEQLGEYKFQ NALLVRYTKK VPQVSTPTLV EVSRNLGKVG SKCCKHPEAK RMPCAEDYLS WLNQLCVLH EKTPVSDRVT KCCTESLVNR RPCFSALEVD ETYVPKEFNA

[0474] ETFTFHADIC TLSEKERQIK KQTALVELVK HKPKATKEQL KAVMDDFAAF VEKCCKADDK ETCFAEEGKK LVAASQAALG LVDEWSHPQF EK

[0475] IL-15RaECD: 1-175

[0476] Linker: 176-190

[0477] HSA: 191-781

[0478] Spacer: 782-784

[0479] STII: 785-792

[0480] SEQ ID NO:47 - IL-15RaSu- (linker)-HSA-STII

[0481] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRGGGGS GGGGSGGGGS RGVFRRDAHK SEVAHRFKDL GEENFKALVL IAFAQYLQQC PFEDHVKLVN EVTEFAKTCV ADESAENCDK SLHTLFGDKL CTVATLRETY GEMADCCAKQ EPERNECFLQ HKDDNPNLPR LVRPEVDVMC TAFHDNEETF LKKYLYE IAR RHPYFYAPEL LFFAKRYKAA FTECCQAADK AACLLPKLDE LRDEGKASSA KQRLKCASLQ KFGERAFKAW AVARLSQRFP KAEFAEVSKL VTDLTKVHTE CCHGDLLECA DDRADLAKYI CENQDS I SSK LKECCEKPLL EKSHCIAEVE NDEMPADLPS LAADFVESKD VCKNYAEAKD VFLGMFLYEY ARRHPDYSW LLLRLAKTYE TTLEKCCAAA DPHECYAKVF DEFKPLVEEP QNLIKQNCEL FEQLGEYKFQ NALLVRYTKK VPQVSTPTLV EVSRNLGKVG SKCCKHPEAK RMPCAEDYLS WLNQLCVLH EKTPVSDRVT KCCTESLVNR RPCFSALEVD ETYVPKEFNA ETFTFHADIC TLSEKERQIK KQTALVELVK HKPKATKEQL KAVMDDFAAF VEKCCKADDK ETCFAEEGKK LVAASQAALG LVDEWSHPQF EK

[0482] IL-15RaSu: 1-65

[0483] Linker: 66-80

[0484] HSA: 81-671

[0485] Spacer: 672-674

[0486] STII: 675-682

[0487] SEQ ID NO:48 - SEQ IL-15RaSu-(linker)-CD133 scFv-STII

[0488] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRGGGGS GGGGSGGGGS ELDIVLSQSP AIMSASPGEK VT I SCSASSS VSYMYWYQQK PGSSPKPWIY RTSNLASGVP ARFSGSGSGT SYSLTI SSME AEDAATYYCQ QYHSYPPTFG AGTKLELKSS GGGGSGGGGG GSSRSSLEVK LVESGPELKK PGETVKI SCK ASGYTFTDYS MHWVNQAPGK GLKWMGWINT ETGEPSYADD FKGRFAFSLE TSASTAYLQI NNLKNEDTAT YFCATDYGDY FDYWGQGTTL TVSSAKTTPP SVTSVDEWSH PQFEK

[0489] IL-15RaSu: 1-65

[0490] Linker: 66-80

[0491] CD133 scFv VL: 81-188

[0492] CD133 scFv linker: 189-207

[0493] CD133 scFv VH: 208-334

[0494] Spacer: 335-337

[0495] STII: 338-345

[0496] CDR1 : 106-115

[0497] CDR2: 131-137

[0498] CDR3: 170-178

[0499] CDR4: 233-239

[0500] CDR5: 259-264

[0501] CDR6: 306-313

[0502] SEQ ID NO:49 - IL-15RaECD-(linker)-CD133 scFv-STII

[0503] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS ELDIVLSQSP AIMSASPGEK VT I SCSASSS VSYMYWYQQK PGSSPKPWIY RTSNLASGVP ARFSGSGSGT SYSLTI SSME AEDAATYYCQ QYHSYPPTFG AGTKLELKSS GGGGSGGGGG GSSRSSLEVK LVESGPELKK PGETVKI SCK ASGYTFTDYS MHWVNQAPGK GLKWMGWINT ETGEPSYADD FKGRFAFSLE TSASTAYLQI NNLKNEDTAT YFCATDYGDY FDYWGQGTTL TVSSAKTTPP SVTSVDEWSH PQFEK

[0504] IL-15RaECD: 1-175

[0505] Linker: 176-190

[0506] CD133 scFv VL: 191-298

[0507] CD 133 scFv linker: 299-317

[0508] CD 133 scFv VH: 318-444

[0509] Spacer: 445-447

[0510] STII: 448-455

[0511] CDR1 : 216-225

[0512] CDR2: 241-247

[0513] CDR3: 280-288

[0514] CDR4: 343-349 CDR5: 369-374 CDR6: 416-423

[0515] SEQ ID NO:50 - IL-15RaECD-(linker)-J591 scFv-STII

[0516] ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTEISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS EVQLVQSGAE VKKPGASVKI SCKTSGYTFT EYTIHWVKQA SGKGLEWIGN INPNNGGTTY NQKFEDRATL TVDKSTSTAY MELSSLRSED TAVYYCAAGW NFDYWGQGTT VTVSSGSTSG GGSGGGSGGG GSSDIVMTQS PSSLSASVGD RVTITCKASQ DVGTAVDWYQ QKPGKAPKLL IYWASTRHTG VPDRFTGSGS GTDFTLTISS LQPEDFADYF CQQYNSYPLT FGGGTKLEIK VDEWSHPQFE K

[0517] IL-15RaECD: 1-175

[0518] Linker: 176-190

[0519] J591 scFv VL: 191-309

[0520] J591 scFv linker: 310-323

[0521] J591 scFv VH: 324-430

[0522] Spacer: 431-433

[0523] STII: 434-441

[0524] CDR1 : 216-222

[0525] CDR2: 242-247

[0526] CDR3: 289-294

[0527] CDR4: 347-357

[0528] CDR5:373-379

[0529] CDR6: 412-420

[0530] SEQ ID NO:51 - IL-15RaSu-(linker)-J591 (PSMA scFv)-STII

[0531] ITCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA

[0532] TNIAHWTTPS LKCIRGGGGS GGGGSGGGGS EVQLVQSGAE VKKPGASVKI

[0533] SCKTSGYTFT EYTIHWVKQA SGKGLEWIGN INPNNGGTTY NQKFEDRATL TVDKSTSTAY MELSSLRSED TAVYYCAAGW NFDYWGQGTT VTVSSGSTSG

[0534] GGSGGGSGGG GSSDIVMTQS PSSLSASVGD RVTITCKASQ DVGTAVDWYQ

[0535] QKPGKAPKLL IYWASTRHTG VPDRFTGSGS GTDFTLTISS LQPEDFADYF

[0536] CQQYNSYPLT FGGGTKLEIK VDEWSHPQFE K

[0537] IL-15RaSu: 1-65

[0538] Linker: 66-80

[0539] PSMA scFv VL: 81-199

[0540] PSMA scFv linker: 200-213

[0541] PSMA scFv VH: 214-320

[0542] Spacer: 321-323

[0543] STII: 324-331

[0544] CDR1 : 106-112

[0545] CDR2: 132-137

[0546] CDR3: 179-184

[0547] CDR4: 237-247

[0548] CDR5:263-269

[0549] CDR6: 302-310

[0550] SEQ ID NO:52 - IL-15RaSU-(linker)-NKp30L ECD-STI1

[0551] ITCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRGGGGS GGGGSGGGGS DLKVEMMAGG TQITPLNDNV TI FCNI FYSQ PLNITSMGIT WFWKSLTFDK EVKVFEFFGD HQEAFRPGAI VSPWRLKSGD ASLRLPGIQL EEAGEYRCEV WTPLKAQGT VQLEWASPA SRLLLDQVGM KENEDKYMCE SSGFYPEAIN ITWEKQTQKF PHPIEISEDV ITGPTIKNMD GTFNVTSCLK LNSSQEDPGT VYQCWRHAS LHTPLRSNFT LTAARHSLSE TEKTDNFSVD EWSHPQFEK

[0552] IL-15RaSu: 1-65

[0553] Linker: 66-80

[0554] NKp60L ECD: 81-318

[0555] Spacer: 319-321

[0556] STII: 322-329

[0557] SEQ ID NO: 53 - IL-15RaECD-(linker)-NKp30L ECD- STII

[0558] ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTEISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS DLKVEMMAGG TQITPLNDNV TI FCNI FYSQ PLNITSMGIT WFWKSLTFDK EVKVFEFFGD HQEAFRPGAI VSPWRLKSGD ASLRLPGIQL EEAGEYRCEV WTPLKAQGT VQLEWASPA SRLLLDQVGM KENEDKYMCE SSGFYPEAIN ITWEKQTQKF PHPIEISEDV ITGPTIKNMD GTFNVTSCLK LNSSQEDPGT VYQCWRHAS LHTPLRSNFT LTAARHSLSE TEKTDNFSVD EWSHPQFEK

[0559] IL-15RaECD: 1- 175

[0560] Linker: 176-190 NKp60L ECD: 191-428 Spacer: 429-431 STII: 432-439

[0561] SEQ ID NO: 54 - IL-15RaSu- (linker)-HER2 scFv (Trastuzumab-based scFv)-STII

[0562] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRGGGGS GGGGSGGGGS EVQLVES GGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFT I SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS GSTSGSGKPG SGEGSTKGDI QMTQSPSSLS ASVGDRVT I T CRASQDVNTA VAWYQQKPGK APKLLIYSAS FLYSGVPSRF SGSRSGTDFT LT I SSLQPED FATYYCQQHY TTPPTFGQGT KVE IKRTWD EWSHPQFEK

[0563] IL-15RaSu: 1-65

[0564] Linker: 66-80

[0565] HER2 scFv VL: 81-200

[0566] HER2 scFv linker: 201-218

[0567] HER2 scFv VH: 219-328

[0568] Spacer: 329-331

[0569] STII: 332-339

[0570] CDR1 : 106-112

[0571] CDR2: 132-137

[0572] CDR3: 179-189

[0573] CDR4: 242-252 CDR5:268-274 CDR6: 307-315

[0574] SEQ ID NO:55 - IL-15RaECD-(linker)-HER2 scFv (Trastuzumab-based scFv)-STII

[0575] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS GSTSGSGKPG SGEGSTKGDI QMTQSPSSLS ASVGDRVT I T CRASQDVNTA VAWYQQKPGK APKLLIYSAS FLYSGVPSRF SGSRSGTDFT LT I SSLQPED FATYYCQQHY TTPPTFGQGT KVE IKRTWD EWSHPQFEK

[0576] IL-15RaECD: 1-175

[0577] Linker: 176-190

[0578] HER.2 scFv VL: 191-310

[0579] HER2 scFv linker: 311-328

[0580] CDHER2133 scFv VH: 329-438

[0581] Spacer: 439-441

[0582] STII: 442-449

[0583] CDR1 : 216-222

[0584] CDR2: 242-247 CDR3: 289-299

[0585] CDR4: 352-362

[0586] CDR5: 378-384

[0587] CDR6: 417-425

[0588] SEQ ID NO:56 - IL-15RaSU-(linker)-HER2 scFv-STH

[0589] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRGGGGS GGGGSGGGGS MDLQLTQSPA ILSASPGEKV TMTCRATPSV SYMHWYQQKP GSSPKPWIYT TSNLASGVPA RFSGGGSGTS YSLTVSRVEA EDAATYYCQQ WSRSPPTFGG GSKLEIKGST SGSGKSSEGK GVQLQESGPE WKPGGSMKI SCKTSGYS FT GHTMNWVKQS HGKNLEWIGL INPYNGDTNY NQKFKGKATF TVDKSSSTAY MELLSLTSED SAVYYCARRV

[0590] TDWYFDVWGA GTTVTVSVDE WSHPQFEK

[0591] IL-15RaSu: 1-65

[0592] Linker: 66-80

[0593] HER2 scFv VL: 81-187

[0594] HER2 scFv linker: 188-201

[0595] HER2 scFv VH: 202-317

[0596] Spacer: 318-320

[0597] STII: 321-328

[0598] CDR1 : 105-114

[0599] CDR2: 130-136

[0600] CDR3: 169-177

[0601] CDR4: 226-232

[0602] CDR5: 252-257

[0603] CDR6: 299-307

[0604] SEQ ID NO:57 - IL-15RaECD-(linker)-HER2 scFv-STII

[0605] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGGGGS GGGGSGGGGS MDLQLTQSPA ILSASPGEKV TMTCRATPSV SYMHWYQQKP GSSPKPWIYT TSNLASGVPA RFSGGGSGTS YSLTVSRVEA EDAATYYCQQ WSRSPPTFGG GSKLEIKGST SGSGKSSEGK GVQLQESGPE WKPGGSMKI SCKTSGYS FT GHTMNWVKQS

[0606] HGKNLEWIGL INPYNGDTNY NQKFKGKATF TVDKSSSTAY MELLSLTSED SAVYYCARRV TDWYFDVWGA GTTVTVSVDE WSHPQFEK

[0607] IL-15RaECD: 1-175

[0608] Linker: 176-190

[0609] HER2 scFv VL: 191-297

[0610] HER2 scFv linker: 298-311

[0611] I IER2 scFv VII: 312-427

[0612] Spacer: 428-430

[0613] STII: 431-438

[0614] CDR1 : 215-224 CDR2: 240-246

[0615] CDR3: 279-287

[0616] CDR4: 336-342

[0617] CDR5: 362-367

[0618] CDR6: 409-417

[0619] SEQ ID NO: 58 - IL-15RaECD-(linker)- TGFprllb arm l-(linker)-TGFprIIb

[0620] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTGSTSG SGKPGSGEGS TKGT IPPHVQ KSVNNDMIVT DNNGAVKFPQ LCKFCDVRFS TCDNQKSCMS NCS ITS ICEK PQEVCVAVWR KNDENI TLET VCHDPKLPYH DFILEDAASP KCIMKEKKKP GETFEMCSCS SDECNDNI I F SEEYNTSNPD LLLVI FQGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG GST IPPHVQK SVNNDMIVTD NNGAVKFPQL CKFCDVRFST CDNQKSCMSN CS I TS ICEKP QEVCVAVWRK NDENITLETV

[0621] CHDPKLPYHD FILEDAASPK CIMKEKKKPG ETFFMCSCSS DECNDNI I FS EEYNTSNPDL LLVI FQVDEW SHPQFEK

[0622] IL-15RaECD: 1-175

[0623] Linker: 176-193

[0624] TGF beta rllb arml : 193-337

[0625] Linker: 338-372

[0626] TGF beta rllb arml : 373-516

[0627] Spacer: 517-519

[0628] STII: 520-527

[0629] SEQ ID NO:59 - IL-15RaSu-(whitlow linker)- TGFprllb arm l-(linker)-TGFPrIIb arm2-STII I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRGSTSG SGKPGSGEGS TKGT IPPHVQ KSVNNDMIVT DNNGAVKFPQ LCKFCDVRFS TCDNQKSCMS NCS ITS ICEK PQEVCVAVWR KNDENI TLET VCHDPKLPYH DFILEDAASP KCIMKEKKKP GETFEMCSCS SDECNDNI IF SEEYNTSNPD LLLVI FQGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG GST IPPHVQK SVNNDMIVTD NNGAVKFPQL CKFCDVRFST CDNQKSCMSN CS I TS ICEKP QEVCVAVWRK NDENITLETV CHDPKLPYHD FILEDAASPK CIMKEKKKPG ETFFMCSCSS DECNDNI I FS EEYNTSNPDL LLVI FQVDEW SHPQFEK

[0630] IL-15RaSu: 1-65

[0631] Linker: 66-83

[0632] TGF beta rllb arm ! : 84-227

[0633] Linker: 228-262

[0634] TGF beta rllb arml : 263-406

[0635] Spacer: 407-409

[0636] STII: 410-417

[0637] VI. Non-TriKE recombinant fusion proteins for binding to the IL-15Ra arm: SEQ ID NO:60 - caml6-(linker)-IL-15-10xhis

[0638] QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS

[0639] I TWSGRDTFY ADSVKGRFT I SRDNSKNTLY LQMNSLRAED TAVYYCAANP

[0640] WPVAAPRSGT YWGQGTLVTV SSSGGGGSGG GGSGGGGSGG GGSGNWVNVI

[0641] SDLKKIEDLI QSMHIDATLY TESDVHPSCK VTAMKCFLLE LQVISLESGD AS IHDTVENL I ILANNSLSS NGNVTESGCK ECEELEEKNI KEFLQS FVHI VQMFINTSVD EHHHHHHHHH H

[0642] Caml6: 1-122

[0643] Linker: 123-144

[0644] IL-15: 145-258

[0645] Spacer: 259-261 lOxHis: 262-271

[0646] CDR1 : 26-32

[0647] CDR2: 52-57

[0648] CDR3: 99-111

[0649] SEQ ID N0:61 - caml6-(linker)-IL-15 (no tag)

[0650] QVQLVESGGGL VQPGGSLRLSC AASGLTFSSYN MGWFRQAPGQG LEAVAS ITWSG RDTFYADSVKG RFTI SRDNSKN TLYLQMNSLRA EDTAVYYCAAN PWPVAAPRSGT YWGQGTLVTVS SSGGGGSGGGG SGGGGSGGGGS GNWVNVISDLK KIEDLIQSMHI DATLYTESDVH PSCKVTAMKCF LLELQVI SLES GDAS IHDTVEN LI ILANNSLSS NGNVTESGCKE CEELEEKNIKE FLQS FVHIVQM FINTS

[0651] Caml6: 1-122

[0652] Linker: 123-144

[0653] IL-15: 145-258

[0654] CDR1 : 26-32

[0655] CDR2: 52-57

[0656] CDR3: 99-111

[0657] SEQ ID NO:62 - B7H3-(linker)-IL-15-(linker)-B7H3-10xhis

[0658] QVQLVESGGG LVQPGGSLRL SCAASGFTFS SYWMYWVRQT PGKGLEWVST INRDGSATWY ADSVKGRFT I SRDNAKNTGY LQMNSLKPDD TAVYYCVSDP DNYSSDEMVP YWGQGTQVTV SSAEAAKEAA KEAAKEAAKA LEAEAAKEAA KEAAKEAAKA NWVNVI SDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDAS IH DTVENLI ILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QS FVHIVQMF INTSAEAAKE AAKEAAKEAA KALEAEAAKE AAKEAAKEAA KAQVQLVESG GGLVQPGGSL RLSCAASGFT FSSYWMYWVR QTPGKGLEWV ST INRDGSAT WYADSVKGRF T I SRDNAKNT GYLQMNSLKP DDTAVYYCVS DPDNYSSDEM VPYWGQGTQV TVSSVDEHHH HHHHHHH

[0659] B7H3: 1-122

[0660] Linker: 123-160

[0661] IL-15: 161-274

[0662] Linker: 275-312 B7H3: 313-434

[0663] Spacer: 435-437 lOxHis: 438-447 CDR1 : 26-32 CDR2:52-57 CDR3:99-111 CDR4:338-344 CDR5: 364-369 CDR6: 411-423

[0664] SEQ ID NO:63 - NKp3OL ECD-(linker)-IL-15-(linker)-B7H3 clone 2-10xhis

[0665] DLKVEMMAGG TQI TPLNDNV T I FCNI FYSQ PLNITSMGI T WFWKSLTFDK EVKVFEFFGD HQEAFRPGAI VSPWRLKSGD ASLRLPGIQL EEAGEYRCEV WTPLKAQGT VQLEWASPA SRLLLDQVGM KENEDKYMCE SSGFYPEAIN I TWEKQTQKF PHPIEI SEDV I TGPTIKNMD GTFNVTSCLK LNSSQEDPGT VYQCWRHAS LHTPLRSNFT LTAARHSLSE TEKTDNFSSG GGGSGGGGSG GGGSGGGGSG NWVNVI SDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDAS IH DTVENLI ILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QS FVHIVQMF INTSAEAAKE AAKEAAKEAA KALEAEAAKE AAKEAAKEAA KAQVQLVESG GGLVQPGGSL RLSCAASGFT FSSYWMYWVR QTPGKGLEWV ST INRDGSAT WYADSVKGRF T I SRDNAKNT GYLQMNSLKP DDTAVYYCVS DPDNYSSDEM VPYWGQGTQV TVSSVDEHHH HHHHHHH

[0666] NKp30L ECD: 1-238

[0667] Linker: 239-260

[0668] IL-15: 261-374

[0669] Linker: 375-412

[0670] B7H3 clone 2: 413-534

[0671] Spacer: 535-537 lOxHis: 538-547

[0672] SEQ ID NO:64 - canine CD16 scFv-(linker)-canine IL15-(linker)-MEDI-10xhis

[0673] QILLTQSPAI MSASPGEKVT MTCSASSSVG YMHWYQQKPG SSPKPWIYDT SDLASGFPAR FSGSRSGTSY SLI ISSMEAE DAATYYCHQR S FYPYTFGGG TKLEIKGGGG SGGGGSGGGG SEVQLQQSGA ELVRSGASVK LSCTASGFNI KDYYMHWVKQ RPEQGLEWIG WLDPENGDTV YAPKFQGRAT MTADTSSNTA YLHLSSLTSE DTAVYYCNAL VYSLLGQDYW GQGTTLTVSS SGGGGSGGGG SGGGGSGGGG SGNWQDVILD LEKIDNLIQS IHMDTTLYTE SDVHPSCKVT AMKCFLLELG VI SLESGSHP IKEAVENLI I LANSDLSSKG NI TETGCKEC EELEEKS IKE FLQSFVHIVQ MFINSSSGGG GSGGGGSGGG GSGGGGSGGS EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYPMNWVRQA PGKGLEWVSY I SPFGGMTDY ATSVKGRFT I SRDNSKNTLY LQMNSLRAED TAVYYCARDA MRGAEVDYWG QGTLVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTI TCRSS QS I PSYLNWY QQKPGKAPKL LIYAASRLQS GVPSRFSGSG SGTDFTLT IS SLQPEDFATY YCQQSYSTPL TFGGGTKVE I KVDEHHHHHH HHHH Canine CD 16 scFv VL: 1-106

[0674] Canine CD16 scFv linker: 107-121

[0675] Canine CD 16 scFv VH: 122-241

[0676] Linker: 242-262

[0677] Canine IL-15: 263-376

[0678] Linker: 377-399

[0679] MEDI VL: 400-519

[0680] MEDI linker: 520-534

[0681] MEDI VH: 535-641

[0682] Spacer: 642-644 lOxHis: 645-654 CDR1 : 24-33 CDR2: 49-55 CDR3:88-96 CDR4: 147-153 CDR5: 173-178 CDR6: 220-229

[0683] SEQ ID NO:65 - canine CD16 scFv-(linker)-human IL15-(linker)-MEDI-10xhis QILLTQSPAI MSASPGEKVT MTCSASSSVG YMHWYQQKPG SSPKPWIYDT SDLASGFPAR FSGSRSGTSY SLI ISSMEAE DAATYYCHQR SFYPYTFGGG TKLEIKGGGG SGGGGSGGGG SEVQLQQSGA ELVRSGASVK LSCTASGFNI KDYYMHWVKQ RPEQGLEWIG WLDPENGDTV YAPKFQGRAT MTADTSSNTA YLHLSSLTSE DTAVYYCNAL VYSLLGQDYW GQGTTLTVSS SGGGGSGGGG SGGGGSGGGG SGNWVNVISD LKKIEDLIQS MHIDATLYTE SDVHPSCKVT AMKCFLLELQ VISLESGDAS IHDTVENLI I LANNSLSSNG NVTESGCKEC EELEEKNIKE FLQSFVHIVQ MFINTSSGGG GSGGGGSGGG GSGGGGSGGS EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYPMNWVRQA PGKGLEWVSY ISPFGGMTDY ATSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDA MRGAEVDYWG QGTLVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCRSS QS IPSYLNWY QQKPGKAPKL LIYAASRLQS GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCQQSYSTPL TFGGGTKVEI KVDEHHHHHH HHHH

[0684] Canine CD 16 scFv VL: 1-106

[0685] Canine CD16 scFv linker: 107-121

[0686] Canine CD 16 scFv VH: 122-241

[0687] Linker: 242-262

[0688] Human IL-15: 263-376

[0689] Linker: 377-399

[0690] MEDI VL: 400-519

[0691] MEDI linker: 520-534

[0692] MEDI VH: 535-641

[0693] Spacer: 642-644 lOxHis: 645-654

[0694] CDR1 : 24-33 CDR2: 49-55

[0695] CDR3:88-96

[0696] CDR4: 147-153

[0697] CDR5: 173-178

[0698] CDR6: 220-229

[0699] SEQ ID NO:66 - 10*His

[0700] HHHHHHHHHH

[0701] VIII. Engager Sequences

[0702] SEQ ID NO: 67 - CD33 scFv

[0703] QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYNMHWVRQA PGQGLEWIGY

[0704] IYPYNGGTGY NQKFKSKATI TADESTNTAY MELSSLRSED TAVYYCARGR

[0705] PAMDYWGQGT LVTVSSGGGG SGGGGSGGGG SDIQMTQSPS SLSASVGDRV

[0706] TITCRASESV DNYGISFMNW FQQKPGKAPK LLIYAASNQG SGVPSRFSGS

[0707] GSGTDFTLTI SSLQPDDFAT YYCQQSKEVP WTFGQGTKVE IK

[0708] VH: 1-116

[0709] Linker: 117-131

[0710] VL: 132-244

[0711] CDRL26-32

[0712] CDR2:52-57

[0713] CDR3: 99-105

[0714] CDR4: 155-169

[0715] CDR5: 185-191

[0716] CDR6: 224-232

[0717] SEQ ID NO: 68 - canine CD 16 scFv

[0718] QILLTQSPAI MSASPGEKVT MTCSASSSVG YMHWYQQKPG SSPKPWIYDT

[0719] SDLASGFPAR FSGSRSGTSY SLI ISSMEAE DAATYYCHQR SFYPYTFGGG

[0720] TKLEIKGGGG SGGGGSGGGG SEVQLQQSGA ELVRSGASVK LSCTASGFNI

[0721] KDYYMHWVKQ RPEQGLEWIG WLDPENGDTV YAPKFQGRAT MTADTSSNTA

[0722] YLHLSSLTSE DTAVYYCNAL VYSLLGQDYW GQGTTLTVSS S

[0723] VH: 1-101

[0724] Linker: 102-121

[0725] VL: 122-241

[0726] CDR1 : 24-33

[0727] CDR2: 49-55

[0728] CDR3: 88-96

[0729] CDR4: 147-153

[0730] CDR5: 173-178

[0731] CDR6: 220-229

[0732] SEQ ID NO: 69 - canine IL 15 NWQDVILDLE KIDNLIQS IH MDTTLYTESD VHPSCKVTAM KCFLLELGVI SLESGSHPIK EAVENLI ILA NSDLSSKGNI TETGCKECEE LEEKS IKEFL QS FVHIVQMF INSS

[0733] SEQ ID NO: 70 - CD33 scFv

[0734] QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYNMHWVRQA PGQGLEWIGY IYPYNGGTGY NQKFKSKAT I TADESTNTAY MELSSLRSED TAVYYCARGR PAMDYWGQGT LVTVSSGGGG SGGGGSGGGG SDIQMTQSPS SLSASVGDRV T I TCRASESV DNYGI S FMNW FQQKPGKAPK LLIYAASNQG SGVPSRFSGS GSGTDFTLTI SSLQPDDFAT YYCQQSKEVP WTFGQGTKVE IK

[0735] VH: 1-116

[0736] Linker: 117-131

[0737] VL: 132-242

[0738] CDR1 : 26-32

[0739] CDR2: 52-57

[0740] CDR3: 99-105

[0741] CDR4: 155-169

[0742] CDR5: 185-191

[0743] CDR6: 224-232

[0744] SEQ ID N0:71 - caml6-(linker)-IL15-(linker)-CD33-10xhis

[0745] QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS I TWSGRDTFY ADSVKGRFT I SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SSSGGGGSGG GGSGGGGSGG GGSGNWVNVI SDLKKIEDLI QSMHIDATLY TESDVHPSCK VTAMKCFLLE LQVISLESGD AS IHDTVENL I ILANNSLSS NGNVTESGCK ECEELEEKNI KEFLQS FVHI VQMFINTSGS TSGSGKPGSG EGSTKGQVQL VQSGAEVKKP GSSVKVSCKA SGYTFTDYNM HWVRQAPGQG LEWIGYIYPY NGGTGYNQKF KSKATI TADE STNTAYMELS SLRSEDTAVY YCARGRPAMD YWGQGTLVTV SSGGGGSGGG GSGGGGSDIQ MTQSPSSLSA SVGDRVT I TC RASESVDNYG I S FMNWFQQK PGKAPKLLIY AASNQGSGVP SRFSGSGSGT DFTLTI SSLQ PDDFATYYCQ QSKEVPWTFG QGTKVE IKVD EHHHHHHHHH H

[0746] Caml6: 1-122 Linker: 123-144 IL-15: 145-258 Linker: 259-276 CD33 VH: 277-392 CD33 Linker: 393-407 CD33 VL: 408-518 Spacer: 519-521 lOxHis: 521-531 CDR1 : 302-308 CDR2: 328-333 CDR3: 375-381 CDR4: 431-445

[0747] CDR5: 461-467

[0748] CDR6: 500-508

[0749] SEQ ID NO:72 - caml61533 TriKE

[0750] MKWVTFI SLL FLFSSAYSQV QLVESGGGLV QPGGSLRLSC AASGLTFSSY NMGWFRQAPG QGLEAVAS I T WSGRDTFYAD SVKGRFT I SR DNSKNTLYLQ MNSLRAEDTA VYYCAANPWP VAAPRSGTYW GQGTLVTVSS SGGGGSGGGG SGGGGSGGGG SGNWVNVI SD LKKIEDLIQS MHIDATLYTE SDVHPSCKVT AMKCFLLELQ VI SLESGDAS IHDTVENLI I LANNSLSSNG NVTESGCKEC EELEEKNIKE FLQSFVHIVQ MFINTSGSTS GSGKPGSGEG STKGQVQLVQ SGAEVKKPGS SVKVSCKASG YTFTDYNMHW VRQAPGQGLE WIGYIYPYNG GTGYNQKFKS KAT ITADEST NTAYMELSSL RSEDTAVYYC ARGRPAMDYW GQGTLVTVSS GGGGSGGGGS GGGGSDIQMT QSPSSLSASV GDRVTI TCRA

[0751] SESVDNYGIS FMNWFQQKPG KAPKLLIYAA SNQGSGVPSR FSGSGSGTDF TLT ISSLQPD DFATYYCQQS KEVPWTFGQG TKVE IKVDE

[0752] SEQ ID NO:73 - CLEC12a.9

[0753] QVQLLESGGG LVQPGGSLRL SCAASGYS I T DQDMSWVRQA PGKGLEWVSG ILATSGSTYY ADSVKGRFT I SRDNSKNTLY LQMNSLRAED TAVYYCAGEV EKSSQSMPFW GQGTLVTVSS

[0754] CDR1 : 26-32

[0755] CDR2: 52-57

[0756] CDR3: 99-109

[0757] SEQ ID NO: 74 - CLEC12a .23

[0758] QVQLLESGGG LVQPGGSLRL SCAASGFRLT DEDMSWVRQA PGKGLEWVST IATRDGSTYY ADSVKGRFT I SRDNSKNTLY LQMNSLRAED TAVYYCAGLR DQLYGNEQLA FWGQGTLVTV SS

[0759] CDR1 : 26-32

[0760] CDR2: 52-57

[0761] CDR3: 99-111

[0762] SEQ ID NO:75 - CLEC12a .33

[0763] QVQLLESGGG LVQPGGSLRL SCAASGDMFS YDDMGWVRQA PGKGLEWVSG IQNTDGSTYY ADSVKGRFT I SRDNSKNTLY LQMNSLRAED TAVYYCATLY DRMVGKEEQL ASWGQGTLVT VSS

[0764] CDR1 : 26-32

[0765] CDR2: 52-57

[0766] CDR3: 99-112

[0767] SEQ ID NO: 76 - CLEC12a.4O QVQLLESGGG LVQPGGSLRL SCAASGDTIN PEDMGWVRQA PGKGLEWVSA IEAQSGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARLT AHQEEPVAYW GQGTLVTVSS

[0768] CDR1 : 26-32

[0769] CDR2: 52-57

[0770] CDR3: 99-109

[0771] SEQ ID NO:77 - CLEC12a.6O

[0772] QVQLLESGGG LVQPGGSLRL SCAASGYMFS ADVMSWVRQA PGKGLEWVSG IWIPDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAAQL NERIAS ITKN MHSWGQGTLV TVSS

[0773] CDR1 : 26-32

[0774] CDR2: 52-57

[0775] CDR3: 99-113

[0776] SEQ ID NO: 78 - CLEC12a.86

[0777] QVQLLESGGG LVQPGGSLRL SCAASGDMLS AQDMGWVRQA PGKGLEWVSG IDSDDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAALW GEDVIMQDSS VGSWGQGTLV TVSS

[0778] CDR1 : 26-32

[0779] CDR2: 52-57

[0780] CDR3: 99-113

[0781] V. Full sequence of PACC-arm recombinant proteins

[0782] SEQ ID NO: 79 - IL-15RaECD-(linker)-CLEC12a.9 -STII

[0783] ITCPPPMSVEH ADIWVKSYSLY SRERYICNSGF KRKAGTSSLTE CVLNKATNVAH

[0784] WTTPSLKCIRD PALVHQRPAPP STVTTAGVTPQ PESLSPSGKEP AASSPSSNNTA

[0785] ATTAAIVPGSQ LMPSKSPSTGT TEISSHESSHG TPSQTTAKNWE LTASASHQPPG

[0786] VYPQGHSDTTA EAAKEAAKEAA KEAAKALEAEA AKEAAKEAAKE AAKAQVQLLES

[0787] GGGLVQPGGSL RLSCAASGYS I TDQDMSWVRQA PGKGLEWVSGI LATSGSTYYAD

[0788] SVKGRFTISRD NSKNTLYLQMN SLRAEDTAVYY CAGEVEKSSQS MPFWGQGTLVT

[0789] VSSVDEWSHPQ FEK

[0790] IL-15RaECD: 1-175

[0791] Linker: 176-213

[0792] CLEC12a.9: 214-333

[0793] Spacer: 334-336

[0794] STII: 337-344

[0795] CDR1 : 239-245

[0796] CDR2: 265-270

[0797] CDR3: 312-322

[0798] SEQ ID NO:80 - IL-15RaSU-(linker)-CLEC12a.9 -STII ITCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA

[0799] TNIAHWTTPS LKCIRAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA

[0800] AKAQVQLLES GGGLVQPGGS LRLSCAASGY S ITDQDMSWV RQAPGKGLEW

[0801] VSGILATSGS TYYADSVKGR FTISRDNSKN TLYLQMNSLR AEDTAVYYCA

[0802] GEVEKSSQSM PFWGQGTLVT VSSVDEWSHP QFEK

[0803] IL-15RaSu: 1-65

[0804] Linker: 66-103

[0805] CLEC12a.9: 104-223

[0806] Spacer: 224-226

[0807] STII: 227-234

[0808] CDR1 : 129-135

[0809] CDR2: 155-160

[0810] CDR3: 202-212

[0811] SEQ ID NO:81 - IL-15RaECD-(linker)-CLEC12a.23 -STII

[0812] ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTEISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLLES GGGLVQPGGS LRLSCAASGF RLTDEDMSWV RQAPGKGLEW VSTIATRDGS TYYADSVKGR FTISRDNSKN TLYLQMNSLR AEDTAVYYCA GLRDQLYGNE QLAFWGQGTL VTVSSVDEWS HPQFEK

[0813] IL-15RaECD: 1-175

[0814] Linker: 176-213

[0815] CLEC12a.23: 214-335

[0816] Spacer: 336-338

[0817] STII: 339-346

[0818] CDR1 : 239-245

[0819] CDR2: 265-270

[0820] CDR3: 312-324

[0821] SEQ ID NO:82 - IL-15RaSU-(linker)-CLEC12a.23 -STII

[0822] ITCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLLES GGGLVQPGGS LRLSCAASGF RLTDEDMSWV RQAPGKGLEW VSTIATRDGS TYYADSVKGR FTISRDNSKN TLYLQMNSLR AEDTAVYYCA GLRDQLYGNE QLAFWGQGTL VTVSSVDEWS HPQFEK

[0823] IL-15RaSu: 1-65

[0824] Linker: 66-103

[0825] CLEC12a.33: 104-225

[0826] Spacer: 226-228

[0827] STII: 229-236

[0828] CDR1 : 129-135

[0829] CDR2: 155-160 CDR3: 202-214

[0830] SEQ ID NO: 83 - IL-15RaECD-(linker)-CLEC12a.33-STII

[0831] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLLES GGGLVQPGGS LRLSCAASGD MFSYDDMGWV RQAPGKGLEW VSGIQNTDGS TYYADSVKGR FT I SRDNSKN TLYLQMNSLR AEDTAVYYCA TLYDRMVGKE EQLASWGQGT LVTVSSVDEW SHPQFEK

[0832] IL-15RaECD: 1-175

[0833] Linker: 176-213

[0834] CLEC12a.33: 214-336

[0835] Spacer: 337-339

[0836] STII: 340-347

[0837] CDR1 : 239-245

[0838] CDR2: 265-270

[0839] CDR3: 312-325

[0840] SEQ ID NO: 84 - IL-15RaSU-(linker)-CLEC12a.33 -STII

[0841] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLLES GGGLVQPGGS LRLSCAASGD MFSYDDMGWV RQAPGKGLEW VSGIQNTDGS TYYADSVKGR FT I SRDNSKN TLYLQMNSLR AEDTAVYYCA TLYDRMVGKE EQLASWGQGT LVTVSSVDEW SHPQFEK

[0842] IL-15RaSu: 1-65

[0843] Linker: 66-103

[0844] CLEC12a.33: 104-226

[0845] Spacer: 227-229

[0846] STII: 230-237

[0847] CDR1 : 129-135

[0848] CDR2: 155-160

[0849] CDR3: 202-215

[0850] SEQ ID NO:85 - IL-15RaECD-(linker)-CLEC12a.40-STII

[0851] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLLES GGGLVQPGGS LRLSCAASGD T INPEDMGWV RQAPGKGLEW VSAIEAQSGS TYYADSVKGR FT I SRDNSKN TLYLQMNSLR AEDTAVYYCA RLTAHQEEPV AYWGQGTLVT VSSVDEWSHP QFEK

[0852] IL-15RaECD: 1-175

[0853] Linker: 176-213 CLEC12a.4O: 214-333

[0854] Spacer: 334-336

[0855] STII: 337-344

[0856] CDR1 : 239-245

[0857] CDR2: 265-270

[0858] CDR3: 312-322

[0859] SEQ ID NO:86 - IL-15RaSu-(linker)-CLEC12a.40-STII

[0860] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLLES GGGLVQPGGS LRLSCAASGD T INPEDMGWV RQAPGKGLEW VSAIEAQSGS TYYADSVKGR FT I SRDNSKN TLYLQMNSLR AEDTAVYYCA RLTAHQEEPV AYWGQGTLVT VSSVDEWSHP QFEK

[0861] IL-15RaSu: 1-65

[0862] Linker: 66-103

[0863] CLEC12a.4O: 104-223

[0864] Spacer: 224-226

[0865] STII: 227-234

[0866] CDR1 : 129-135

[0867] CDR2: 155-160

[0868] CDR3: 202-212

[0869] SEQ ID NO: 87 - IL-15RaECD-(linker)-CLEC12a.60-STII

[0870] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLLES GGGLVQPGGS LRLSCAASGY MFSADVMSWV RQAPGKGLEW VSGIWI PDGS TYYADSVKGR FT I SRDNSKN TLYLQMNSLR AEDTAVYYCA AQLNERIAS I TKNMHSWGQG TLVTVSSVDE WSHPQFEK

[0871] IL-15RaECD: 1-175

[0872] Linker: 176-213

[0873] CLEC12a.6O: 214-337

[0874] Spacer: 338-340

[0875] STII: 341-348

[0876] CDR1 : 239-245

[0877] CDR2: 265-270

[0878] CDR3: 312-326

[0879] SEQ ID NO:88 - IL-15RaSu-(linker)-CLEC12a.60-STII

[0880] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLLES GGGLVQPGGS LRLSCAASGY MFSADVMSWV RQAPGKGLEW VSGIWI PDGS TYYADSVKGR FT I SRDNSKN TLYLQMNSLR AEDTAVYYCA AQLNERIAS I TKNMHSWGQG TLVTVSSVDE WSHPQFEK IL-15RaSu: 1-65

[0881] Linker: 66-103

[0882] CLEC12a.6O: 104-227

[0883] Spacer: 228-230

[0884] STII: 231-238

[0885] CDR1 : 129-135

[0886] CDR2: 155-160

[0887] CDR3: 202-216

[0888] SEQ ID NO: 89 - IL-15RaECD-(linker)-CLEC12a.86-STII

[0889] I TCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA

[0890] TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTE ISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLLES GGGLVQPGGS LRLSCAASGD MLSAQDMGWV RQAPGKGLEW VSGIDSDDGS TYYADSVKGR FT I SRDNSKN TLYLQMNSLR

[0891] AEDTAVYYCA ALWGEDVIMQ DSSVGSWGQG TLVTVSSVDE WSHPQFEK

[0892] IL-15RaECD: 1-175

[0893] Linker: 176-213

[0894] CLEC12a.86: 214-337

[0895] Spacer: 338-340

[0896] STII: 341-348

[0897] CDR1 : 239-245

[0898] CDR2: 265-270

[0899] CDR3: 312-326

[0900] SEQ ID NO:90 - IL-15RaSU-(linker)-CLEC12a.86-STII

[0901] I TCPPPVSVE HADIRVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNIAHWTTPS LKCIRAEAAK EAAKEAAKEA AKALEAEAAK EAAKEAAKEA AKAQVQLLES GGGLVQPGGS LRLSCAASGD MLSAQDMGWV RQAPGKGLEW VSGIDSDDGS TYYADSVKGR FT I SRDNSKN TLYLQMNSLR AEDTAVYYCA ALWGEDVIMQ DSSVGSWGQG TLVTVSSVDE WSHPQFEK

[0902] IL-15RaSu: 1-65

[0903] Linker: 66-103

[0904] CLEC12a.86: 104-227

[0905] Spacer: 228-230

[0906] STII: 231-238

[0907] CDR1 : 129-135

[0908] CDR2: 155-160

[0909] CDR3: 202-216

Claims

What is claimed is:

1. A multispecific compound comprising: a first protein comprising: an IL- 15 domain; a first functional domain; and a second functional domain; and a second protein comprising: an IL-15Ra domain; and a third functional domain.

2. The multispecific compound of claim 1, wherein the first functional domain comprises a first targeting domain and the second functional domain comprises a second targeting domain.

3. The multispecific compound of claim 2, wherein the third functional domain comprises a third targeting domain.

4. The multispecific compound of claim 2, wherein the third functional domain comprises albumin.

5. The multispecific compound of claim 1, wherein the first functional domain comprises a first targeting domain, the second functional domain comprises albumin, and the third functional domain comprises a second targeting domain.

6. The multispecific compound of any preceding claim, wherein the second protein further comprises a fourth functional domain.

7. The multispecific compound of claim 6, wherein the fourth functional domain comprises a targeting domain.

8. The multispecific compound of claim 6, wherein the fourth functional domain comprises albumin.

9. The multispecific compound of any preceding claim, wherein the IL- 15 domain comprises an amino acid sequence having at least 70% identity with SEQ ID NO: 18.

10. The multi specific compound of any preceding claim, wherein the IL-15Ra domain comprises the extracellular domain of IL-15Ra.

11. The multispecific compound of claim 10, wherein the IL-15Ra domain comprises an amino acid sequence having at least 70% identity with SEQ ID NO:2.

12. The multispecific compound of any preceding claim, wherein the IL-15Ra domain comprises the IL-15Ra sushi domain.

13. The multispecific compound of claim 12, wherein the IL-15Ra domain comprises an amino acid sequence having at least 70% identity with SEQ ID NO: 1.

14. The multispecific compound of any preceding claim, wherein the first functional domain, the second functional domain, the third functional domain, and / or the fourth functional domain each independently comprises albumin, such as human serum albumin, an anti-CD33 antibody, an anti-B7H3 antibody, an anti-CLEC12A antibody, an anti-CD16 antibody, an anti-CD3, an anti-ADAM17 antibody, an anti-mesothelin antibody, an anti -PSM A antibody, an anti-CD133 antibody, anti-CD83, a TGFp RI ECD, a TGFp RII ECD, a CTLA-4 ECD, a PD-1 ECD, NKp30L ECD, CD27 ECD, CLEC12 or a variant thereof, or HSA.

15. The multispecific compound of any preceding claim, wherein the first functional domain, the second functional domain, the third functional domain, and / or the fourth functional domain each independently comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NOs: 19-31 and 73-78.

16. The multispecific compound of any preceding claim, wherein the first protein comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NOs:34-59 and 79- 90 and wherein the second protein comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NOs:60-63 and 73-78.

17. An isolated nucleic acid encoding the amino acid sequence of any preceding claim.

18. The isolated nucleic acid of claim 17, wherein the isolated nucleic acid sequence encodes an amino acid sequence having at least 70% identity with any one of SEQ ID NOs: 34-59 and 79-90and wherein the second protein comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NOs:60-63 and 73-78.

19. A host cell comprising the isolated nucleic acid of claim 17.

20. A pharmaceutical composition comprising: the multispecific compound of any one of claims 1-16; and a pharmaceutically acceptable carrier.

21. A method compri sing : administering to a subject the multispecific compound of any one of claims 1-16 in an amount effective to induce natural killer (NK)-mediated killing of a cell.

22. A method for stimulating expansion of natural killer (NK) cells in vivo comprising: administering to a subject the multispecific compound of any one of claims 1-16 in an amount effective to stimulated expansion of NK cells in the subject.

23. A method of treating a subject having, or at risk of having cancer comprising: administering to the subject the multispecific compound of any one of claims 1-16 in an amount effective to: ameliorate at least one symptom or clinical sign of cancer; or decrease the likelihood that the subject develops cancer compared to an untreated individual.

24. The method of claim 23, wherein the cancer comprises prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematopoietic cancer.

25. The method of any one of claims 22-24, wherein the multispecific compound is administered prior to, simultaneously with, or following chemotherapy, surgical resection of a tumor, or radiation therapy.

26. The method of claim 25, wherein the chemotherapy comprises altretamine, amsacrine, L- asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphane, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamaide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, tioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, or vinorelbine.

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