Multispecific polypeptide for immune cell immunotherapy of cancer

Abstract

Provided herein is a multispecific polypeptide that includes (i) an immune cell targeting moiety, ii) a tumor targeting moiety, (iii) a cytokine molecule, and (iv) a chemokine. Also provided are methods of using said polypeptide, such as to treat cancer.

Description

[0001] MULTISPECIFIC POLYPEPTIDE FOR IMMUNE CELL IMMUNOTHERAPY OF CANCER

[0002] PRIORITY

[0003] This application claims the benefit of priority to U.S. Provisional Serial No. 63 / 675,322, filed Jul 25, 2024, which is incorporated by reference as if fully set forth herein.

[0004] INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0005] This application contains a Sequence Listing which has been submitted electronically in ST26 format and hereby incorporated by reference in its entirety. Said ST26 file, created on July 25, 2025, is named 1165200W01.xml and is 50,312 bytes in size.

[0006] BACKGROUND

[0007] The dichotomy between the need for immune, such as NK, cell presence and the inevitable dysfunction that impairs these cells’ activity once inside the tumor involves a synergism between recruitment and activation that simultaneously enhances both. Inadequacy of NK cell activity against solid tumors, such as GBM,1has been noted pre-clinically and clinically, with multiple trials resulting in no noteworthy improvements in patient survival.2,3And though genetic engineering has been pursued as a means to either knock in desired anti -tumor specificity - namely via chimeric antigen receptor (CAR) engineering, or by knocking out inhibitory signaling,4such edits have so far resulted in only incremental improvements in NK cell anti -tumor capacity.

[0008] Absent from previous engineering approaches is a consideration for the trafficking of NK cells to complex solid tumors. Intravenous infusions of NK cells have a limited capacity to penetrate brain tumors, with the blood-brain barrier posing the most severe, though not exclusive, barrier to their trafficking. Intratumoral injections of NK cells have, thus, been the preferred method of administration of adoptive NK cell therapies to patients.5However, even in cases of locoregional administration of NK cells, inefficient chemokine receptor-ligand gradients disrupt the movement of NK and bystander immune cells to and within the tumor, resulting in limited efficacy of the infused cells.6,7Many tumors are characterized by low levels of chemokines for trafficking of immune cells, despite adequate levels of chemokine receptors on effector cells.

[0009] Synaptic engagement between NK and tumor cells has been achieved with the engineering of antibody-based engagers, which have the unique capacity to ligate local NK cells to tumors via single or dual tumor antigen specificity. Engineering such engagers has promoted the recruitment of NK cells via activating receptors such as CD16a, NKG2D, NKp30, NKp80, or NKp46;8 10such variety being attractive due to being independent of heterogeneous expression levels of CD 16a or NKG2D or CD 16a polymorphism in tumors. Such NK cell engagers have often incorporated the endogenous production of a cytokine, most notably IL-15,11to further enhance the persistence of otherwise short-lived NK cells in the tumor. Pre-clinical responses have been achieved in tumors including colorectal cancer, breast cancer, ovarian cancer, and lung cancer, among others.12SUMMARY

[0010] In one embodiment, the disclosure includes a multispecific polypeptide comprising an immune cell targeting moiety, a tumor targeting moiety, a cytokine molecule, and a chemokine. The targeting moieties can be antibodies, ligands, or receptors that bind immune cell markers - such as NK cell markers (NKp30, NKp44, NKG2D, CD16a / FCGR3A) or T cell markers (CD3, CD28) and cancer antigens on hematological or solid tumors (for example, PDL1, mesothelin, CA9, CA12, or GLUT1 on pancreatic, breast, colorectal, lung, ovarian, glioblastoma, or liver cancer cells). The cytokine can be selected from GM-CSF, IL-la / p, IL-2, IL-4, IL-7, IL-15 (including IL-15 mutants), IFN-a / p / y, TNFa / p, or others, while the chemokine can be chosen from CCL1-CCL28, CXCL1-CXCL12, XCL1 / 2, or CS3CLl (for example, CXCL10). The chemokine can be joined to the polypeptide via a tumor protease-sensitive linker, such as a uPA or MMP- cleavable sequence.

[0011] In another embodiment, the polypeptide comprises paired heavy and light IgG chains with variable domains derived from antibodies against the immune cell and tumor targets, with cytokine and chemokine coding sequences incorporated as peptide linkers. The chains can form at least two non-contiguous polypeptide chains, which can optionally be connected by peptide linkers, and one or more of i) to iv) can exhibit at least 90% sequence identity to any of SEQ ID NOs:l-13.

[0012] In another embodiment, the disclosure includes a host cell engineered to express the multispecific polypeptide.

[0013] In another embodiment, the disclosure includes a pharmaceutical composition comprising the multispecific polypeptide and a pharmaceutically acceptable carrier.

[0014] In another embodiment, the disclosure includes a method of treating cancer by administering an effective amount of the multispecific polypeptide to a subject in need thereof. Another embodiment provides a method to method increase immune cell infiltration in solid tumors or metastatic lesions (including pancreatic, breast, colorectal, lung, skin, ovarian, glioblastoma, or liver cancers). The administering can be by, for example, intratumoral or intravenous injection, and can be combined with chemotherapeutic agents, biological agents, radiation, or surgery.

[0015] In another embodiment, the disclosure includes a method of tethering immune cells to cancer cells by contacting immune cells and tumor cells with the multispecific polypeptide. Another embodiment provides for the use of the polypeptides described herein to treat cancer, increase immune cell infiltration in a tumor or to tether immune cells to cancer cells.

[0016] These and other features will be more clearly understood from the following detailed description and accompanying claims.

[0017] DESCRIPTION OF THE DRAWINGS

[0018] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed herein.

[0019] FIGS. 1A-1F. The CXCL10-CXCR3 axis is a target for increased NK cell infiltration into GBM. Fig. 1 A) Scheme of tumor-infiltrating NK cell isolation and CXCR3 expression analysis. Tumor-infiltrating lymphocytes isolated from fresh GBM tumor tissues and peripheral blood were analyzed by flow cytometry. Fig. IB) CXCR3 expression on NK cells isolated from healthy donors and / or GBM patients. n=6. Fig. 1C) Correlation between CXCL10 concentration and infiltration of major immune cell types in GBM. The data were analyzed by TIMER2.0. Fig. ID) CXCL10 expression levels on GBM patient tumors and normal tissues from GEPIA. Fig. IE) CXCL10 secretion is deficient in GBM. Patient-derived primary GBM43 cell culture medium was collected after 24 hours and chemokines in the supernatant were measured by multiplex ELISA. Fig. IF) H4C staining of CXCL10 in tumor tissues from GBM patients. *p<0.005 **p<0.001 ***p<0.0005.

[0020] FIGS. 2A-2H. Exogeneous CXCL10 induces NK cell migration but does not affect NK cell functions. Fig. 2A) CXCR3 expression on freshly isolated and ex vivo expanded NK cells. Fig. 2B) CXCL 10 -mediated induction of migration of freshly isolated and ex vivo expanded NK cells in vitro. To measure migration, NK cells were seeded in the upper chamber in the presence of CXCL10 (500 ng / mL) in the bottom chamber. Fig. 2C) CXCLIO-mediated induction of migration of expanded NK cells in a dose-dependent manner. Fig. 2D) Effect of CXCR3 knockdown on NK cell migration induced by CXCL10. (Fig. 2E) Effect of CXCL10 on the viability of expanded NK cells at 4 and 24 hours measured by the CCK-8 assay. (Fig. 2F) & (Fig. 2G) & (Fig. 2H) Effect of CXCL 10 on the cytotoxicity of NK cells. Expanded NK cells were pretreated with various concentrations of CXCL 10 for 24 hours and then co-cultured with GBM43 cells. (Fig. 2F). IFN-g (Fig. 2G) and CD 107a (Fig. 2H) expression on NK cells were measured by flow cytometry. (Fig. 2G) and (Fig. 2H) were measured at the E:T ratio of 1 : 1. *p<0.005 **p<0.00 , ***p<0.0005

[0021] FIGS. 3A-3M. CXCL10 improves the recruitment of NK cells into GBM tumors in vivo. Fig. 3 A) The schema of the in vivo GBM xenograft study. WT and CXCLIO-overexpressing GBM (GBM43CXCL10) cells were subcutaneously injected into NSG mice and treated with ex vivo expanded human NK cells by tail vein once a week. Mice also received i.p. injections of recombinant human IL-2 three times a week during the course of treatment. Fig. 3B) H&E and IHC staining for human CXCL10 of tumor samples from mice with WT or GBMCXCL1° tumors. Fig. 3C) Tumor sizes of the mice with WT or GBMCXCL1° tumors. Fig. 3D) NK cell recruitment into flank tumors of GBM-bearing mice. NK cells were pre-stained with Xenolight dye and injected into the mice intravenously. Fluorescence images of the mice were taken by the Spectrum AMII at specified timepoints. Fig. 3E) IHC staining of NKp46 in the tumor samples. After treatment, mice were sacrificed, and tumor samples were isolated for IHC staining of human NK cell marker NKp46. Fig. 3F) Tumor size and Fig. 3G) the survival rate of mice bearing WT- GBM43 and the GBM43CXCL10tumors (n=5). Fig. 3H- Fig. 31 -Fig. 3 J) Phenotyping of immune cells isolated from C57BL / 6 mice with flank murine GL261 tumors. Fig. 3K- Fig. 3L) Phenotyping of immune cells isolated from C57BL / 6 mice with orthotopic intracranial murine GL261 tumors. *p<0.005 **p<0.00r, ***p<0.0005

[0022] FIGS. 4A-4J. Design, expression and characterization of multifunctional NKCEs. Fig. 4A) Scheme of the functional blocks for the design of NKCEs. Fig. 4B) Scheme of the designed IgG-like NKCEs in various formats. Fig. 4C) Binding results of NKCEs in different formats to target cells. NK cells, U87MG, and GBM43 cells were harvested and incubated in the presence of each NKCE at 4°C for 30 min. After incubation, cells were washed and further stained by APC- anti-His mAb. Binding was measured by flow cytometry. Fig. 4D) Binding of NKCE-3 to target cells in a dose-dependent manner. Fig. 4E) & Fig. 4F) Effect of NKCE-3 on NK cell proliferation and survival. Freshly isolated NK cells were pre-stained with Cell Tracer Yellow dye and then cultured in medium containing NKCE or recombinant IL- 15 (rhIL-15). After 7 days, cells were stained with Live / Dead dye and measured by flow cytometry. Experiments were performed on NK cells from 3 different donors. Fig. 4G) The cleavage of CXCL10 in vitro detected by anti-His tag Western Blot. Fig. 4H) Induction of NK cell migration by NKCE-3 in vitro. NK cells were seeded in the upper chamber of a Transwell system in the presence of either CXCL10 (500 ng / mL) or NKCE-3 loaded in the bottom chamber. Fig. 41) Effect of NKCE-3 pretreatment on NK cell cytotoxicity against GBM cells. Fig. 4J) NKCE pretreatment-mediated stimulation of IFN-g release from NK cells. *p<0.005 **p<0.00r, ***p<0.0005

[0023] FIGS. 5A-5G. NKCE enhances the anti-tumor activity of NK cells against GBM. Fig. 5A) Ability of NKCE-3 to promote the conjugation of NK cells and GBM cells. Cell Tracer far-red labeled NK cells and CFSE-labeled U87MG or GBM43 cells were co-cultured in the presence of NKCE-3 or vehicle control to establish binding. Fig. 5B) Effect of NKCE-3 on the cytotoxicity of NK cells against GBM cells. Ex vzvo-expanded NK cells were co-cultured with target tumor cells in the presence or absence of NKCE-3. The cytotoxicity was measured by LDH released into the medium. Fig. 5C) Detection of NKCE-3 -mediated release of IFN-g from NK cells by ELISA. Fig. 5D) Lytic degranulation of NK cells in the presence of NKCE-3 detected as CD107a+cells. Ex vzvo-expanded NK cells were co-cultured with target tumor cells in the presence of NKCE-3. CD107a-expressing cells were evaluated within the gated NKp46+cell population by flow cytometry. Fig. 5E) and Fig. 5F) Effect of NKCE-3 on the cytotoxicity of NK cells against GBM in a 3D spheroid model. NK cells were co-cultured with mCherry-GBM43 3D spheroids in the presence of NKCE-3 or vehicle control. The total area of mCherry fluorescence was measured and recorded for quantification of tumor spheroid lysis, as a measure of the cytotoxicity of NK cells against GBM spheroids. *p<0.005 **p<0.001 ***p<0.0005.

[0024] FIGS. 6A-6G. NKCE-3 induces NK cell anti-tumor activity against orthotopic GBM in vivo. Fig. 6A) Scheme of the GBM43 intracranial study. Fig. 6B) Bodyweight of mice measured throughout the course of the study (n=5). Fig. 6C) Bioluminescence intensity for individual mice. Fig. 6D) Average tumor volume measured by the total ROI of bioluminescence. Fig. 6E) Kaplan- Meier survival of mice in each treatment group. F&G) Representative IHC staining images and quantification of IHC staining in tumor sections from mice receiving the indicated treatments: Fig. 6F) CXCL10 and Fig. 6G) Granzyme B. *p<0.005 **p<0.00 , ***p<0.0005

[0025] FIGS. 7A-7H. CXCR3-CXCL10 do not affect NK cell functions. Fig. 7 A) CXCR3 expression on NK cells after co-culture with GBM43 cells. Fig. 7B- Fig. 7D) Effect of CXCR3- knockdown on NK cell proliferation and cytotoxicity against GBM43 cells. Fig. 7B) Measurement of CXCR3 expression by flow cytometry. Fig. 7C) Proliferation of NK cells measured by the CCK-8 assay for 96 hours. Fig. 7D) The cytotoxicity of engineered NK cells measured by the LDH assay. Fig. 7E) The effect of CXCL9 / 10 / 11 on NK cell proliferation in the medium without serum and cytokines for 24 hours. Fig. 7F) & Fig. 7G) The effect of CXCL9 and CXCL11 on NK cell viability for 4 and 24 hours. Fig. 7H) The effect of CXCL10 (500 ng / mL) on the activating receptor expression on NK cells. Expanded NK cells were pretreated with various concentrations of CXCL10 for 24 hours and then co-cultured with GBM43 cells at the required E: T ratio for 4 hours. The expressions of NKG2D, NKp46, CD 16, and DNAM-1 were measured by flow cytometry. Both percentage and MFI are shown. *p<0.005; **p<0.001; ***p<0.0005.

[0026] FIGS. 8A-8F. does not promote tumor cell growth in vitro. Fig. 8A) The effect of CXCL9 / 10 / 11 on tumor cell proliferation. GBM43 cells were cultured in the presence of chemokines CXCL9 / 10 / 11 at various concentrations for 72 h. Fig. 8B) The effect of mouse CXCL10 on the viability of mouse glioma GL261cells. Fig. 8C) CXCL10 secretion by WT and CXCLIO-overexpressing GBM43 cells (CXCL10 / GFP-GBM43). Fig. 8D) Cell proliferation measured via trypan blue staining for 2 weeks. Fig. 8E) The migration of NK cells induced by the conditioned medium collected from control GBM43 cells and CXCLIO-ov erexpressing GBM43 cells. Fig. 8F) The cytotoxicity of NK cells against engineered GBM43 cells measured by the LDH assay. *p<0.005; **p<0.001; ***p<0.0005.

[0027] FIGS. 9A-9E. Positive feedback between CXCLIO-NK-IFN-Y-GBM cells. Fig. 9A) IFN-

[0028] Y induced CXCL10 production by GBM cells. GBM43 or U87MG cells were pretreated with IFN-

[0029] Y (10 ng / mL) for 24 hrs in serum -free medium, the supernatant was harvested and the CXCL10 concentration was measured by ELISA. Fig. 9B) The induction by IFN-y of CXCL10 production by GBM43 cells occurs in a dose-dependent manner. GBM43 cells were pretreated with IFN-y at various concentrations for 24 hrs. Fig. 9C) & Fig. 9D) NK cell activated by the tumor cells induced the production of CXCL10 by GBM43 cells via released IFN-y. These data were collected from 3 independent donor NK cells in duplicates. Fig. 9E) The scheme of the potential positive CXCLIO-NK-IFN-Y-GBM feedback loop. *p<0.005; **p<0.001; ***p<0.0005.

[0030] FIGS. 10A-10E. NKp46 and IL- 15 play a role in NK cell activation and proliferation. Fig.10A) NKp46 expression on NK cells from GBM patients and healthy donors. Fig.1 OB) NKp46 expression on ex vivo-expanded NK cells after co-culture with GBM cells. NK cells were cultured with GBM43 cells and the expression of NKp46 was detected by flow cytometry. Fig.10C) NKp46 blockade inhibited the cytotoxicity of NK cells against GBM cells. Expanded NK cells were cocultured with GBM43 cells in the presence of anti-NKp46 neutralizing antibody (5 ng / mL) at the indicated E:T ratios for 4 hours. The cytotoxicity was measured by the LDH assay. Fig.10D) NK cells’ proliferation under various cytokine stimulation conditions. NK cells isolated from the same donor were expanded in feeder medium for 1 week and then cultured in the medium with various cytokines for another 7 days. The NK cell numbers were counted by Trypan-blue staining on days 1, 4, and 7. Fig.lOE) Killing assay of NK cells stimulated with various cytokines against GBM cells. *p<0.005; **p<0.001; ***p<0.0005.

[0031] FIGS. 11A-11C. IL-13Ra2 expression on GBM cells. Fig.11 A) IL-13Ra2 expression on various cell lines detected by flow cytometry. U87MG, GBM43, SJ-GBM2, GBM10, and K562 cells were stained with PE-anti-human IL-13Ra2 antibody. K562 cells were included as negative control. Fig.1 IB) and Fig.l 1C) The effect of CXCL10 or IFN-y on the IL-13Ra2 expression level on GBM cells. GBM cells were pre-treated with CXCL10 (500 ng / mL) or IFN-y (5 ng / mL) for 24 hours. IL-13Ra2 expression levels on these cells were measured by flow cytometry. *p<0.005; **p<0.001; ***p<0.0005.

[0032] FIGS. 12A-12E. NKCE-3 promotes the anti-tumor activity of NK cells against GBM. Fig. l2A) The effect of NKCE-3 pretreatment on NK cell cytotoxicity against SJ-GBM2 and GBM10 cells. Fig. l2B) The quantification of the conjugation assay in 3 donors. The conjugation of NK and GBM cells mediated by NKCE-3 was quantified by measuring double-positive events (CFSE / Cell Tracer far red) using a flow cytometer. Fig.12C) Effect of NKCE-3 on the cytotoxicity of NK cells against GBM cells. Ex vivo-expanded NK cells were co-cultured with target tumor cells in the presence or absence of NKCE-3. The cytotoxicity was measured by LDH released into the medium. Fig.12D) NKCE-3 mediated the release of IFN-y from NK cells measured by ELISA. Fig. l2E) Lytic degranulation of NK cells in the presence of NKCE-3 detected as CD107a+ cells. *p<0.005; **p<0.001; ***p<0.0005.

[0033] FIG. 13. Depicts general schema of design of protein construct.

[0034] FIG. 14. Depicts Amino Acid Sequence of a designed protein construct. (SEQ ID NO: 1) FIG. 15. Depicts example of an expression vector construct. scFv works well in both HEK293T and ExpiCHO cells.

[0035] FIG. 16. Depicts functional domain construction.

[0036] FIG. 17. Depicts Optimization of the expression target protein: Option 3 GenScript. Shows diagram and amino acid sequence. (SEQ ID NO: 2)

[0037] FIG. 18. Depicts Construct Design Format - 1. Shows diagram and amino acid sequence. (SEQ ID NO: 3)

[0038] FIG. 19. Depicts Format - 1 Schema of construction.

[0039] FIG. 20. Depicts Construct Design Format - 2. Shows representation of construct and amino acid sequence of domains. (SEQ ID NOs: 4-6)

[0040] FIG. 21. Depicts Construct Design Format - 3. Shows representation of construct and amino acid sequence of domains. (SEQ ID NOs: 7-10)

[0041] FIG. 22. Depicts Construct Design Format - 4. Shows representation of construct and amino acid sequence of domains. (SEQ ID NOs: 11-13)

[0042] DETAILED DESCRIPTION

[0043] Despite success of NK cell therapy in hematologic malignancies, the use of immune cells, such as NK cells, for the treatment of solid tumors remains challenging, largely limited by the immunosuppressive tumor microenvironment (TME) and the inadequate infiltration of the therapeutic (e.g., NK) cells into the tumor sites.

[0044] Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM, and other cancers are urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy by linking a single-chain variable fragment (scFv) from the heavy chain and the light variable chains of an antibody, which is specific for a tumor antigen to a second scFv that activates immune (e.g., NK) cells via a short peptide linker.

[0045] However, engager-triggered activation of immune cells in tumors has so far not addressed the inadequacy of activation of immune cells not directly engaged via receptor ligation, such as bystander immune cells. Local recruitment of immune cells via the modulation of the TME can be beneficial, especially in non-immune-privileged tumors such as GBM.

[0046] Provided herein is the identification of the CXCR3-CXCL10 axis as a major chemokine interaction regulating, for example, NK cell infiltration into GBM without affecting their activation and cytotoxicity.13However, despite the high expression of CXCR3 on activated NK cells in GBM, local concentrations of CXCL10 are low. To restore this disrupted gradient, a strategy of triggering the local production of CXCL10 in the tumor from a novel NK cell engager (NKCE) / multispecific polypeptide bearing multiple functional moieties was adopted, thereby facilitating NK cell migration to tumor sites. One embodiment provides a tetravalent NKCE / multispecific polypeptide that contains two moieties targeting NKp46 (an immune cell targeting moiety) on NK cells and IL-13Ra2 (a tumor targeting moiety) on GBM cells, an IL-15 mutein (a cytokine molecule), and a cleavable CXCL10 (a chemokine) domain to stimulate NK cell proliferation and persistence. The local delivery of CXCL10 via cleavage of a tumor proteasesensitive linker in the tumor restored disrupted CXCR3-CXCL10 gradients, and tethered NK cells to GBM to stimulate their elimination. Finally, the elevated presence of CXCL10 had the added ability to recruit bystander effector immune cells including CD8+T cells. By incorporating these functional moieties, the NKCE / multispecific polypeptide was shown to have superior and potent cytotoxicity against GBM in vivo. The development of a chemokine-producing engager / multispecific polypeptide is a powerful approach to immune recruitment and retention in the tumor and an advancement in adoptive therapy with immune cells, such as NK cells. Definitions

[0047] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 16th Edition, by M. Larranga, R Lewis Sr., and R Lewis, New York, N.Y., 2016. References in the specification to "one embodiment," "an embodiment," etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described. As used herein, the term "in some embodiments" refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0048] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0049] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is di -substituted.

[0050] As used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating a listing of items, “and / or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0051] As used herein, the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof, are intended to be inclusive similar to the term “comprising.” The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. The term about can also modify the endpoints of a recited range as discuss above in this paragraph.

[0052] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

[0053] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more," and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

[0054] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0055] The term "at least" prior to a number or series of numbers (e.g., "at least two") is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that could logically be included, as clear from context. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range.

[0056] As used herein, the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof, are intended to be inclusive similar to the term “comprising.”

[0057] The terms “comprises,” “comprising,” and the like can have the meaning ascribed to them in U. S. Patent Law and can mean “includes,” “including” and the like. As used herein, “including” or “includes” or the like means including, without limitation.

[0058] In some embodiments, the multispecific polypeptide includes a tumor targeting moiety. A “tumor targeting moiety,” as used herein, refers to a binding agent that recognizes or associates with, e.g., binds to, a target in or on a cancer cell. The tumor targeting moiety can be an antibody molecule, a receptor molecule (e.g., a full-length receptor, receptor fragment, or fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule (e.g., a full-length ligand, ligand fragment, or fusion thereof (e.g., a ligand-Fc fusion)) that binds to the cancer antigen (e.g., the tumor).

[0059] In some embodiments, the multispecific polypeptide includes an immune cell targeting moiety. An “immune cell targeting moiety” refers to one or more binding specificities that bind and / or activate an immune cell, e.g., a cell involved in an immune response. In embodiments, the immune cell is chosen from a T cell, an NK cell, a B cell, a dendritic cell, and / or a macrophage cell. The immune cell targeting moiety can be an antibody molecule, a receptor molecule (e.g., a full length receptor, receptor fragment, or fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule (e.g., a full length ligand, ligand fragment, or fusion thereof (e.g., a ligand-Fc fusion)) that binds to the immune cell antigen (e.g., the NK cell antigen, the B cell antigen, the dendritic cell antigen, and / or the macrophage cell antigen).

[0060] In some embodiments, the multispecific polypeptide includes a cytokine molecule. As used herein, a “cytokine molecule” refers to full length, a fragment or a variant of a cytokine that elicits at least one activity of a naturally occurring cytokine. In some embodiments the cytokine molecule is chosen from interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin- 12 (IL-12), interleukin- 15 (IL-15), interleukin- 18 (IL-18), interleukin-21 (IL-21), or interferon gamma, or a fragment or variant thereof, or a combination thereof. The cytokine molecule can be a monomer or a dimer. In embodiments, the cytokine molecule can further include a cytokine receptor dimerizing domain.

[0061] In some embodiments, the multispecific polypeptide includes a chemokine molecule. Chemokines are a family of small signaling proteins (cytokines) that that play a role in immune responses, such as directing the movement of immune cells. They guide immune cells to specific locations in the body, playing a role in both innate and adaptive immunity. In some embodiments, the chemokine is chosen from CCL1, CCL2 (MCP-1), CCL3 (MIP-la), CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 / CCL10, CCL11 (Eotaxin), CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1 (Fractalkine), CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8 (IL-8), CXCL9, CXCL10, CXCL11, CXCL12 (SDF-1), XCL1 (Lymphotactin), XCL2, CS3CL1 or a fragment or variant thereof, or a combination thereof.

[0062] As used herein, the term “molecule” as used in, e.g., antibody molecule, cytokine molecule, receptor molecule, chemokine molecule includes full-length, naturally occurring molecules, as well as variants, e.g., functional variants (e.g., truncations, fragments, mutated (e.g., substantially similar sequences) or derivatized form thereof), so long as at least one function and / or activity of the unmodified (e.g., naturally occurring) molecule remains.

[0063] The term “functional variant” refers to polypeptides that have a substantially identical amino acid sequence to the naturally occurring sequence, or are encoded by a substantially identical nucleotide sequence, and are capable of having one or more activities of the naturally occurring sequence.

[0064] “Antibody molecule” as used herein refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. An antibody molecule encompasses antibodies (e.g., full-length antibodies) and antibody fragments. In an embodiment, an antibody molecule comprises an antigen binding or functional fragment of a full- length antibody, or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that is naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes). In embodiments, an antibody molecule refers to an immunologically active, antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment, e.g., functional fragment, is a portion of an antibody, e.g., Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single chain variable fragment (scFv). A functional antibody fragment binds to the same antigen as that recognized by the intact (e.g., full-length) antibody. The terms “antibody fragment” or “functional fragment” also include isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”). In some embodiments, an antibody fragment does not include portions of antibodies without antigen binding activity, such as Fc fragments or single amino acid residues. Exemplary antibody molecules include full length antibodies and antibody fragments, e.g., dAb (domain antibody), single chain, Fab, Fab', and F(ab')2 fragments, and single chain variable fragments (scFvs).

[0065] As used herein, an “immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain. For example, the sequence can include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two, or more N- or C-terminal amino acids, or may include other alterations that are compatible with formation of the protein structure.

[0066] In embodiments, an antibody molecule is monospecific, e.g., it comprises binding specificity for a single epitope. In some embodiments, an antibody molecule is multispecific, e.g., it comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, an antibody molecule is a bispecific antibody molecule. “Bispecific antibody molecule” as used herein refers to an antibody molecule that has specificity for more than one (e.g., two, three, four, or more) epitope and / or antigen.

[0067] “Antigen” (Ag) as used herein refers to a molecule that can provoke an immune response, e.g., involving activation of certain immune cells and / or antibody generation. Any macromolecule, including almost all proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinant or DNA. For example, any DNA comprising a nucleotide sequence or a partial nucleotide sequence that encodes a protein capable of eliciting an immune response encodes an “antigen.” In embodiments, an antigen does not need to be encoded solely by a full-length nucleotide sequence of a gene, nor does an antigen need to be encoded by a gene at all. In embodiments, an antigen can be synthesized or can be derived from a biological sample, e.g., a tissue sample, a tumor sample, a cell, or a fluid with other biological components. As used, herein a “tumor antigen” or interchangeably, a “cancer antigen” includes any molecule present on, or associated with, a cancer, e.g., a cancer cell or a tumor microenvironment that can provoke an immune response. As used, herein an “immune cell antigen” includes any molecule present on, or associated with, an immune cell, such as obe that can provoke an immune response.

[0068] The “antigen-binding site,” or “binding portion” of an antibody molecule refers to the part of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that participates in antigen binding. In embodiments, the antigen binding site is formed by amino acid residues of the variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the variable regions of the heavy and light chains, referred to as hypervariable regions, are disposed between more conserved flanking stretches called “framework regions,” (FRs). FRs are amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface, which is complementary to the three-dimensional surface of abound antigen. The three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” The framework region and CDRs have been defined and described, e.g., in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) is typically made up of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0069] “Cancer” as used herein can encompass all types of oncogenic processes and / or cancerous growths. In embodiments, cancer includes primary tumors as well as metastatic tissues or malignantly transformed cells, tissues, or organs. In embodiments, cancer encompasses all histopathologies and stages, e.g., stages of invasiveness / severity, of a cancer. In embodiments, cancer includes relapsed and / or resistant cancer. The terms “cancer” and “tumor” can be used interchangeably. For example, both terms encompass solid and liquid tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.

[0070] As used herein, an “immune cell” refers to any of various cells that function in the immune system, e.g., to protect against agents of infection and foreign matter. In embodiments, this term includes leukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate leukocytes include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer (NK) cells. Innate leukocytes identify and eliminate pathogens, either by attacking larger pathogens through contact or by engulfing and then killing microorganisms and are mediators in the activation of an adaptive immune response. The cells of the adaptive immune system are special types of leukocytes, called lymphocytes. B cells and T cells are types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response, whereas T cells are involved in cell- mediated immune response. The term “immune cell” includes immune effector cells.

[0071] “Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NK T) cells, and mast cells.

[0072] The term “effector function” or “effector response” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.

[0073] The compositions and methods of the present invention encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 85%, 90%, 95%, 96%, 97% identical or higher to the sequence specified. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and / or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.

[0074] In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.

[0075] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[0076] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, 60%, or even more at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[0077] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0078] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In one embodiment, a set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0079] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using, for example, a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0080] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0081] It is understood that the molecules of the present invention may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.

[0082] The term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives and congeners thereof, amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. As used herein the term “amino acid” includes both the D- or L-optical isomers and peptidomimetics.

[0083] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0084] The terms “polypeptide”, “peptide” and “protein” (if single chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. The polypeptide can be isolated from natural sources, can be a produced by recombinant techniques from a eukaryotic or prokaryotic host, or can be a product of synthetic procedures.

[0085] The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence,” and “polynucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide can be either single-stranded or double-stranded, and if single-stranded may be the coding strand or non-coding (antisense) strand. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The nucleic acid can be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.

[0086] The term “isolated,” as used herein, refers to material that is removed from its original or native environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated by human intervention from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotides can be part of a vector and / or such polynucleotides or polypeptides can be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature.

[0087] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0088] As used herein, an “effective amount” or “therapeutically effective amount” (terms can be used interchangeably) means an amount sufficient to produce a selected effect, such as alleviating symptoms of a disease or disorder. A "therapeutically effective amount" of a treatment is a predetermined amount calculated to achieve the desired effect, i.e., to treat, combat, ameliorate, prevent, or improve one or more symptoms of, for example, cancer. The activity contemplated by the present methods includes both medical therapeutic and / or prophylactic treatment, as appropriate. The specific dose of a compound administered according to the present disclosure to obtain therapeutic and / or prophylactic effects will, of course, be determined by the circumstances surrounding the case, including, for example, the compound / composition / agent / fusion protein administered, the route of administration, and the condition being treated or prevented. It will be understood that the effective amount administered will be determined by the physician in the light of the relevant circumstances including the condition to be treated, the choice of compound to be administered, and the chosen route of administration, and therefore any dosage ranges provided herein are not intended to limit the scope of the present disclosure in any way. The term “delivery vehicle” or “carrier” refers to any kind of device or material which can be used to deliver the invention in vivo.

[0089] The term "treat," "treated," or "treating" refers to therapeutic treatment and / or prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. For purposes of the embodiments described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease. Treatment can also include eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0090] A "preventive" or "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs, or exhibits only early signs, of a disease or disorder. A prophylactic or preventative treatment is administered for the purpose of decreasing the risk of developing pathology associated with developing the disease or disorder.

[0091] As used herein, the term "therapeutic" means an agent utilized to treat, combat, ameliorate, prevent, or improve an unwanted condition or disease of a subject / patient.

[0092] The term "in need thereof means that the subject has been identified or suspected as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis or observation. In any of the methods described herein, the subject can be in need thereof.

[0093] As used herein “injecting, administering or applying” includes administration of the invention by any number of routes and means including, but not limited to, topical, oral, buccal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, ophthalmic, or pulmonary.

[0094] The term "subject" refers to an animal, such as a mammalian species (e.g., human). More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian, or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual that needs therapy or suspected of needing therapy. The terms "individual" or "patient" are intended to be interchangeable with "subject." For example, a subject can be an individual who has been diagnosed with having cancer, is going to receive a therapy for cancer, or has received at least one cancer therapy prophylactic / preventative (e.g., vaccine) treatment.

[0095] A disease, condition, or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a subject, or both, are reduced.

[0096] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this technology belongs. Although exemplary methods, devices and materials are described herein, any methods and materials similar or equivalent to those expressly described herein can be used in the practice or testing of the present technology. For example, the reagents described herein are merely exemplary and that equivalents of such are known in the art. The practice of the present technology can employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition: the series Ausubel etal. eds. (2007) Current Protocols in Molecular Biology: the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR I: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1 995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999); Antibodies. A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); and Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells (Cold Spring Harbor Laboratory).

[0097] Various aspects of the invention are described in further detail below. Additional definitions are set out throughout the specification.

[0098] Antibody Molecules

[0099] In one embodiment, the antibody molecule binds to a cancer antigen, e.g., a tumor antigen. In some embodiments, the cancer antigen is, e.g., a mammalian, e.g., a human, cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, e.g., a mammalian, e.g., a human, immune cell antigen. For example, the antibody molecule binds specifically to an epitope, e.g., linear or conformational epitope, on the cancer antigen or the immune cell antigen. One embodiment provides for a multispecific antibody molecule, such as a bispecific antibody molecule. A bispecific antibody has specificity for two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In an embodiment the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a scFv or a Fab, or fragment thereof, have binding specificity for a first epitope and a scFv or a Fab, or fragment thereof, have binding specificity for a second epitope.

[0100] In an embodiment, an antibody molecule comprises a diabody, and a single-chain molecule, as well as an antigen-binding fragment of an antibody (e.g., Fab, F(ab')2, and Fv). For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In an embodiment an antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (scFv for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies. A preparation of antibody molecules can be monoclonal or polyclonal. An antibody molecule can also be a human, humanized, CDR-grafted, or in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from, e.g., kappa or lambda. The term “immunoglobulin” (Ig) is used interchangeably with the term “antibody” herein.

[0101] Examples of antigen-binding fragments of an antibody molecule include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0102] Antibody molecules include intact molecules as well as functional fragments thereof. Constant regions of the antibody molecules can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).

[0103] Antibody molecules can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. According to another aspect of the invention, a single domain antibody is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention. The VH and VL regions can be subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed “framework regions” (FR or FW).

[0104] The extent of the framework region and CDRs has been precisely defined by a number of methods (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0105] The terms “complementarity determining region,” and “CDR,” as used herein refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3).

[0106] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme).

[0107] Each VH and VL typically includes three CDRs and four FRs, arranged from aminoterminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0108] The antibody molecule can be a polyclonal or a monoclonal antibody.

[0109] The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. A monoclonal antibody can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0110] The antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

[0111] In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse or rabbit which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91 / 00906, Kucherlapati et al. PCT publication WO 91 / 10741; Lonberg et al. International Application WO 92 / 03918; Kay et al. International Application 92 / 03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7: 13-21; Morrison, S. L. et al. 1994 roc. Natl. Acad. Set. USA 81 :6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21 : 1323-1326).

[0112] An antibody molecule can be one in which the variable region, or a portion thereof, e.g., the CDRs, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibody molecules generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[0113] An “effectively human” protein is a protein that substantially does not evoke a neutralizing antibody response, e.g., the human anti-murine antibody (HAMA) response. HAMA can be problematic in a number of circumstances, e.g., if the antibody molecule is administered repeatedly, e.g., in treatment of a chronic or recurrent disease condition. A HAMA response can make repeated antibody administration potentially ineffective because of an increased antibody clearance from the serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32: 180-190 (1990)) and also because of potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0114] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication PCT / US86 / 02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240: 1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80: 1553-1559).

[0115] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDRs (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen. In an example, the donor can be a rodent antibody, e.g., a rat or mouse antibody, and the recipient can be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.”

[0116] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[0117] An antibody molecule can be humanized by methods known in the art (see e.g., Morrison, S. L., 1985, Science 229: 1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, the contents of all of which are hereby incorporated by reference).

[0118] Humanized or CDR-grafted antibody molecules can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See, e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321 :552-525; Verhoeyan et al. 1988 Science 239: 1534; Beidler et al. 1988 J. Immunol. 141 :4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.

[0119] The antibody molecule can be a single chain antibody. A single-chain antibody (scFv) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target protein.

[0120] In yet other embodiments, the antibody molecule has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE. In another embodiment, the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and / or complement function). In one embodiment the antibody has: effector function; and can fix complement. In other embodiments the antibody does not; recruit effector cells; or fix complement. In another embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[0121] Methods for altering an antibody constant region are known in the art. Antibodies with altered function, e.g. altered affinity for an effector ligand, such as FcR on a cell, or the Cl component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388,151 Al, U.S. Pat. Nos. 5,624,821 and 5,648,260, the contents of all of which are hereby incorporated by reference). Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.

[0122] An antibody molecule can be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a “derivatized” antibody molecule is one that has been modified. Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinity ligand such as biotin. Accordingly, the antibody molecules of the invention are intended to include derivatized and otherwise modified forms of the antibodies described herein. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0123] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).

[0124] Multispecific Antibody Molecules

[0125] In embodiments, multispecific antibody molecules can comprise more than one antigenbinding site(s), where different sites are specific for different antigens. In embodiments, multispecific antibody molecules comprise an antigen-binding site specific for a cancer cell and a different antigen-binding site specific for an immune cell (e.g., NK cell). In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0126] BsIgG is a format that is monovalent for each antigen. Exemplary BsIgG formats include but are not limited to crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs- in-holes common LC, knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, dk-body, orthogonal Fab. See Spiess et al. Mol. Immunol. 67(2015):95- 106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, BsIgG comprises heavy chains that are engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a “knobs-into-holes” strategy, a SEED platform, a common heavy chain (e.g., in Kk-bodies), and use of heterodimeric Fc regions. See Spiess et al. Mol. Immunol. 67(2015):95-106. Strategies that have been used to avoid heavy chain pairing of homodimers in BsIgG include knobs-in-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity. See Id. BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly into a BsIgG. BsIgG can also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, e.g., using protein A and sequential pH elution.

[0127] IgG appended with an additional antigen-binding moiety is another format of bispecific antibody molecules. For example, monospecific IgG can be engineered to have bispecificity by appending an additional antigen-binding unit onto the monospecific IgG, e.g., at the N- or C- terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chain or variable light chain), engineered protein scaffolds, and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). Examples of appended IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (Abb Vie), which binds IL-la and IL-1P; and ABT-122 (Abb Vie), which binds TNF and IL- 17 A. Bispecific antibody fragments (BsAb) are a format of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAb lack an Fc region. In embodiments, bispecific antibody fragments include heavy and light chain regions that are connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include but are not limited to nanobody, nanobody- HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody. For example, the BiTE format comprises tandem scFvs, where the component scFvs bind to CD3 on T cells and a surface antigen on cancer cells.

[0128] Bispecific fusion proteins include antibody fragments linked to other proteins, e.g., to add additional specificity and / or functionality. An example of a bispecific fusion protein is an immTAC, which comprises an anti-CD3 scFv linked to an affinity-matured T-cell receptor that recognizes HLA-presented peptides. In embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. Also, fusions to albumin binding proteins or human serum albumin can be extend the serum half-life of antibody fragments.

[0129] In embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to create BsAb molecules. An exemplary bispecific antibody conjugate includes the CovX-body format, in which a low molecular weight drug is conjugated site-specifically to a single reactive lysine in each Fab arm or an antibody or fragment thereof. In embodiments, the conjugation improves the serum half-life of the low molecular weight drug.

[0130] The antibody molecules can be produced by recombinant expression, e.g., of at least one or more component, in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, e.g., CHO cells, or insect cells, e.g., SF9 or S2 cells) and prokaryotic cells (e.g., E. colt). Bispecific antibody molecules can be produced by separate expression of the components in different host cells and subsequent purification / assembly. Alternatively, the antibody molecules can be produced by expression of the components in a single host cell. Purification of bispecific antibody molecules can be performed by various methods such as affinity chromatography, e.g., using protein A and sequential pH elution. In other embodiments, affinity tags can be used for purification, e.g., histidine-containing tag, myc tag, or streptavidin tag.

[0131] Antibody-Based Fusions

[0132] A variety of formats can be generated which contain additional binding entities attached to the N or C terminus of antibodies. These fusions with single chain or disulfide stabilized Fvs or Fabs result in the generation of tetravalent molecules with bivalent binding specificity for each antigen. Combinations of scFvs and scFabs with IgGs enable the production of molecules which can recognize three or more different antigens.

[0133] Antibody-Fab Fusion

[0134] Antibody-Fab fusions are bispecific antibodies comprising a traditional antibody to a first target and a Fab to a second target fused to the C terminus of the antibody heavy chain. Commonly the antibody and the Fab will have a common light chain. Antibody fusions can be produced by (1) engineering the DNA sequence of the target fusion, and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. The antibody-scFv fusion can be linked by a (Gly)- Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv, as described by Coloma, J. et al. (1997) Nature Biotech 15: 159.

[0135] Antibody-scFv Fusion

[0136] Antibody-scFv Fusions are bispecific antibodies comprising a traditional antibody and a scFv of unique specificity fused to the C terminus of the antibody heavy chain. The scFv can be fused to the C terminus through the Heavy Chain of the scFv either directly or through a linker peptide. Antibody fusions can be produced by (1) engineering the DNA sequence of the target fusion, and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. The antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv, as described by Coloma, J. et al. (1997) Nature Biotech 15: 159.

[0137] Fc-Containing Multispecific Molecules

[0138] In some embodiments, the multispecific molecules disclosed herein includes an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be chosen from the heavy chain constant regions of IgGl, IgG2, IgG3 or IgG4.

[0139] In some embodiments, the immunoglobulin chain constant region (e.g., the Fc region) is altered, e.g., mutated, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function.

[0140] In other embodiments, an interface of a first and second immunoglobulin chain constant regions (e.g., a first and a second Fc region) is altered, e.g., mutated, to increase or decrease dimerization, e.g., relative to a non-engineered interface, e.g., a naturally occurring interface. For example, dimerization of the immunoglobulin chain constant region (e.g., the Fc region) can be enhanced by providing an Fc interface of a first and a second Fc region with one or more of: a paired protuberance-cavity (“knob-in-a hole”), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer to homomultimer forms, e.g., relative to a nonengineered interface.

[0141] Heterodimerized Antibody Molecules & Methods of Making

[0142] Various methods of producing multispecific antibodies have been disclosed. Exemplary multispecific antibody formats and methods of making multispecific antibodies are disclosed in e.g., Speiss et al. Molecular Immunology 67 (2015) 95-106; and Klein et al mAbs 4:6, 653-663; November / December 2012; the entire contents of each of which are incorporated by reference herein.

[0143] Heterodimerized bispecific antibodies are based on the natural IgG structure, wherein the two binding arms recognize different antigens. IgG derived formats that enable defined monovalent (and simultaneous) antigen binding are generated by forced heavy chain heterodimerization, combined with technologies that minimize light chain mispairing (e.g., common light chain). Forced heavy chain heterodimerization can be obtained using, e.g., knobin-hole or strand exchange engineered domains (SEED).

[0144] Knob-In-Hole

[0145] Knob-in-Hole as described in U.S. Pat. Nos. 5,731,116, 7,476,724 and Ridgway, J. et al. (1996) Prot. Engineering 9(7): 617-621, broadly involves: (1) mutating the CH3 domain of one or both antibodies to promote heterodimerization; and (2) combining the mutated antibodies under conditions that promote heterodimerization. “Knobs” or “protuberances” are typically created by replacing a small amino acid in a parental antibody with a larger amino acid; “holes” or “cavities” are created by replacing a larger residue in a parental antibody with a smaller amino acid.

[0146] For bispecific antibodies including an Fc domain, introduction of specific mutations into the constant region of the heavy chains to promote the correct heterodimerization of the Fc portion can be utilized. Several such techniques are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety. These techniques include the “knobs-into-holes” (KiH) approach which involves the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary “hole” in the other CH3 domain of the paired heavy chain so as to promote correct pairing of heavy chains (see e.g., U.S. Pat. No. 7,642,228).

[0147] Common Light Chain

[0148] Light chain mispairing can be avoided to generate homogenous preparations of bispecific IgGs. One way to achieve this is through the use of the common light chain principle, i.e. combining two binders that share one light chain but still have separate specificities. An exemplary method of enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable light chain to interact with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions and methods of producing bispecific antibodies with a common light chain as disclosed in, e.g., U.S. Pat. No. 7,183,076B2, US20110177073 Al, EP2847231A1, W02016079081A1, and EP3055329A1, the contents of each of which is incorporated by reference herein.

[0149] CrossMab

[0150] Another option to reduce light chain mispairing is the CrossMab technology which avoids non-specific L chain mispairing by exchanging CHI and CL domains in the Fab of one half of the bispecific antibody. Such crossover variants retain binding specificity and affinity but make the two arms so different that L chain mispairing is prevented. The CrossMab technology (as reviewed in Klein et al.) involves domain swapping between heavy and light chains so as to promote the formation of the correct pairings. Briefly, to construct a bispecific IgG-like CrossMab antibody that could bind to two antigens by using two distinct light chain-heavy chain pairs, a two-step modification process is applied. First, a dimerization interface is engineered into the C-terminus of each heavy chain using a heterodimerization approach, e.g., Knob-into-hole (KiH) technology, to ensure that only a heterodimer of two distinct heavy chains from one antibody (e.g., Antibody

[0151] A) and a second antibody (e.g., Antibody B) is efficiently formed. Next, the constant heavy 1 (CHI) and constant light (CL) domains of one antibody are exchanged (Antibody A), keeping the variable heavy (VH) and variable light (VL) domains consistent. The exchange of the CHI and CL domains ensured that the modified antibody (Antibody A) light chain would only efficiently dimerize with the modified antibody (antibody A) heavy chain, while the unmodified antibody (Antibody B) light chain would only efficiently dimerize with the unmodified antibody (Antibody

[0152] B) heavy chain; and thus only the desired bispecific CrossMab would be efficiently formed (see e.g., Cain, C. SciBX 4(28); doi: 10.1038 / scibx.2011.783, the contents of which are incorporated by reference herein).

[0153] Common Heavy Chain

[0154] An exemplary method of enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable heavy chain to interact with each of the heteromeric variable light chain regions of the bispecific antibody. Compositions and methods of producing bispecific antibodies with a common heavy chain are disclosed in, e.g., US20120184716, US20130317200, and US20160264685 Al, the contents of each of which is incorporated by reference herein.

[0155] Amino Acid Modifications Alternative compositions and methods of producing multispecific antibodies with correct light chain pairing include various amino acid modifications. For example, Zymeworks describes heterodimers with one or more amino acid modifications in the CHI and / or CL domains, one or more amino acid modifications in the VH and / or V domains, or a combination thereof, which are part of the interface between the light chain and heavy chain and create preferential pairing between each heavy chain and a desired light chain such that when the two heavy chains and two light chains of the heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains rather than the other (see e.g., WO2015181805). Other exemplary methods are described in WO2016026943 (Argen-X), US20150211001, US20140072581A1, US20160039947A1, and US20150368352.

[0156] Tumor Targeting Moieties

[0157] The present disclosure provides, inter alia, multispecific polypeptides, that include, e.g., are engineered to contain, one or more tumor specific targeting moieties that direct the molecule to a tumor cell.

[0158] In certain embodiments, the multispecific polypeptides disclosed herein include a tumor targeting moiety. The tumor targeting moiety can be chosen from an antibody molecule (e.g., an antigen binding domain as described herein), a receptor or a receptor fragment, or a ligand or a ligand fragment, or a combination thereof. In some embodiments, the tumor targeting moiety associates with, e.g., binds to, a cancer cell (e.g., a molecule, e.g., antigen, present on the surface of the cancer cell). In certain embodiments, the tumor targeting moiety targets, e.g., directs the multispecific polypeptides disclosed herein to a cancer (e.g., a cancer or tumor cells). In certain embodiments, the tumor targeting moiety targets and associates with a cancer cell. In some embodiments, the cancer is chosen from a hematological cancer, a solid cancer, a metastatic cancer, or a combination thereof.

[0159] In some embodiments, the multispecific polypeptide, e.g., the tumortargeting moiety, binds to a solid tumor antigen. The solid tumor antigen can be present on a solid tumor, or a metastatic lesion thereof. In some embodiments, the solid tumor is chosen from one or more of pancreatic (e.g., pancreatic adenocarcinoma), breast, colorectal, lung (e.g., small or non-small cell lung cancer), skin, ovarian, or liver cancer.

[0160] In certain embodiments, the solid tumor antigen is chosen from one or more of: PDL1, CD47, mesothelin, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, IL-13Ra2, Telomerase, SAP-1, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, TRP-1 / -2, MC1R, P-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, P-catenin, CDK4, CDC27, CD47, a actinin-4, TRPl / gp75, TRP2, gplOO, Melan-A / MARTl, gangliosides, WT1, EphA3, Epidermal growth factor receptor (EGFR), CD20, MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, 0A1, OGT, RCC, RUI1, RUI2, SAGE, TRG, TRP1, TSTA, Folate receptor alpha, Ll-CAM, CAIX, EGFRvIII, gpA33, GD3, GM2, VEGFR, Intergrins (Integrin alpha Vbeta3, Integrin alpha5Betal), Carbohydrates (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, carbonic anhydrase IX (CA9; such as an antibody comprising sequences (such as heavy and light chain variable sequences) from commercially available antibodies such as the CA9 antibodies from Bio-techne / R&D Systems, Aero Biosystems, ThermoFisher Scientific, Invitrogen, Abeam, Novus Biologicals, Biocompare, Proteintech, or Miltenyi Biotec, including but not limited to: Heavy chain:

[0161] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVQQAPGKGLKWMGWINTYTGE PTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARGGIATPTSYWGQGTTLTV SS (SEQ ID NO: 39) and Light chain:

[0162] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLD SGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCCQGTHFPWTFGGGTKLEIK (SEQ ID NO: 40)), carbonic anhydrase 12 (CA12; such as an antibody comprising sequences (such as heavy and light chain sequences) from commercially available antibodies such as the CA12 antibodies from Bio-techne / R&D Systems, Aero Biosystems, ThermoFisher Scientific, Sino Biological, LSBio, Invitrogen, Abeam, Novus Biologicals, Biocompare, Proteintech, or Miltenyi Biotec)), glucose transporter type 1 (GLUTl / solute carrier family 2 (such as an antibody comprising sequences (such as heavy and light chain variable sequences) from commercially available antibodies such as the glucose transporter type 1 (GLUTl / solute carrier family 2 antibodies from Bio-techne / R&D Systems, Aero Biosystems, ThermoFisher Scientific, Invitrogen, Abeam, Novus Biologicals, Biocompare, Proteintech, or Miltenyi Biotec), facilitated glucose transporter member 1 (SLC2A1)) or RANKE.

[0163] In one embodiment, the tumor-targeting moiety includes an antibody molecule (e.g., Fab or scFv) that binds to IL-13Ra2 (e.g., IL13Ra scFv: DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASRQGS GVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIKGGGGSGG GGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFSNYLMNWVKQRPEQDLDWIG RIDPYDGDIDYNQNFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCARGYGTAYGVD YWGQGTSVTVSS (SEQ ID NO: 41), carbonic anhydrase IX (CA9), carbonic anhydrase 12 (CAI 2), or glucose transporter type 1 (GLUT 1 / solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)).

[0164] Cytokine Molecules

[0165] Cytokines are generally polypeptides that influence cellular activity, for example, through signal transduction pathways. Accordingly, a cytokine of the multispecific or multifunctional polypeptide is useful and can be associated with receptor-mediated signaling that transmits a signal from outside the cell membrane to modulate a response within the cell. Cytokines are proteinaceous signaling compounds that are mediators of the immune response. They control many different cellular functions including proliferation, differentiation and cell survival / apoptosis. Cytokines are synthesized under various stimuli by a variety of cells of both the innate (monocytes, macrophages, dendritic cells) and adaptive (T- and B-cells) immune systems.

[0166] In one embodiment the cytokine of the multispecific or multifunctional polypeptide is a cytokine selected from the group of GM-CSF, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-a, IFN- , IFN-y, MIP-la, MIP-1 , TGF-P, TNF-a, and TNFp.

[0167] The present disclosure provides, inter alia, multispecific polypeptides that include, e.g., are engineered to contain, one or more cytokine molecules, e.g., immunomodulatory (e.g., proinflammatory) cytokines or variants, e.g., functional variants, thereof. Accordingly, in some embodiments, the cytokine molecule is an interleukin or a variant, e.g., a functional variant thereof. In some embodiments the interleukin is a proinflammatory interleukin. In some embodiments the interleukin is chosen from interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), interleukin- 18 (IL- 18), interleukin-21 (IL-21) or IFN-y.

[0168] In an embodiment, the cytokine of the multispecific or multifunctional polypeptide is IL- 15. In one embodiment, the cytokine molecule is human IL-15 comprising the amino acid sequence: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI ILA DNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 14), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 14). In a specific embodiment said IL-15 cytokine is a mutant IL-15 cytokine. In one embodiment the mutant IL-15 cytokine of the multispecific or multifunctional polypeptide according to the invention comprises at least one amino acid mutation. In one embodiment the amino acid mutation is an amino acid substitution. In a specific embodiment, the mutant IL-15 cytokine comprises an amino acid substitution at the position corresponding to residue 72 of human IL-15. In a more specific embodiment, the mutant IL- 15 cytokine is human IL- 15 comprising the amino acid substitution N72D.

[0169] In other embodiments, the cytokine molecule is IL-2, e.g., human IL-2 comprising the amino acid sequence (accession number NP_000577):

[0170] 1 MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML 61 TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE 121 TTFMCEYADE TATIVEFLNR iTFCQSiis TLT (SEQ ID NO: 15), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 15).

[0171] In other embodiments, the cytokine molecule is IL-18, e.g., human IL-18 comprising the amino acid sequence (accession number: NP 001553):

[0172] 1 MAAEPVEDNC INFVAMKFID NTLYFIAEDD ENLESDYFGK LESKLSVIRN LNDQVLFIDQ

[0173] 61 GNRPLFEDMT DSDCRDNAPR TIFIISMYKD SQPRGMAVTI SVKCEKISTL SCENKIISFK

[0174] 121 EMNPPDNIKD TKSDII FFQR SVPGHDNKMQ FESSSYEGYF LACEKERDLF KLILKKEDEL

[0175] 181 GDRSIMFTVQ NED (SEQ ID NO: 16), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 16).

[0176] In other embodiments, the cytokine molecule is IL-21, e.g., human IL-21 comprising the amino acid sequence (accession number NP 068575):

[0177] 1 MRSSPGNMER IVICLMVI FL GTLVHKSSSQ GQDRHMIRMR QLIDIVDQLK NYVNDLVPEF 61 LPAPEDVETN CEWSAFSCFQ KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL 121 TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DS (SEQ ID NO: 17), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 17). In yet other embodiments, the cytokine molecule is interferon gamma, e.g., human interferon gamma comprising the amino acid sequence (accession number: NP 000610):

[0178] 1 MKYTSYILAF QLCIVLGSLG CYCQDPYVKE AENLKKYFNA GHSDVADNGT LFLGILKNWK 61 EESDRKIMQS QIVSFYFKLF KNFKDDQSIQ KSVETIKEDM NVKFFNSNKK KRDDFEKLTN 121 YSVTDLNVQR KAIHELIQVM AELSPAAKTG KRKRSQMLFR GRRASQ (SEQ ID NO: 18), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 18).

[0179] Mutant cytokine molecules useful as effector moieties in the multispecific or multifunctional polypeptide can be prepared by deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include sitespecific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing. Substitution or insertion may involve natural as well as non-natural amino acid residues. Amino acid modification includes well known methods of chemical modification such as the addition or removal of glycosylation sites or carbohydrate attachments, and the like.

[0180] Immune Cell Targeting Moieties

[0181] The immune cell targeting moieties of the multispecific polypetides disclosed herein can mediate binding to, and / or activation of, an immune cell, e.g., an immune effector cell. In some embodiments, the immune cell is chosen from an NK cell, a B cell, a dendritic cell, or a macrophage cell targeting moiety, or a combination thereof. In some embodiments, the immune cell targeting moiety is chosen from one, two, three, or all of a T cell targeting moiety, NK cell targeting moiety, a B cell targeting moiety, a dendritic cell targeting moiety, or a macrophage cell targeting moiety, or a combination thereof. In some embodiments, the immune cell targeting moiety can be an antibody molecule, a ligand molecule, a small molecule, a nucleotide molecule.

[0182] Natural Killer Cell Targeting Moieties

[0183] Natural Killer (NK) cells recognize and destroy tumors and virus-infected cells in an antibody-independent manner. The regulation of NK cells is mediated by activating and inhibiting receptors on the NK cell surface. One family of activating receptors is the natural cytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46. The NCRs initiate tumor targeting by recognition of heparan sulfate on cancer cells. NKG2D is a receptor that provides both stimulatory and costimulatory innate immune responses on activated killer (NK) cells, leading to cytotoxic activity. DNAM1 is a receptor involved in intercellular adhesion, lymphocyte signaling, cytotoxicity and lymphokine secretion mediated by cytotoxic T-lymphocyte (CTL) and NK cell. DAP10 (also known as HCST) is a transmembrane adapter protein which associates with KLRK1 to form an activation receptor KLRK1-HCST in lymphoid and myeloid cells; this receptor plays a role in triggering cytotoxicity against target cells expressing cell surface ligands such as MHC class I chain-related MICA and MICB, and U (optionally Ll)6-binding proteins (ULBPs); the KLRK1-HCST receptor plays a role in immune surveillance against tumors and is needed for cytolysis of tumors cells; indeed, melanoma cells that do not express KLRK1 ligands escape from immune surveillance mediated by NK cells. CD16 is a receptor for the Fc region of IgG, which binds complexed or aggregated IgG and also monomeric IgG and thereby mediates antibodydependent cellular cytotoxicity (ADCC) and other antibody-dependent responses, such as phagocytosis.

[0184] The present disclosure provides, inter alia, multi-specific proteins, that are engineered to contain one or more NK cell targeting moieties that mediate binding to and / or activation of an NK cell. Accordingly, in some embodiments, the NK cell targeting moiety is selected from an antigen binding domain or ligand that binds to (e.g., activates): NKp30, NKp40, NKp44, NKp46 (NKp46: MSSTLPALLCVGLCLSQRISAQQQTLPKPFIWAEPHFMVPKEKQVTICCQGNYGAVEYQ LHFEGSLFAVDRPKPPERINKVQFYIPDMNSRMAGQYSCIYRVGELWSEPSNLLDLVVT EMYDTPTLSVHPGPEVISGEKVTFYCRLDTATSMFLLLKEGRSSHVQRGYGKVQAEFPL GPVTTAHRGTYRCFGSYNNHAWSFPSEPVKLLVTGDIENTSLAPEDPTFPADTWGTYLL TTETGLQKDHALWDHTAQNLLRMGLAFLVLVALVWFLVEDWLSRKRTRERASRAST WEGRRRLNTQTL (SEQ ID NO: 42); Anti-NKp46:

[0185] DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPS RFSGSGSGTDFTFTISSLQPEDIATYFCQQGNTRPWTFGGGTKVEIKRGGGGSGGGGSGG GGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSDYVINWVRQAPGQGLEWMGEIYPG SGTNYYNEKFKAKATITADKSTSTAYMELSSLRSEDTAVYYCARRGRYGLYAMDYWG QGTTVTVSS (SEQ ID NO: 43), KLRK1 / CD214 / NKG2D, DNAM1, DAP10, CD16 (e.g, CD16a (CD16a / FCGR3A), CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160.

[0186] In one embodiment, the NK cell targeting moiety is a ligand of NKp30, e.g., comprises the amino acid sequence of:

[0187] DLKVEMMAGGTQITPLNDNVTIFCNIFYSQPLNITSMGITWFWKSLTFDKEVKVFEFFGD HQEAFRPGAIVSPWRLKSGDASLRLPGIQLEEAGEYRCEVVVTPLKAQGTVQLEVVASP ASRLLLDQVGMKENEDKYMCESSGFYPEAINITWEKQTQKFPHPIEISEDVITGPTIKNM DGTFNVTSCLKLNSSQEDPGTVYQCVVRHASLHTPLRSNFTLTAARHSLSETEKTDNFS (SEQ ID NO: 19), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 19).

[0188] In other embodiments, the NK cell targeting moiety is a ligand of NKG2D chosen from MICA, MICB, or ULBPl, e.g., wherein:

[0189] (i) MICA comprises the amino acid sequence:

[0190] EPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNK TWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGEL FLSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLK SGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSSHDTQQWG DVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHW (SEQ ID NO: 20), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 20);

[0191] (ii) MICB comprises the amino acid sequence:

[0192] AEPHSLRYNLMVLSQDESVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAEDVLGA KTWDTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCEHiEDSSTRGSRHFYYDGEL FLSQNLETQESTVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLK SGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGD VLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKVLVLQSQRTD (SEQ ID NO:

[0193] 21), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 21); or

[0194] (iii) ULBP1 comprises the amino acid sequence:

[0195] GWVDTHCLCYDFI ITPKSRPEPQWCEVQGLVDERPFLHYDCVNHKAKAFASLGKKVNV TKTWEEQTETLRDWDFLKGQLLDIQVENLIPIEPLTLQARMSCEHEAHGHGRGSWQFL FNGQKFLLFDSNNRKWTALHPGAKKMTEKWEKNRDVTMFFQKISLGDCKMWLEEFL

[0196] MYWEQMLDPTKPPSLAPG (SEQ ID NO: 22), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 22). In other embodiments, the NK cell targeting moiety is a ligand of DNAM1 chosen from NECTIN2 or NECL5, e.g., wherein:

[0197] (i) NECTIN2 comprises the amino acid sequence: QDVRVQVLPEVRGQLGGTVELPCHLLPPVPGLYISLVTWQRPDAPANHQNVAAFHPKM GPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDATLALHGLTVEDEGNYTCEFATFPKGS VRGMTWLRVIAKPKNQAEAQKVTFSQDPTTVALCISKEGRPPARISWLSSLDWEAKETQ VSGTLAGTVTVTSRFTLVPSGRADGVTVTCKVEHESFEEPALIPVTLSVRYPPEVS ISGYD DNWYLGRTDATLSCDVRSNPEPTGYDWSTTSGTFPTSAVAQGSQLVIHAVDSLFNTTFV CTVTNAVGMGRAEQVI FVRETPNTAGAGATGG (SEQ ID NO: 23), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 23); or

[0198] (ii) NECL5 comprises the amino acid sequence: WPPPGTGDVWQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAV FHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVD IWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPG FLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVS ISGYDNN WYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICN VTNALGARQAELTVQVKEGPPSEHSGISRN (SEQ ID NO: 24), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 24).

[0199] In yet other embodiments, the NK cell targeting moiety is a ligand of DAP 10, which is an adapter for NKG2D (see e.g., Proc Natl Acad Sci USA. 2005 May 24; 102(21): 7641-7646; and Blood, 15 Sep. 2011 Volume 118, Number 11, the full contents of each of which is incorporated by reference herein).

[0200] In other embodiments, the NK cell targeting moiety is a ligand of CD 16, which is a CD16a / b ligand (the primary ligand for CD16 (also known as FcyRIII) is the Fc portion of IgG antibodies (e.g., IgGl, IgG2, IgG3 orIgG4, such as IgGl); specifically, CD16a (FcyRIIIA) binds to IgG), e.g., a CD16a / b ligand further comprising an antibody Fc region (see e.g., Front Immunol. 2013; 4: 76 discusses how antibodies use the Fc to trigger NK cells through CD16, the full contents of which are incorporated herein). In other embodiments, the NK cell targeting moiety is a ligand of CRTAM, which is NECL2, e.g., wherein NECL2 comprises the amino acid sequence: QNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSS ELKVSLTNVS ISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNC TAMASKPATTIRWFKGNTELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQVE HPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQPVMVTWV RVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDYMLYVYDPPTTIPPPTTTTTTTTTTTTTILTI ITDSRAGEEGS IRAVDH SEQID NQ. 25),afragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 25).

[0201] In other embodiments, the NK cell targeting moiety is a ligand of CD27, which is CD70, e.g., wherein CD70 comprises the amino acid sequence: QRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQ LRI IHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRS ISLLRLSFHQGCTIASQR LTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP (SEQ ID NO: 26), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 26).

[0202] In other embodiments, the NK cell targeting moiety is a ligand of PSGL1, which is L- selectin (CD62L), e.g., wherein L-selectin comprises the amino acid sequence: WTYHYSEKPMNWQRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYYWIGIRKIGGI WT WVGTNKS L TEEAENWGDGE PNNKKNKE DCVE I Y I KRNKDAGKWNDDACHKLKAA LCYTASCQPWSCSGHGECVEI INNYTCNCDVGYYGPQCQFVIQCEPLEAPELGTMDCTH PLGNFSFSSQCAFSCSEGTNLTGIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDLGIMNCSH PLASFSFTSACTFICSEGTELIGKKKTICESSGIWSNPSPICQKLDKSFSMIKEGDYN (SEQ ID NO: 27), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 27).

[0203] In other embodiments, the NK cell targeting moiety is a ligand of CD96, which is NECL5, e.g., wherein NECL5 comprises the amino acid sequence: WPPPGTGDVWQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAV FHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVD IWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPG FLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVS ISGYDNN WYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICN VTNALGARQAELTVQVKEGPPSEHSGISRN (SEQ ID NO: 28), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 28).

[0204] In other embodiments, the NK cell targeting moiety is a ligand of CD 100 (SEMA4D), which is CD72, e.g., wherein CD72 comprises the amino acid sequence:

[0205] RYLQVSQQLQQTNRVLEVTNSSLRQQLRLKITQLGQSAEDLQGSRRELAQSQEALQVEQ RAHQAAEGQLQACQADRQKTKETLQSEEQQRRALEQKLSNMENRLKPFFTCGSADTCC PSGWIMHQKSCFYISLTSKNWQESQKQCETLSSKLATFSEIYPQSHSYYFLNSLLPNGGS GNSYWTGLSSNKDWKLTDDTQRTRTYAQSSKCNKVHKTWSWWTLESESCRSSLPYICE MTAFRFPD (SEQ ID NO: 29), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 29).

[0206] In other embodiments, the NK cell targeting moiety is a ligand of NKp80, which is CLEC2B (AICL), e.g., wherein CLEC2B (AICL) comprises the amino acid sequence: KLTRDSQSLCPYDWIGFQNKCYYFSKEEGDWNSSKYNCSTQHADLTI IDNIEEMNFLRR YKCSSDHWIGLKMAKNRTGQWVDGATFTKSFGMRGSEGCAYLSDDGAATARCYTER KWICRKRIH (SEQ ID NO: 30), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 30).

[0207] In other embodiments, the NK cell targeting moiety is a ligand of CD244, which is CD48, e.g., wherein CD48 comprises the amino acid sequence: QGHLVHMTWSGSNVTLNISESLPENYKQLTWFYTFDQKIVEWDSRKSKYFESKFKGR VRLDPQSGALYISKVQKEDNSTYIMRVLKKTGNEQEWKIKLQVLDPVPKPVIKIEKIEDM DDNCYLKLSCVIPGESVNYTWYGDKRPFPKELQNSVLETTLMPHNYSRCYTCQVSNSVS SKNGTVCLSPPCTLARS (SEQ ID NO: 31), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 31).

[0208] T Cell Targeting Moieties

[0209] The present disclosure provides, inter alia, multispecific polypeptides, that are engineered to contain one or more T cell targeting moieties that mediate binding to and / or activation of a T cell. Accordingly, in some embodiments, the T cell targeting moiety is selected from an antigen binding domain or ligand that binds to (e.g., and in some embodiments activates) one or more of CD3, TCRa, TCRp, TCRy, TCR^, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226.

[0210] B Cell, Macrophage & Dendritic Cell Targeting Moieties

[0211] B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system by secreting antibodies. Additionally, B cells present antigen (they are also classified as professional antigen-presenting cells (APCs)) and secrete cytokines. Macrophages are a type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, cancer cells via phagocytosis. Besides phagocytosis, they play important roles in nonspecific defense (innate immunity) and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. For example, they are important as antigen presenters to T cells. Beyond increasing inflammation and stimulating the immune system, macrophages also play an anti-inflammatory role and can decrease immune reactions through the release of cytokines. Dendritic cells (DCs) are antigen-presenting cells that function in processing antigen material and present it on the cell surface to the T cells of the immune system.

[0212] The present disclosure provides, inter alia, multispecific polypeptides, that include, e.g., are engineered to contain, one or more B cell, macrophage, and / or dendritic cell targeting moieties that mediate binding to and / or activation of a B cell, macrophage, and / or dendritic cell.

[0213] Accordingly, in some embodiments, the immune cell targeting moiety comprises a B cell, macrophage, and / or dendritic cell targeting moiety chosen from one or more of CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule that binds to 0X40; an 0X40 ligand (OX40L); an agonist of a Toll-like receptor; a 41BB; a CD2; a CD47; or a STING agonist, or a combination thereof.

[0214] In some embodiments, the B cell targeting moiety is a CD40L, an OX40L, or a CD70 ligand, or an antibody molecule that binds to 0X40, CD40 or CD70. In some embodiments, the macrophage targeting moiety is a CD2 agonist. In some embodiments, the macrophage targeting moiety is an antigen binding domain that binds to: CD40L or antigen binding domain or ligand that binds CD40, a Toll like receptor (TLR) agonist, CD47, or a STING agonist. In some embodiments, the STING agonist is a cyclic dinucleotide, e.g., cyclic di-GMP (cdGMP) or cyclic di-AMP (cdAMP).

[0215] In some embodiments, the dendritic cell targeting moiety is a CD2 agonist. In some embodiments, the dendritic cell targeting moiety is a ligand, a receptor agonist, or an antibody molecule that binds to one or more of: OX40L, 4 IBB, a TLR agonist, CD47, or and a STING agonist. In some embodiments, the STING agonist is a cyclic dinucleotide, e.g., cyclic di-GMP (cdGMP) or cyclic di-AMP (cdAMP).

[0216] In other embodiments, the immune cell targeting moiety mediates binding to, or activation of, one or more of a B cell, a macrophage, and / or a dendritic cell. Exemplary B cell, macrophage, and / or dendritic cell targeting moieties can be chosen from one or more of CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule to 0X40; an 0X40 ligand (OX40L); a Toll-like receptor agonist; a 41BB agonist; a CD2; a CD47; or a STING agonist, or a combination thereof.

[0217] In one embodiment, the OX40L comprises the amino acid sequence: QVSHRYPRIQSIKVQF TEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQ EVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGE LILIHQNPGEFCVL (SEQ ID NO: 32), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 32).

[0218] In another embodiment, the CD40L comprises the amino acid sequence: MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLY YIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFE LQPGASVFVNVTDPSQVSHGTGFTSFGLLKL (SEQ ID NO: 33), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 33).

[0219] In yet other embodiments, the STING agonist comprises a cyclic dinucleotide, e.g., a cyclic di-GMP (cdGMP), a cyclic di-AMP (cdAMP), or a combination thereof, optionally with 2', 5' or 3', 5' phosphate linkages. In one embodiment, the immune cell targeting moiety includes 4 IBB ligand, e.g., comprising the amino acid sequence:

[0220] ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLS WYSDPGLAGVSLTGGLSYKEDTKELWAKAGVYYVFFQLELRRWAGEGSGSVSLALH LQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARH AWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 34), a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 34).

[0221] Chemokines

[0222] Chemokines or chemotactic cytokines are a family of small cytokines or signaling proteins secreted by cells that induce directional movement of cells, such as immune cells. In addition to playing a role in the activation of host immune responses, chemokines have a role in biological processes, including morphogenesis and wound healing, as well as in the pathogenesis of diseases like cancers.

[0223] Chemokines have been classified into four main subfamilies: CXC, CC, CX3C and C. These proteins exert their biological effects by interacting with G protein-linked transmembrane receptors called chemokine receptors, that are selectively found on the surfaces of their target cells.

[0224] A role of chemokines is to act as a chemoattractant to guide the migration of cells. Cells that are attracted by chemokines follow a signal of increasing chemokine concentration towards the source of the chemokine. Some chemokines control cells of the immune system during processes of immune surveillance, such as directing lymphocytes to the lymph nodes so they can screen for invasion of pathogens by interacting with antigen-presenting cells residing in these tissues. These are known as homeostatic chemokines and are produced and secreted without any need to stimulate their source cells. Some chemokines have roles in development; they promote angiogenesis (the growth of new blood vessels), or guide cells to tissues that provide specific signals critical for cellular maturation. Other chemokines are inflammatory and are released from a wide variety of cells in response to bacterial infection, viruses and agents that cause physical damage such as silica or the urate crystals that occur in gout. Their release is often stimulated by pro-inflammatory cytokines such as interleukin 1. Inflammatory chemokines function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils and other effector cells from the blood to sites of infection or tissue damage. Certain inflammatory chemokines activate cells to initiate an immune response or promote wound healing. They are released by many different cell types and serve to guide cells of both innate immune system and adaptive immune system.

[0225] In some embodiments, the chemokine molecule is chosen from CCL1, CCL2 (MCP-1), CCL3 (MIP-la), CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 / CCL10, CCL11 (Eotaxin), CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1 (Fractalkine), CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8 (IL-8), CXCL9, CXCL10, CXCL11, CXCL12 (SDF-1), XCL1 (Lymphotactin), XCL2, CS3CL1 or a fragment or variant thereof, or a combination thereof.

[0226] Linkers

[0227] The multispecific polypeptide disclosed herein can further include a linker, e.g., a linker between one or more of: the cancer targeting moiety and the cytokine or chemokine molecule, the cancer targeting moiety and the immune cell targeting moiety, the cytokine or chemokine molecule and the immune cell targeting moiety, the cytokine molecule and the immunoglobulin chain constant region (e.g., the Fc region), the cancer targeting moiety and the immunoglobulin chain constant region, or the immune cell targeting moiety and the immunoglobulin chain constant region. In embodiments, the linker chosen from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker, or a combination thereof.

[0228] In one embodiment, the multispecific polypeptide can include one, two, three or four linkers, e.g., a peptide linker. In one embodiment, the peptide linker includes Gly and Ser. Exemplary peptide linkers can include, but are not limited to, GGGGS (SEQ ID NO: 35); GGGGSGGGGS (SEQ ID NO: 36); GGGGS GGGGS GGGGS (SEQ ID NO: 37); or DVPSGPGGGGGSGGGGS (SEQ ID NO: 38).

[0229] In one embodiment, the chemokine can be cleaved from a polypeptide sequence with the aid of an enzymatically cleavable linker, such as a protease-sensitive linker, such as those selected from the group consisting of urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMPs).

[0230] Matrix metalloproteinases (MMPs), also known as matrix metallopeptidases or matrixins, are metalloproteinases that are calcium-dependent zinc-containing endopeptidases; other family members are adamalysins, serralysins, and astacins. The MMPs belong to a larger family of proteases known as the metzincin superfamily, which can be organized into six groups based on domain organization and substrate preference: Collagenases (MMP-1, -8 and -13), Gelatinases (MMP-2 and MMP-9), Stromelysins (MMP-3, -10 and -11), Matrilysin (MMP-7 and MMP-26), Membrane-type (MT)-MMPs (MMP-14, -15, -16, -17, -24 and -25) and others (MMP-12, -19, - 20, -21, -23, -27 and -28).

[0231] MMPs include, but are not limited to, MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP- 9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-21, MMP-23, MMP-23A, MMP-23B, MMP-24, MMP-25, MMP-26, MMP-27, MMP-28, functional fragment thereof, functional variants thereof, or a combination thereof.

[0232] Exemplary Multispecific Configurations

[0233] In some embodiments, the multispecific molecule includes a first and a second contiguous or non-contiguous polypeptide, wherein:

[0234] (i) the first polypeptide includes, e.g., in the N- to C-orientation, a tumor targeting moiety, e.g., an antibody molecule (e.g., a first portion of a first antigen domain, e.g., a first VH-CH1 of a Fab molecule), that binds to, e.g., a tumor antigen (a solid tumor, or a hematological antigen), connected, optionally, via a linker to, a first domain that promotes association between the first and the second polypeptide (e.g., a first immunoglobulin constant domain (e.g., a first Fc molecule as described herein); and

[0235] (ii) the second polypeptide includes, e.g., in the N- to C-orientation, an immune cell targeting moiety (e.g., an antibody molecule, e.g., a scFv, that binds to an immune cell antigen), connected, optionally, via a linker to, a second domain that promotes association between the first and the second polypeptide (e.g., a second immunoglobulin constant domain (e.g., a second Fc molecule as described herein), wherein either the first or the second polypeptide further comprise a cytokine molecule, optionally covalently linked to the C-terminus of the first or second immunoglobulin constant domain, wherein either the first or the second polypeptide further comprise a chemokine molecule, optionally covalently linked to the C-terminus of the first or second immunoglobulin constant domain, wherein the chemokine molecule is cleavable from the first or second immunoglobulin.

[0236] One embodiment comprises a (i) an IgG heavy chain (CH1-CH2-CH3) with a VH domain derived from an antibody targeting an immune cell and a linker with a sequence coding for a cytokine; (ii) an IgG light chain with a VL domain derived from an antibody targeting the immune cell; (iii) an IgG heavy chain (CH1-CH2-CH3) with a VH domain derived from an antibody targeting a cancer cell and a cleavable linker followed by a chemokine; and (iv) an IgG light chain with a VL domain derived from an antibody targeting the cancer cell.

[0237] One embodiment comprises a multispecific polypeptide comprising (i) an IgG heavy chain (CH1-CH2-CH3) with a VH domain derived from an antibody targeting NKp46 and a flexible linker linking a sequence encoding IL-15, such as IL-15N72D; (ii) an IgG light chain with a VL domain derived from an antibody targeting NKp46; (iii) an IgG heavy chain (CH1-CH2-CH3) with a VH domain derived from an antibody targeting IL-13Ra2 and a cleavable linker followed by human CXCL10 and an optional 6'His tag; (iv) an IgG light chain with a VL domain derived from an antibody targeting IL-13Ra2. In some embodiments, the construct further comprises an IgGl-like Fc domain to promote expression, folding, and stability, such as IgG Fc domain modified with the knob-into-hole approach.

[0238] One embodiment provides any combination of at least one of each: 1) an immune cell targeting moiety targeting NKp30, NKp40, NKp44, NKp46, KLRK1 / CD214 / NKG2D, DNAM1, DAP10, CD16a / FCGR3A, CD16b, CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160 and 2) a tumor cell targeting moiety targeting PDL1, CD47, mesothelin, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, IL- 13Ra2, Telomerase, SAP-1, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART- 1, Gpl00 / pmell7, Tyrosinase, TRP-1 / -2, MC1R, P-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, P-catenin, CDK4, CDC27, CD47, a actinin-4, TRPl / gp75, TRP2, gplOO, Melan- A / MART1, gangliosides, WT1, EphA3, Epidermal growth factor receptor (EGFR), CD20, MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUI2, SAGE, TRG, TRP1, TSTA, Folate receptor alpha, Ll-CAM, CAIX, EGFRvIII, gpA33, GD3, GM2, VEGFR, Intergrins (Integrin alphaVbeta3, Integrin alpha5Betal), Carbohydrates (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, carbonic anhydrase IX (CA9), carbonic anhydrase 12 (CA12), glucose transporter type 1 (GLUTl / solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)) or RANKL.

[0239] One embodiment provides any combination of at least one of each: 1) an immune cell targeting moiety targeting, for example, KLRK1 / CD314 or CD16 / FCGR3A and 2) a tumor cell targeting moiety targeting carbonic anhydrase IX (CA9), carbonic anhydrase 12 (CA12), or glucose transporter type 1 (GLUTl / solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)).

[0240] Nucleic Acids

[0241] The invention also features nucleic acids comprising nucleotide sequences that encode heavy and light chain variable regions and CDRs or hypervariable loops of the antibody molecules, as described herein, as well as linker(s), immune cell targeting moieties, tumor targeting moieties, cytokine(s) and / or chemokine(s).

[0242] Vectors

[0243] Further provided herein are vectors comprising the nucleotide sequences described herein. The vectors include, but are not limited to, a virus, plasmid, cosmid, lambda phage or a yeast artificial chromosome (YAC).

[0244] Numerous vector systems can be employed. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, lentivirus, retroviruses (Rous Sarcoma Virus, MMTV or MOMLV) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern Equine Encephalitis virus and Flaviviruses.

[0245] Additionally, cells which have stably integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow for the selection of transfected host cells. The marker may provide, for example, prototropy to an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper, or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0246] Once the expression vector or DNA sequence containing the constructs has been prepared for expression, the expression vectors may be transfected or introduced into an appropriate host cell. Various techniques may be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are grown in media and screened for the appropriate activity.

[0247] Methods and conditions for culturing the resulting transfected cells and for recovering the antibody molecule produced are known to those skilled in the art and may be varied or optimized depending upon the specific expression vector and mammalian host cell employed, based upon the present description.

[0248] Cells

[0249] In another aspect, the application features host cells and vectors containing the nucleic acids described herein. The nucleic acids may be present in a single vector, or separate vectors present in the same host cell or separate host cell. The host cell can be a eukaryotic cell, e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E. colt. For example, the mammalian cell can be a cultured cell or a cell line. Exemplary mammalian cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII cells.

[0250] The invention also provides host cells comprising a nucleic acid(s) as described herein.

[0251] In one embodiment, the host cells are genetically engineered by using an expression cassette. The phrase “expression cassette,” refers to nucleotide sequences, which are capable of affecting expression of a gene in hosts compatible with such sequences. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in effecting expression may also be used, such as, for example, an inducible promoter.

[0252] The invention also provides host cells comprising the nucleotide sequences, vectors and proteins described herein.

[0253] Uses

[0254] Methods described herein include treating a cancer in a subject by using a multispecific polypeptide described herein, e.g., using a pharmaceutical composition described herein. Also provided are methods for reducing or ameliorating a symptom of a cancer in a subject, as well as methods for inhibiting the growth of a cancer and / or killing one or more cancer cells. In embodiments, the methods described herein decrease the size of a tumor and / or decrease the number of cancer cells in a subject administered with a multispecific polypeptide described herein or a pharmaceutical composition described herein.

[0255] In embodiments, the cancer is a hematological cancer. In embodiments, the hematological cancer is a leukemia or a lymphoma. As used herein, a “hematologic cancer” refers to a tumor of the hematopoietic or lymphoid tissues, e.g., a tumor that affects blood, bone marrow, or lymph nodes. Exemplary hematologic malignancies include, but are not limited to, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, acute monocytic leukemia (AMoL), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), or large granular lymphocytic leukemia), lymphoma (e.g., AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma (e.g., classical Hodgkin lymphoma or nodular lymphocyte-predominant Hodgkin lymphoma), mycosis fungoides, non-Hodgkin lymphoma (e.g., B-cell non-Hodgkin lymphoma (e.g., Burkitt lymphoma, small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, or mantle cell lymphoma) or T-cell non- Hodgkin lymphoma (mycosis fungoides, anaplastic large cell lymphoma, or precursor T- lymphoblastic lymphoma)), primary central nervous system lymphoma, Sezary syndrome, Waldenstrom macroglobulinemia), chronic myeloproliferative neoplasm, Langerhans cell histiocytosis, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, or myelodysplastic / myeloproliferative neoplasm.

[0256] In embodiments, the cancer is a solid cancer. Exemplary solid cancers include, but are not limited to, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, cancer of the anal region, uterine cancer, colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, Kaposi's sarcoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, brain stem glioma, pituitary adenoma, epidermoid cancer, carcinoma of the cervix squamous cell cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the vagina, sarcoma of soft tissue, cancer of the urethra, carcinoma of the vulva, cancer of the penis, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, spinal axis tumor, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, metastatic lesions of said cancers, or combinations thereof.

[0257] In embodiments, the multispecific polypeptides (or pharmaceutical composition) are administered in a manner appropriate to the disease to be treated or prevented. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease. Appropriate dosages can be determined by clinical trials. For example, when “an effective amount” or “a therapeutic amount” is indicated, the precise amount of the pharmaceutical composition (or multispecific polypeptides) to be administered can be determined by a physician with consideration of individual differences in tumor size, extent of infection or metastasis, age, weight, and condition of the subject. In embodiments, the pharmaceutical composition described herein can be administered at a dosage of 104to 109cells / kg body weight, e.g., 105to 106cells / kg body weight, including all integer values within those ranges. In embodiments, the pharmaceutical composition described herein can be administered multiple times at these dosages. In embodiments, the pharmaceutical composition described herein can be administered using infusion techniques described in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

[0258] In embodiments, the multispecific polypeptides or pharmaceutical composition is administered to the subject parenterally. In embodiments, the multispecific polypeptides or pharmaceutical compositions are administered to the subject intravenously, subcutaneously, intratumorally, intranodally, intramuscularly, intradermally, or intraperitoneally. In embodiments, the multispecific polypeptides or pharmaceutical compositions are administered, e.g., injected, directly into a tumor or lymph node. In embodiments, the cells are administered as an infusion (e.g., as described in Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988) or an intravenous push. In embodiments, the multispecific polypeptides or pharmaceutical compositions are administered as an injectable depot formulation.

[0259] In embodiments, the subject is a mammal. In embodiments, the subject is a human, monkey, pig, dog, cat, cow, sheep, goat, rabbit, rat, or mouse. In embodiments, the subject is a human. In embodiments, the subject is a pediatric subject, e.g., less than 18 years of age, e.g., less than 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less years of age. In embodiments, the subject is an adult, e.g., at least 18 years of age, e.g., at least 19, 20, 21, 22, 23, 24, 25, 25-30, 30- 35, 35-40, 40-50, 50-60, 60-70, 70-80, or 80-90 years of age.

[0260] Combination Therapies

[0261] The multispecific polypeptides disclosed herein can be used in combination with a second therapeutic agent or procedure.

[0262] In embodiments, the multispecific polypeptide and the second therapeutic agent or procedure are administered / performed after a subject has been diagnosed with a cancer, e.g., before the cancer has been eliminated from the subject. In embodiments, the multispecific polypeptide and the second therapeutic agent or procedure are administered / performed simultaneously or concurrently. For example, the delivery of one treatment is still occurring when the delivery of the second commences, e.g., there is an overlap in administration of the treatments. In other embodiments, the multispecific polypeptide and the second therapeutic agent or procedure are administered / performed sequentially. For example, the delivery of one treatment ceases before the delivery of the other treatment begins.

[0263] In embodiments, combination therapy can lead to more effective treatment than monotherapy with either agent alone. In embodiments, the combination of the first and second treatment is more effective (e.g., leads to a greater reduction in symptoms and / or cancer cells) than the first or second treatment alone. In embodiments, the combination therapy permits use of a lower dose of the first or the second treatment compared to the dose of the first or second treatment normally required to achieve similar effects when administered as a monotherapy. In embodiments, the combination therapy has a partially additive effect, wholly additive effect, or greater than additive effect. In one embodiment, the multispecific polypeptide is administered in combination with a therapy, e.g., a cancer therapy (e.g., one or more of anti-cancer agents, immunotherapy, photodynamic therapy (PDT), surgery and / or radiation). The terms “chemotherapeutic,” “chemotherapeutic agent,” and “anti-cancer agent” are used interchangeably herein. Anti-Cancer Therapies

[0264] In other embodiments, the multispecific polypeptide is administered in combination with a low or small molecular weight chemotherapeutic agent. Exemplary low or small molecular weight chemotherapeutic agents include, but not limited to, 13-cis-retinoic acid (isotretinoin, ACCUTANE®), 2-CdA (2-chlorodeoxyadenosine, cladribine, LEUSTATIN™), 5-azacitidine (azacitidine, VID AZ A®), 5 -fluorouracil (5-FU, fluorouracil, ADRUCIL®), 6-mercaptopurine (6- MP, mercaptopurine, PURINETHOL®), 6-TG (6-thioguanine, thioguanine, THIOGUANINE TABLOID®), abraxane (paclitaxel protein-bound), actinomycin-D (dactinomycin, COSMEGEN®), alitretinoin (PANRETIN®), all-transretinoic acid (ATRA, tretinoin, VESANOID®), altretamine (hexamethylmelamine, HMM, HEXALEN®), amethopterin (methotrexate, methotrexate sodium, MTX, TREXALL™, RHEUMATREX®), amifostine (ETHYOL®), arabinosylcytosine (Ara-C, cytarabine, CYTOSAR-U®), arsenic trioxide (TRISENOX®), asparaginase (Erwinia L-asparaginase, L-asparaginase, ELSPAR®, KIDROLASE®), BCNU (carmustine, BiCNU®), bendamustine (TREANDA®), bexarotene (TARGRETIN®), bleomycin (BLENOXANE®), busulfan (BUSULFEX®, MYLERAN®), calcium leucovorin (Citrovorum Factor, folinic acid, leucovorin), camptothecin-11 (CPT-11, irinotecan, CAMPTOSAR®), capecitabine (XELODA®), carboplatin (PARAPLATIN®), carmustine wafer (prolifeprospan 20 with carmustine implant, GLIADEL® wafer), CCI-779 (temsirolimus, TORISEL®), CCNU (lomustine, CeeNU), CDDP (cisplatin, PLATINOL®, PLATINOL-AQ®), chlorambucil (leukeran), cyclophosphamide (CYTOXAN®, NEOSAR®), dacarbazine (DIC, DTIC, imidazole carboxamide, DTIC-DOME®), daunomycin (daunorubicin, daunorubicin hydrochloride, rubidomycin hydrochloride, CERUBIDINE®), decitabine (DACOGEN®), dexrazoxane (ZINECARD®), DHAD (mitoxantrone, NOVANTRONE®), docetaxel (TAXOTERE®), doxorubicin (ADRIAMYCIN®, RUBEX®), epirubicin (ELLENCE™), estramustine (EMCYT®), etoposide (VP- 16, etoposide phosphate, TOPOSAR®, VEPESID®, ETOPOPHOS®), floxuridine (FUDR®), fludarabine (FLUDARA®), fluorouracil (cream) (CARAC™, EFUDEX®, FLUOROPLEX®), gemcitabine (GEMZAR®), hydroxyurea (HYDREA®, DROXIA™, MYLOCEL™), idarubicin (IDAMYCIN®), ifosfamide (IFEX®), ixabepilone (IXEMPRA™), LCR (leurocristine, vincristine, VCR, ONCOVIN®, VINCASAR PFS®), L-PAM (L-sarcolysin, melphalan, phenylalanine mustard, ALKERAN®), mechlorethamine (mechlorethamine hydrochloride, mustine, nitrogen mustard, MUSTARGEN®), mesna (MESNEX™), mitomycin (mitomycin-C, MTC, MUTAMYCIN®), nelarabine (ARRANON®), oxaliplatin (ELOXATIN™), paclitaxel (TAXOL®, ONXAL™), pegaspargase (PEG-L-asparaginase, ONCOSPAR®), PEMETREXED (ALIMTA®), pentostatin (NIPENT®), procarbazine (MATULANE®), streptozocin (ZANOSAR®), temozolomide (TEMODAR®), teniposide (VM-26, VUMON®), TESPA (thiophosphoamide, thiotepa, TSP A, THIOPLEX®), topotecan (HYCAMTIN®), vinblastine (vinblastine sulfate, vincaleukoblastine, VLB, ALKABAN-AQ®, VELBAN®), vinorelbine (vinorelbine tartrate, NAVELBINE®), and vorinostat (ZOLINZA®).

[0265] In another embodiment, the multispecific polypeptide is administered in conjunction with a biologic. Biologies useful in the treatment of cancers are known in the art and a polypeptide of the invention may be administered, for example, in conjunction with such known biologies. For example, the FDA has approved the following biologies for the treatment of breast cancer: HERCEPTIN® (trastuzumab, Genentech Inc., South San Francisco, Calif.; a humanized monoclonal antibody that has anti-tumor activity in HER2 -positive breast cancer); FASLODEX® (fulvestrant, AstraZeneca Pharmaceuticals, LP, Wilmington, Del.; an estrogen-receptor antagonist used to treat breast cancer); ARIMIDEX® (anastrozole, AstraZeneca Pharmaceuticals, LP; a nonsteroidal aromatase inhibitor which blocks aromatase, an enzyme needed to make estrogen); Aromasin® (exemestane, Pfizer Inc., New York, N.Y.; an irreversible, steroidal aromatase inactivator used in the treatment of breast cancer); FEMARA® (letrozole, Novartis Pharmaceuticals, East Hanover, N.J.; a nonsteroidal aromatase inhibitor approved by the FDA to treat breast cancer); and NOLVADEX® (tamoxifen, AstraZeneca Pharmaceuticals, LP; a nonsteroidal antiestrogen approved by the FDA to treat breast cancer). Other biologies with which the polypeptides of the invention may be combined include: AVASTIN® (bevacizumab, Genentech Inc.; the first FDA-approved therapy designed to inhibit angiogenesis); and ZEVALIN® (ibritumomab tiuxetan, Biogen Idee, Cambridge, Mass.; a radiolabeled monoclonal antibody currently approved for the treatment of B-cell lymphomas).

[0266] In addition, the FDA has approved the following biologies for the treatment of colorectal cancer: AVASTIN®; ERBITUX® (cetuximab, ImClone Systems Inc., New York, N.Y., and Bristol-Myers Squibb, New York, N.Y.; is a monoclonal antibody directed against the epidermal growth factor receptor (EGFR)); GLEEVEC® (imatinib mesylate; a protein kinase inhibitor); and ERGAMISOL® (levamisole hydrochloride, Janssen Pharmaceutica Products, LP, Titusville, N. J.; an immunomodulator approved by the FDA in 1990 as an adjuvant treatment in combination with 5 -fluorouracil after surgical resection in patients with Dukes' Stage C colon cancer). For the treatment of lung cancer, exemplary biologies include TARCEVA® (erlotinib HCL, OSI Pharmaceuticals Inc., Melville, N.Y.; a small molecule designed to target the human epidermal growth factor receptor 1 (HER1) pathway).

[0267] For the treatment of multiple myeloma, exemplary biologies include VELCADE® Velcade (bortezomib, Millennium Pharmaceuticals, Cambridge Mass.; a proteasome inhibitor). Additional biologies include THALIDOMID® (thalidomide, Clegene Corporation, Warren, N.J.; an immunomodulatory agent and appears to have multiple actions, including the ability to inhibit the growth and survival of myeloma cells and anti-angiogenesis).

[0268] Additional exemplary cancer therapeutic antibodies include, but are not limited to, 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab pegol, alemtuzumab (CAMPATH®, MAB CAMPATH®), altumomab pentetate (HYBRI-CEAKER®), anatumomab mafenatox, anrukinzumab (IMA-638), apolizumab, arcitumomab (CEA-SCAN®), bavituximab, bectumomab (LYMPHOSCAN®), belimumab (BENLYSTA®, LYMPHOSTAT-B®), besilesomab (SCINTIMUN®), bevacizumab (AVASTIN®), bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide (PROSTASCINT®), catumaxomab (REMOVAB®), CC49, cetuximab (C225, ERBITUX®), citatuzumab bogatox, eixutumumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, denosumab (PROLIA®), detumomab, ecromeximab, edrecolomab (P ANOREX®), elotuzumab, epitumomab cituxetan, epratuzumab, ertumaxomab (REXOMUN®), etaracizumab, farletuzumab, figitumumab, fresolimumab, galiximab, gemtuzumab ozogamicin (MYLOTARG®), girentuximab, glembatumumab vedotin, ibritumomab (ibritumomab tiuxetan, ZEVALIN®), igovomab (INDIMACIS-125®), intetumumab, inotuzumab ozogamicin, ipilimumab, iratumumab, labetuzumab (CEA-CIDE®), lexatumumab, lintuzumab, lucatumumab, lumiliximab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, nacolomab tafenatox, naptumomab estafenatox, necitumumab, nimotuzumab (THERACIM®, THERALOC®), nofetumomab merpentan (VERLUMA®), ofatumumab (ARZERRA®), olaratumab, oportuzumab monatox, oregovomab (OVAREX®), panitumumab (VECTIBIX®), pemtumomab (THERAGYN®), pertuzumab (OMNITARG®), pintumomab, pritumumab, ramucirumab, ranibizumab (LUCENTIS®), rilotumumab, rituximab (MABTHERA®, RITUXAN®), robatumumab, satumomab pendetide, sibrotuzumab, siltuximab, sontuzumab, tacatuzumab tetraxetan (AFP-CIDE®), taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, tositumomab (BEXXAR®), trastuzumab (HERCEPTIN®), tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab (HUMASPECT®), zalutumumab (HUMAX-EGFR®), and zanolimumab (HUMAX- CD4®).

[0269] EXAMPLE

[0270] The following example is intended to be illustrative and is not meant in any way to be limiting.

[0271] Materials and Methods

[0272] Reagents

[0273] DMEM with high glucose medium, RPMI-1640 medium, IMDM medium, GlutaMAX, trypsin / EDTA solution, penicillin and streptomycin, and ACK Lysis Buffer (RBC Lysis) were purchased from Gibco (New York, NY, USA). Fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) were purchased from Coming (New York, NY, USA). Recombinant human CXCL10 and recombinant mouse CXCL10 were purchased from BioLegend (San Diego, CA, USA). Collagenase IV (Cat No. LS002006) and DNase I (Cat No. LS002006) were from Worthington Biochemical (Lakewood, NY, USA). Recombinant human IL-2 (rhIL-2) was gifted from Akron Biotech (Boca Raton, FL, USA). rhIL-21, rh4-lBB ligand, and rh IFN-g were purchased from Proteintech (Rosemont, IL, USA). The CCK-8 assay kit was purchased from Glpbio (Montclair, CA, USA). Antibodies including PerCP-Cy5.5-conjugated anti-human IFN-y antibody (clone B27), PE-conjugated anti-human CD107a antibody (clone H4A3), GolgiPlug solution, GolgiStop solution, Cytofix / Cy toperm solution, and Perm / Wash buffer were purchased from BD Biosciences (San Jose, CA, USA). Disposable Safety Scalpel, Cell Strainers (both 70 pm and 40 pm), Debris Removal Solution (Cat No. 130-109-398), and Tissue Storage Buffer (Cat No. 130- 100-008) were from Miltenyi Biotec. (San Diego, CA, USA). Lymphoprep (Cat No. 07801) and NK cell isolation kit were ordered from StemCell Technologies (Vancouver, Canada) CountBright™ Sytox-Blue Live / Dead dye, Absolute Counting Beads (Cat No. C36950), neutralization antibody for human CXCL10, and Lipofectamine™ 3000 Transfection Reagent were obtained from Thermo Fisher (New York, NY, USA). Antibodies for patient samples, including FITC-anti-human CD3, FITC-anti-human CD14, FITC-anti-human CD19, BUV396- anti-human CD 16, APC-Cy7 anti -human CD56, BV605-anti -human CD45, PE-Cy7-anti-human CXCR3, B V785 -anti -human NKp46, antibodies for activating receptors phenotyping ofNK cells, including PE-Cy5.5-anti -human CD56, PE-Cy7-anti-human CD3, PE-anti-human NKp46, FITC- anti-human CD 16, BV421 -anti-human NKG2D, APC-anti-human DNAM-1, and other antibodies including PE-anti-human CXCR3, were purchased from Biolegend (San Diego, CA, USA). Transwell® polycarbonate membrane cell culture inserts with a pore size of 5.0 mm (Cat No. CLS3421) were purchased from Millipore Sigma (St. Louis, MI, USA). RetroNectin was ordered from Takara (kusatsu, Japan). Xenolight DiR 750 Fluorescent Cell Labeling Dye (Catalog: 125964) for in vivo study was purchased from Perkin Elmer (Waltham, MA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MI, USA) unless specifically noted otherwise.

[0274] Antibodies including PE-anti-human NKG2D, FITC-anti-human CD 16, BV785-anti- human NKp46, and APC-anti-human 4- IBB, APC-conjugated-anti-His tag antibody (clone J095G46), and recombinant human u-Plasminogen activator (Urokinase) (Cat: #755302) were purchased from BioLegend (San Diego, CA, USA). SYTOX blue dead cell stain, CellTracer Far- Red dye, CellTrace Violet Proliferation Dye, and CFSE were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

[0275] Cells

[0276] The human erythroleukemic cell line K562 was obtained from ATCC and maintained in an IMDM medium supplemented with 10% FBS, 100 U / ml penicillin, and 100 pg / mL streptomycin. Human patient-derived glioblastoma cell line GBM43 cell, Uppsala 87 Malignant Glioma (U87MG) cell line, were kindly provided by Karen Pollok at Indiana University School of Medicine. GBM43 cells were cultured in DMEM medium (without sodium pyruvate) supplemented with 10% FBS and 1% HEPS. U87MG cells were cultured in IMDM medium containing 10% FBS plus 100 U / mL penicillin and 100 pg / mL streptomycin. Murine glioma 261 (GL261) cells were purchased from the National Cancer Institute (Frederick, MD, USA) and cultured in DMEM medium supplemented with 10% FBS.

[0277] Isolation of tumor-infiltrating lymphocytes from GBM patients

[0278] GBM patient tumors and peripheral blood samples were collected by IU Health ECRO Biorepository at IU Health Methodist Hospital under lU’s Institutional Review Board (IRB) protocol #1011004282. Informed consent was obtained from all participating patients before undergoing surgery and procedures of blood retrieval. Fresh peripheral blood from GBM patients was collected in Na heparin tubes and transported to our lab for processing. Whole blood was diluted with an equal volume of 1 *PBS containing 2% FBS and top-layered with the same volume of Lymphoprep solution in 50 mL conical tubes. After centrifugation at 800 g for 30 min at room temperature, the interface between plasma and Lymphoprep solution was collected and washed once with FACS buffer (2% FBS in 1 *PBS). The collected peripheral blood mononuclear cells (PBMCs) were counted and visualized under the microscope.

[0279] Freshly resected human tumor tissue from GBM patients was stored in the tissue storage solution and transported to the lab on the same day. The tumor tissue was washed with 1 *PBS buffer, minced into small pieces using a scalpel, and suspended in digestion buffer containing 1 mg / mL of collagenase IV and 400 pg / mL DNase I. The dissociated tissue mixture was digested for 1 hour at 37°C with agitation. After enzymatic digestion, the mixture was diluted with an equal volume of 1 *PBS and strained using 70 mm cell strainers. After brief centrifugation, the pellet was resuspended in I PBS and passed through a 40 mm cell trainer. After another round of centrifugation, the pellet was resuspended in an appropriate volume of 1 *PBS and mixed with an appropriate volume of debris removal solution, then overlayed with 1 *PBS. The tube was centrifuged at 3,000 g for 10 minutes at 4°C. After centrifugation, the cell pellet on the bottom of the tube was collected and washed once with I PBS to remove the cell debris removal buffer. If necessary, the pellet was resuspended in ACK lysis buffer and incubated at room temperature for 5 minutes to lyse the red blood cells. The isolated cells were washed, then counted, and visualized using the microscope.

[0280] Isolation and culture of primary NK cells from healthy donors

[0281] Primary NK (pNK) cells isolated from healthy donors were obtained under Purdue University's IRB-approved consent forms (IRB-approved protocol #1804020540). Healthy donors were recruited, and the consent forms were collected and signed by each donor before the blood was drawn. Peripheral blood was collected in Na-heparin tubes. NK cells were purified from the blood by negative selection, using the EasySep Direct Human NK cell Isolation Kit (StemCell Technologies). Isolated NK cells were resuspended in the expansion medium, consisting of RPMH640 supplemented with 10% FBS, 1% penicillin / streptomycin and soluble cytokine cocktail including rhIL-2 (500 Unit / mL), rhIL-21(50 ng / mL) and rh4-lBB ligand (50 ng / mL) at a cell density of around 1 million / mL. Meanwhile, mitomycin C-pretreated K562 cells were utilized as feeders. These cells were added into the expansion system at a feeder cell:NK ratio of 1 : 1 on day 0. Every 3 days fresh medium was added into the expansion system. After 7 days of expansion, NK cell purity was measured by staining the cells with anti-human CD3 and anti-human CD56 antibody using flow cytometry. NK cells were ex vivo expanded and used for further experiments for 4-5 weeks.

[0282] CXCL 10-CXCR3 -induced NK cell migration

[0283] NK cell migration induced by chemokines was measured using a Transwell system. To do so, 600 pL of RPMH640 basal medium with and without various concentrations of the chemokine CXCL10 were added to the bottom layer of a 24-well plate Transwell culture system having a membrane pore size of 5 mm. 3* 105NK cells / well were seeded into the upper layer of the Transwell inserts in 100 pL of RPMH640 basal medium and cultured for 4 hours at 37°C. After culture, all of the cells that had migrated into the bottom layer were harvested and washed once with 1 *PBS. The migrated harvested cells were further resuspended in 300 pL of FACS buffer containing diluted Live / Dead dye and mixed with 50 uL of CountBright™ Absolute Counting Beads. Samples were analyzed by flow cytometry. The number of migrated cells was calculated using the equation: Cell number = [assigned bead count of the lot (beads / 50 pL) x (number of live cell events)] / (number of beads events)]. The migration (%) of NK cells induced by the chemokine was calculated using the equation: Migration (%) = 100 x (migrated cell number) / (originally seeded cell number in the upper chamber).

[0284] NKCE design, assembly, and expression

[0285] The synthesis and expression of the NKCEs involved a multi-step process. The design of NKCE constructs in different modular formats included specific chains representing various components of the engager, such as IgG heavy and light chains with variable heavy (VH) and variable light (VL) domains using sequences for antibodies targeting NKp46, IL-13Ra2, and IL- 15N72D. These hybrid genes were then synthesized by Wuxi Biologicals (Wuxi, Jiangsu, China). Specifically, the NKCE represented by format 3 consists of four chains: (i) an IgG heavy chain (CH1-CH2-CH3) with a VH domain derived from a known antibody targeting NKp46 and a flexible linker with a sequence encoding IL-15N72D; (ii) an IgG light chain with a VL domain derived from a reported antibody targeting NKp46; (iii) an IgG heavy chain (CH1-CH2-CH3) with a VH domain derived from a reported antibody targeting IL-13Ra2 and a cleavable linker followed by human CXCL10 and a 6'His tag; (iv) an IgG light chain with a VL domain derived from a reported antibody targeting IL-13Ra2. The IgG Fc domain was modified with the knob- into-hole approach to allow for the correct assembly of the NKCE. A crossMAb was included into the IL-13Ra2 Fab to prevent aggregation. All of these 4 plasmids encoding the various functional domains of NKCEs were co-transfected into ExpiCHO cells at a ratio of 1 : 1 : 1 : 1 for expression and assembly. Following a 7-day incubation, the supernatant containing the expressed NKCEs was harvested, and the target proteins were purified through affinity and size exclusion chromatography for further analysis and application.

[0286] NKCE binding to human NK and GBM cells

[0287] Target cells (primary NK cells, GBM43, and U87MG) were harvested and washed once with lx PBS. NKCE was added to the cells at the required final concentration in 100 pL lx PBS and the cells were allowed to incubate at 4°C for 30 min. After binding, cells were washed twice with lx PBS to remove the unbound protein and stained with APC-conjugated anti-His Tag antibody at 4°C for 30 min in the dark. After washing, the cells were resuspended in FACS buffer containing SYTOX-blue dead cell stain for live / dead staining. The bound APC-anti-His antibody was measured by flow cytometry on a BD Fortessa Cell Analyzer. Data were analyzed using FlowJo software. Target cells (primary NK cells, GBM43, and U87MG) were harvested and washed once with lx PBS. NKCE was added to the cells at the required final concentration in 100 pL lx PBS and the cells were allowed to incubate at 4°C for 30 min. After binding, cells were washed twice with lx PBS to remove the unbound protein and stained with APC-conjugated anti-His Tag antibody at 4°C for 30 min in the dark. After washing, the cells were resuspended in FACS buffer containing SYTOX-blue dead cell stain for live / dead staining. The bound APC-anti-His antibody was measured by flow cytometry on a BD Fortessa Cell Analyzer. Data were analyzed using FlowJo software.

[0288] NKCE-mediated conjugation between NK cells and GBM

[0289] To assess the co-engagement of NK cells and GBM tumor cells induced by the NKCEs, flow cytometry was used. Briefly, NK cells were pre-labeled with Cell Tracer Far-Red dye, while GBM43 and U87MG cells were labeled using CFSE staining according to kit specifications. After labeling, 2* 105NK cells / test were co-cultured with GBM43 or U87MG cells at an E: T ratio of 2: 1 in the presence of NKCE-3 or vehicle control at 4°C for 30 min. Following incubation, cells were harvested and fixed using CytoFix solution at 4°C for 20 min. The conjugation capacity of NKCEs was quantified by measuring double-positive events using a flow cytometer (BD Biosciences, San Jose, CA, USA).

[0290] Cell Tracer-based Proliferation assay

[0291] Freshly isolated NK cells from the peripheral blood of healthy donors were resuspended in 1 'PBS at a density of 1 ' 107cells / mL. Cell Tracer Yellow dye was added to the cells at a 1000' dilution and incubated at RT for 10 min, protected from light. After incubation, the staining process was stopped by adding a 10x volume of complete culture medium to the cells, and the cells were incubated at RT for another 5 min. The cells were then washed twice and resuspended in the complete culture medium at a density of IxlO6cells / mL for 5 min. The recovered prelabeled NK cells were divided into 3 groups: the control group with no supplements; the rhIL-15 group with 50 ng / mL of rhIL- 15 added to the medium; the NKCE group with a final concentration of 3.34 nM of NKCE in the medium, which was equivalent to the concentration of IL-15. The cells were cultured in a 24-well plate in a humidified atmosphere containing 5% CO2 at 37 °C for 7 days. After the 7-day culture, all cells in each well were collected and resuspended in FACS buffer containing S YTOX-Blue Live / Dead dye. The fluorescent intensity of cell tracer yellow was measured by flow cytometry and the data were analyzed using FlowJo software.

[0292] Cytotoxicity assay

[0293] To measure the effect of NKCE-3 on the functional killing ability of NK cells, target tumor cells were pre-seeded into 96-well plate at a density of 2* 104cells per well and allowed to attach overnight. NK cells were added to the wells containing target cells to achieve E: T ratios of 1 : 1, 5: 1, and 10: 1 in the NK expansion medium in the presence of NKCE-3 or not. Following a 4-hour incubation at 37°C, NK cell cytotoxicity was measured using the Cytoscan-LDH assay. Briefly, 50 pL of supernatant from the co-culture plate was mixed with an equal volume of LDH reaction mixture and incubated at 37°C for 30 min, protected from light. After the substrate reaction, 50 pL of stop solution was added to the wells to stop the reaction. The absorbance at 490 nm and 680 nm was measured using a plate reader. Cytotoxicity (%) for each E:T ratio was calculated using the following equation with corrected values: Cytotoxicity (%) = 100 / [(Experimental value - Effector cells spontaneous control - Target cells spontaneous control) / (Target cell maximum control - Target cells spontaneous control).

[0294] Degranulation assay

[0295] To detect NK cell degranulation, CD107a surface staining was performed. Target tumor cells were seeded into a 24-well plate at a density of 3* 105cells per well and allowed to attach overnight. Ex vivo expanded NK cells were added to the wells at an E:T ratio of 1 : 1 for co-culture in the NK cell expansion medium. NK cells, without targets, were included as the negative control. APC-conjugated anti-human CD107a antibody (clone H4A3, BioLegend) was added to each well at a concentration of 5 pL / mL. After 1 hour, monensin (BD GolgiStop™) was added to the cells to inhibit intracellular protein transport. Following an additional 3-hour incubation, the cells were harvested, washed and stained with antibodies including PE-Cy5.5-anti-human NKp46, and APC- anti-human CD107a. Cells were resuspended into the FACS buffer containing Cytox-Blue Live / Dead stain and analyzed by flow cytometry on a BD Fortessa Cell Analyzer (Becton Dickinson). Data were analyzed using FlowJo.

[0296] IFN-g ELISA assay

[0297] Target tumor cells and expanded NK cells were co-cultured as described in the LDH-based cytotoxicity assay. After the coculture, 20 pL of supernatant from each well was collected and stored at -80 °C for further ELISA measurement on the IFN-y level according to the manual. 3D GBM spheroid model and killing assay

[0298] A GBM 3D spheroid model was established using mCherry-engineered GBM43 cells. To form the spheroids, mCherry-GBM43 cells were harvested from the monolayer culture and resuspended in DMEM medium with 10% FBS and 1% MEM.150 pL of cell suspension containing 5000 cells were seeded into a 96-well ultra-low attachment plate and briefly centrifuged at 300g for 3 min. 3-5 days after being cultured in the incubator at 37°C, spheroids had formed and were observable with a size of -300 pm. Once the tumor spheroids had formed, ex vzvo-expanded human NK cells were added into the wells containing the spheroids in the presence of the NKCE in NK cell growth medium (without phenol red). The E:T ratio was 10: 1. The plate was then incubated at 37 °C in the IncuCyte S3 to acquire brightfield and orange field area (indicating mCherry fluorescence) images every 2 hours for 96 hours. Subsequently, the mCherry fluorescence area of the recorded spheroid images was used to quantify NK cell-induced cytotoxicity against 3D GBM cells.

[0299] In vivo GBM mouse models and efficacy studies

[0300] All animal experiments described in this study were approved by the Purdue University Animal Care and Use Committee. Female and male 6- to 8-week-old NOD.Cg- Ragltm !MomIL2rgtm IWjU zS (NRG) mice and C57BL / 6 mice were purchased from Jackson Laboratory and housed at the Purdue Center for Cancer Research.

[0301] The subcutaneous GBM xenograft mice model was used to evaluate the infiltration of adoptively transferred NK cells into tumors and their anti -tumor activity. 3* 106wildtype (WT)- GBM43 or CXCLIO-overexpressing (GBM43CXCL10) cells, were subcutaneously injected into the right flank of NRG mice (6- to 8-week-old, female and male). When tumors reached a volume of approximately 150 mm3, mice were randomly assigned to 5 groups (n = 5 / group). Mice in the NK cell treatment group received intravenous (i.v.) injection of 107ex vzvo-expanded primary NK cells once a week for 3 weeks, alongside rhIL-2 (105units / mouse) by intraperitoneal (i.p.) injection every other day. Mice in the CXCL10 blockade + NK cell treatment group received, in addition to the NK cell treatment as described above, an anti-human CXCL10 neutralization antibody (50 pg / mice / week) administered I.P. one day before the NK cell injection. Mice in the control group received the injection of the same volume of vehicle (1 *PBS). To visualize the recruitment of adoptively transferred NK cells in mice, NK cells were pre-stained with Xenolight dye (320 mg / mL in 1 *PBS) at RT for 30 min before injection. Cell trafficking in mice was measured and recorded by AMI spectrum at various time points. Tumor growth was measured and recorded by caliper measurements and tumor volumes were calculated using the formula: V = 0.52 x Lx W x H. The body weights of the mice were also recorded during the treatment. At the end of the therapy, the mice were sacrificed, and tumors were isolated for histologic analyses.

[0302] Immunocompetent mouse models bearing GL261 tumors were established to investigate the effect of chemokines on immune cell infiltration into tumors. For the subcutaneous GBM model, female C57BL / 6 mice (6-8 weeks) were injected with Luc-GL261 cells (5 x io6 / mouse) into the right flank. After the tumors reached a size of ~50 mm3, mice were randomly divided into 2 groups. Mice in the rmCXCLIO group received an intra-tumoral injection of rmCXCLIO (0.1 mg in 20 mL 1 *PBS per mouse) 3 times a week, while mice in the control group received an intra- tumoral injection of 1 *PBS of the same volume and frequency.

[0303] For the orthotopic intracranial GBM model, Luc-GL261 cells (5 x l05 / mice in 5 mL of 1 *DPBS) were stereotactically implanted in the right forebrain of C57BL / 6 mice (female, 6-8 weeks via a burr hole created with a stereotactic device at a depth of 3.5 mm, as described previously14. After the tumor had been established as confirmed by imaging, mice were randomly divided into 2 groups. Mice in the rmCXCLIO group received an intra-tumoral injection of rmCXCLIO (0.1 mg in 5 pL 1 xDPBS per mouse) once a week. After 2 weeks of treatment, mice were sacrificed, and the blood, spleen, and tumor were harvested and sampled for immune cell phenotyping by flow cytometry and histological analysis.

[0304] To establish intracranial xenograft GBM tumors, GBM43-Luc cells (1 x io5 / mice in 5 mL of 1 xDPBS) were stereotactically implanted into the NRG mice (female, 6-8 weeks) in the right forebrain. After the tumors had formed as confirmed by imaging, mice were randomly divided into 4 groups (n=8 / group). Mice in the NK treatment group received an intra-tumoral injection of 2 x 106ex vzvo-expanded primary NK cells (in 5 pL 1 xDPBS per mouse) once a week for 3 weeks. Mice in the NKCE-3(i.t.) + NK cell treatment group received the same number of NK cells together with 5 pL of NKCE-3 administered intratum orally. Mice in the NKCE-3(i.v.) + NK treatment group received, in addition to NK intracranial injections as above (in 5 pL 1 xDPBS per mouse), intravenously administration of NKCE (in 50 pL 1 xDPBS per mouse) three times a week. The treatment lasted for 3 weeks, and the bioluminescence and body weight of mice were measured twice a week until the mice reached a predetermined endpoint. The survival of the mice was recorded, and tumors were isolated at the endpoint for histologic analyses. Immunohistochemical (IHC) Staining

[0305] Immunohistochemical (IHC) staining was carried out at the Purdue Histology Research Laboratory. Briefly, the harvested tumors from mice were fixed in 10% neutral -buffered formalin, embedded in paraffin, and cut into 3-5 pm sections. To measure the levels of CXCL10 in the tumors and CXCR3+NK cell recruitment, tumor sections were stained with anti-human CXCL10 antibody (1 mg / mL, R&D system), anti-human CXCR3 antibody (MAB160, 10 mg / mL, R&D system), anti-human NKp46 antibody (5 mg / mL, R&D system), respectively before immunohistochemical imaging. Quantification of histological analysis was performed on four random intratum oral sections from each treatment group at 200 x magnification. Positively stained areas were calculated by the Imaged software for each frame and the percentage of positive area over the total area of each frame was used to indicate staining intensity as readouts for expression levels, NK cell activation, or NK cell trafficking. Chemokine secretion profile from GBM cells

[0306] GBM43 cells were seeded into a 6-well plate at a cell density of l >< 106cells / well and cultured until the confluency was around 90%. After attachment, the medium was changed with fresh GBM43 culture medium, and cells were cultured for 48 hrs. The conditioned medium was harvested as a sample to measure the chemokine secretion profile by the human cytokine / chemokine panel discovery assay array (Eve Technologies, Calgary, AB Canada). Proliferation assay

[0307] The effect of chemokines, including CXCL9, CXCL10, and CXCL11, on NK and GBM cells' proliferation was assessed using the CCK-8 assay according to the manufacturer's instructions. Briefly, for GBM43 and U87MG cells, 5000 cells per well were seeded in a 96-well plate with 100 pL of culture medium. 10 pL of various concentrations of chemokine (in I / PBS) were added to appropriate wells to final chemokine concentrations ranging from 0, 0.5, 5, 50, and 500 ng / mL. The cells were incubated for 48 and 72 hours at 37°C with 5% CO2. For primary NK cells, 2.5>< 104cells per well were seeded into a 96-well plate and incubated in the presence of CXCL10 at concentrations of 0, 0.5, 5, 50, and 500 ng / mL for 4 and 24 hours. At pre-determined exposure times, 10 pL of CCK-8 reagent (Glpbio, Montclair, CA, USA) were added to the wells, and the cells were further incubated for 4 hours at 37°C. Absorbance (A) at 450 nm was measured using a microplate reader. Cell viability for each sample was calculated using the formula: Cell viability (%) = [(As - Ab) / (Ac - Ab)] x 100 (%).

[0308] IFN-y intracellular staining assay

[0309] Target tumor cells were seeded into a 24-well plate at a density of 3x l05cells / well and allowed to attach overnight. Expanded NK cells were added to the wells containing target GBM43 cells at an E:T ratio of 1 : 1. NK cells, without targets, were included as the negative control. After 1 hour, brefeldin A (BD GolgiPlug™, final concentration of 0.5 mg / mL) was added into each well to block the intracellular protein transport processes. Cells were incubated for another 3 hours. After a total of 4-hours of co-culture, NK cells were harvested and washed with UPBS. Cells were stained with Live / Dead fixable violet stain on ice for 30 min. After Live / Dead staining, cells were fixed and permeabilized with Cytofix / Cytoperm (BD Biosciences) solution and further stained by APC-anti-human IFN-g antibody on ice for another 30 min. After washing twice with Perm / Wash buffer, the intracellular IFN-g expression levels on NK cells were measured and analyzed by flow cytometry on a BD Fortessa Cell Analyzer (Becton Dickinson). Data were analyzed using FlowJo software.

[0310] Phenotyping of NK cells To assess the effect of chemokines on NK cell activation, flow cytometry was used to analyze the expression of key activating receptors on NK cells. In detail, ex vivo expanded NK cells were subjected to pretreatment with or without CXCL10 at a final concentration of 500 ng / mL for 24 hours. Following the 24-hour pretreatment, NK cells were washed twice with 1 PBS and co-cultured with GBM43 cells at Effector: Target (E: T) ratios of 10: 1 and 2.5: 1 at 37°C for 4 hours in NK expansion medium without chemokine. For each co-culture, 3^ 105 NK cells were seeded per sample. The required number of GBM43 cells were pre-seeded into a tissue culture- treated 24-well plate one day before the co-culture according to the specified E:T ratios. Control samples of NK cells, without target tumor cells, were also included. After the 4-hour incubation, NK cells were harvested and washed twice with 1 xPBS. NK cells were then resuspended in FACS buffer and stained with the following antibodies at 4°C for 30 min: PE-Cy5.5-anti-human CD56, PE-Cy7-anti-human CD3, PE-anti-human NKp46, FITC-anti-human CD16, BV421 -anti-human NKG2D, APC-anti -human DNAM-1. After staining, NK cells were washed and resuspended in FACS buffer and the expression levels of these activating receptors on NK cells were measured and analyzed using a BD Fortessa Cell Analyzer (Becton Dickinson). Data analysis was carried out using Flow Jo.

[0311] Lentivirus packaging and titering

[0312] To produce lentiviral particles for genetic editing, 18 x 106HEK293T cells were initially seeded into cell culture dishes with a 15 cm diameter in 25 mL of culture medium and allowed to attach overnight. After 24 hours, at least 70% of cell confluency was observed. At that point, the medium was changed to culture medium without 1% penicillin / streptomycin for transfection. HEK293T cells were transfected with Lipofectamine 3000 using 2nd generation lentiviral plasmids as follows: 18 pg of transfer plasmid (customized pCMV-CXCLIO / GFP plasmid or CXCR3-shRNA from Vector Builder), 4.5 pg of envelope plasmid (pMD2.G), and 13.5 pg of packaging plasmid (psPAX2). These were mixed well with 72 pL of p3000 reagent in 7 mL of Opti-MEM medium. The mixture was then combined with 54 pL of lipo3000 in 7 mL of Opti- MEM medium. After a 10-minute incubation at room temperature, the transfection mixture was added to cells dropwise. 16 hours post-transfection, the medium was replaced with HEK293T complete culture medium and incubated for an additional 72 hours. Subsequently, the supernatant containing the produced lentivirus was harvested by centrifugation at 500 g for 5 min and filtered through a 0.45 pm pore-sized filter membrane to remove cell debris. To increase the virus titer, 4x lentivirus concentration solution (40% (W / V) of PEG-8000, 1.2M ofNaCl in I xPBS solution) was added to the supernatant and incubated overnight at 4°C with shaking. After centrifugation at 1600xg for 60 min at 4°C, the lentivirus was pelleted, resuspended in 1 *PBS, and stored at -80°C until needed.

[0313] The titer of the harvested lentiviral particles was determined by HEK293T cell transduction. 4* 104cells per well of HEK293T were pre-seeded in a 48-wells plate with complete culture medium. After 24 hours, the harvested lentivirus was diluted into 200 pL of culture medium (withoutl% penicillin / streptomycin) at dilution factors of 10-2, 10-3, 10-4, 10-5, 10-6, and 10-7. The diluted virus was added to the cells in the presence of 8 pg / mL of polybrene and centrifuged at 1000 g for 30 min at room temperature. Non-transduced cells were included as controls. Cells were then incubated at 37°C with 5% CO2 and the medium was replaced with complete culture medium after 24 hours. 72 hours after the transduction, HEK293T cells were detached and harvested for flow cytometry to measure GFP expression. The titer of the lentivirus was calculated using the following equation: Titer (Unit / mL) = Dilution factor x (Total cell number x GFP positive cell percentage) / Volume of virus added).

[0314] Transduction of NK and GBM cells

[0315] To establish GBM cells overexpressing CXCL10, patient-derived GBM43 cells (2x l05cells / well) were pre-seeded into a 6-wells plate to allow for overnight attachment. Lentiviral particles expressing a CXCL10 / GFP gene were added to the cells at a multiplicity of infection (MOI) of 10 in the presence of 8 pg / mL of polybrene. Lentiviral particles expressing only GFP were also included as vector controls. Cells were spun down at 1000 g for 30 min at room temperature and then incubated in a 37°C incubator with 5% CO2. 3 days after the transduction, GBM43 cells were selected with puromycin, added at a final concentration of 5 pg / mL in GBM43 culture medium for 3 days. The puromycin-resistant GBM43 cells were collected and cultured for further study.

[0316] To knock down the expression of chemokine receptor CXCR3 on primary NK cells (pNK cells), we performed a two-round lentiviral transduction. In brief, ex vivo expanded pNK cells were plated in RetroNectin pre-coated 24-well plates at a density of 2x l05cells / well in 200 pL NK expansion medium. Then, the CXCR3 -targeting shRNA lentiviral particles at an MOI of 10 were added into the wells in the presence of 8 pg / mL of protamine sulfate. The plate was centrifuged at 1000 g for 90 min at 32°C to improve the co-localization of lentiviral particles and cells. After overnight incubation in a 37°C incubator with 5% CO2, the cells were harvested and transduced using the same methods. NK cells were then transferred to the NK expansion medium and expanded until they were ready for future use. 3 days after the two-round transduction, some of the NK cells were harvested and stained with PE-anti-human CXCR3 antibody to measure the transduction efficiency. uPA Cleavage assay

[0317] To validate the cleavage of CXCL10 via the cleavable linker presented within the NKCE structures, GBM43 cells were incubated in the presence of NKCE at a final concentration of 183 nM at 4°C for 30 min to allow for binding. After washing twice, urokinase plasminogen activator (uPA) was added to the cells at concentrations of 100 nM or 400 nM in 1 *PBS and incubated at RT for 1 hour. During the incubation, the mixture was gently mixed every 20 min. After incubation and washing, cells were stained with APC-anti-His antibody at 4°C for an additional 30 min. Cell samples were then harvested and analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA). Flow cytometric analysis was carried out using FlowJo vlO (FlowJo LLC., Ashland, OR, USA).

[0318] Statistical analysis

[0319] Data were presented as mean ± SEM. Statistical analyses were conducted using GraphPad Prism version 10.0.2 (GraphPad Prism Software, Inc., La Jolla, CA). A p-value of 0.05 was considered statistically significant in all studies. A comparison between two normally distributed test groups was performed using the two-tailed Student’s t-test. For the analysis of three or more groups, the comparison was performed using a one-way ANOVA analysis. p< 0.05 was considered to be statistically significant. *p < 0.05, **p< 0.01. ***p< 0.001.

[0320] Results

[0321] CXCR3-CXCL10 is a target to improve NK cell infiltration into GBM

[0322] Chemokines, alongside their receptors, guide leukocyte infiltration into solid tumors. To identify potential chemokine interactions contributing to NK cell recruitment to GBM tumors, chemokine receptor CXCR3 expression levels on NK cells from GBM patients was measured using freshly surgically resected tumors and healthy donors via multi-color flow cytometry (Figures 1A&B). Peripheral NK cells from healthy donors, in a resting state without activation, exhibited low expression of CXCR3. Despite 43.29% of the NK cell population being CXCR3+, the MFI was low (MFI=1370). Interestingly, NK cells in the peripheral blood of GBM patients showed similar CXCR3 expression. However, the CXCR3 levels on NK cells from healthy donors were significantly higher at 69.63% (MFI=8693) after one week of ex vivo expansion stimulated by feeder cells and cytokines (IL-2 / IL-21 / 4-1BBL). Moreover, a comparison between tumorinfiltrating and peripheral NK cells from GBM patients, indicated that, despite GBM patients having a lower percentage of tumor-infiltrating than peripheral CXCR3+NK cells (42.19% to 18.11%), the MFI on tumor-infiltrating CXCR3+NK cells was significantly higher (3609 vs. 990.9). The higher CXCR3 expression on tumor-infiltrating NK cells points to a correlation between CXCR3 expression and NK cell infiltration into GBM tumors. Additionally, CXCR3 expression remained upregulated on activated NK cells even after co-culture with tumor cells (Figure 7).

[0323] Analysis of GBM patient data using TIMER2.0 (http: / / timer.cistrome.org / ) (Figure 1C) indicated a positive correlation between CXCL10 (one of the CXCR3 ligands) concentration and multiple anti-tumor immune cells, to not only include NK cells, but also CD8+T cells, neutrophils, and Ml macrophages. However, despite its overexpression in GBM over normal tissues, the CXCR3 ligand CXCL10 was secreted at low levels by GBM cells (Figures 1D-1F), demonstrating that the paucity of CXCL10 in the tumor could be disrupting NK cell migration via the CXCR3 axis. These data suggest that locally increasing CXCL10 in tumors which naturally lack this chemokine could be beneficial to enhance NK cell recruitment and infiltration into GBM via CXCR3.

[0324] Exogenous CXCL10 induces NK cell migration but does not affect its function in vitro

[0325] To evaluate the functional consequence of CXCL10-CXCR3 interactions on NK cells, the chemotaxis of NK cells in response to exogenous CXCL10 was measured in vitro. Exogeneous CXCL10 was able to induce the migration of ex vivo expanded NK cells, with a concentration of 500 ng / mL of CXCL10 able to significantly induce NK cell chemotaxis (Figure 2C). Given the difference in expression of CXCR3 between freshly isolated NK cells and ex vivo expanded NK cells (Figure 2A), the migration of NK cells induced by exogenous CXCL10 was measured using both cell populations. In the presence of the same concentration of CXCL10, ex vivo expanded NK cells, characterized by high CXCR3 expression, exerted a more potent trafficking ability than freshly isolated NK cells (41.41% vs 9.405%, Figure 2B). At the same time, CXCR3 knockdown on NK cells disabled their chemotaxis induced by exogenous CXCL10 (Figure 2D). These CXCR3KDNK cells showed a similar proliferative and cytotoxic capacity against GBM43 cells (Figure 7) as expanded NK cells from the same donor. Collectively, these data show that exogenous CXCL10 induces NK cell migration via CXCR3 expression on NK cells.

[0326] Additionally, the presence of CXCL10 did not exert a detrimental effect on NK cell viability and proliferation in the presence of cytokines (Figure 2E). In the absence of cytokines, exogenous CXCL10 both activated NK cells and stimulated their proliferation (Figure 7). Moreover, pretreatment of NK cells with CXCL10 for 24-hour did not affect either the expression of activating receptors on NK cells, including NKG2D, NKp46, CD16, and DNAM-1 (Figure 7), nor IFN-g / CD107A expression on NK cells or their cytotoxic capacity against GBM (Figures 2F- 2H). Thus, it was observed that exogenous CXCL10 does not affect the activation and function of NK cells but induces NK cell migration in response to CXCR3. High expression of CXCL10 induces NK cell infiltration into GBM in vivo

[0327] To evaluate the effect of CXCL10 on the infiltration and anti -tumor activity of NK cells in vivo, wild-type (IF7)-GBM43 or CXCL10 overexpressing-GBM43 (GBM43CXCL10) cells were subcutaneously injected into the right flank of immunodeficient mice (Figure 3 A). Comparable tumor growth rates (Figure 3C) and body weights (data not shown) were observed in mice injected with either WT GBM43 or GBM43CXCL10cells, indicating that increased levels of rhCXCLIO (confirmed by IHC staining, Figure 3B) in the tumor did not promote tumor growth in vivo, aligning with the in vitro data showing no effect of CXCL10 on tumor cell proliferation. (Figure 8).

[0328] The effect of CXCL10 on NK cell infiltration into the tumor was measured by labeling NK cells and tracking their in vivo recruitment via the AMI spectrum. Mice with increased levels of CXCL10 showed significantly more NK cells colocalized in the tumors 48 hours post-injection, and these NK cells persisted for 7 days (Figure 3D). Blockade of CXCL10 engagement using a neutralizing antibody injected before NK cell administration significantly suppressed NK cell recruitment. These data were further confirmed by IHC staining of isolated tumor samples (NKp46, Figure 3E). However, no significant difference was observed in terms of tumor growth or survival among these groups, despite the fact that increased CXCL10 leads to more NK cells infiltrating the tumor.

[0329] It was also observed that high levels of CXCL10 in the tumors altered the immune cell constituency in an immunocompetent C57BL / 6 mouse model bearing GL261 glioma tumors (Figures 3H and 31). GBM tumors with increased levels of CXCL10 showed higher leukocyte recruitment, including NK1.1+cells, CD3+cells, CD11C+ / HLA-DR+cells, while fewer neutrophils (Ly6G+CDl lb+) and MDSCs (Ly6G+Ly6C+ / CD45+) were detected. These tumorinfiltrating NK1.1+cells expressed elevated levels of IFN-g and CD69, and lower levels of exhaustion makers PD-1, LAG-3, and NKG2A, suggesting NK cells in GBM tumors to be both activated and exhausted. Taken together, the in vivo data showed that locally increased CXCL10 concentrations in GBM tumors induced more NK cell trafficking and infiltration, although elevated NK cell presence did not lead to significant improvement in tumor control, possibly because of the exhausted nature of NK cells inside the tumor.

[0330] Given that CXCL10 is known to be induced by IFN-y and activated NK cells release abundant IFN-y upon stimulation, it was hypothesized that there exists a CXCL 10 -related positive feedback loop between NK cells and GBM cells. CXCL10 production significantly increased in both GBM43 and U87MG cells after 24 hours of stimulation with IFN-y in a dose-dependent manner (Figure 9). Additionally, the data revealed that NK cells alone, without tumor cells, induced weak CXCL10 production in GBM43 cells via the spontaneous release of IFN-y. However, upon direct cell-cell contact by co-culturing NK cells with tumor cells, NK cells induced a significant increase in CXCL10 production by GBM43 cells. Furthermore, the observed enhancement in CXCL10 production was completely abolished when IFN-y signaling was blocked by adding a neutralizing antibody to the medium. This evidence suggests the presence of positive feedback between NK and tumor cells via CXCL10. In summary, increased CXCL10 at tumor sites leads to NK cell recruitment to the tumor, activation by tumor cells, IFN-y release, and subsequent promotion of CXCL10 production by tumor cells, potentially inducing further immune cell infiltration.

[0331] Multifunctional NKCEs can be expressed and successfully engage NK and GBM cells

[0332] To specifically deliver CXCL10 to GBM, which lacks this chemokine ligand, in order to improve NK cell infiltration while preserving NK cell anti-tumor activity in the immunosuppressive TME, a novel multi-functional NKCE was designed and generated with four functional moieties: one module targeting the NK cell activating receptor NKp46, a second domain targeting the GBM-associated antigen IL-13Ra2, the cytokine IL-15 carrying the N72D mutation to facilitate the persistence of NK cells, and a CXCLIO-releasing moiety to promote the recruitment of NK and other immune cells to tumor sites. NKp46 was selected as the NK-binding receptor owing to the NK cells’ ability to sustain its expression both after co-culture with GBM target cells in vitro as well as on tumor-infiltrating NK cells from GBM patients (Figure 10). In addition, IL-13Ra2, a clinically validated GBM-associated antigen, was measured to be highly and persistently expressed in different types of GBM, including patient-derived primary adult (GBM43), pediatric (SJ-GBM2), and recurrent adult (GBM10) brain tumor cells (Figure 11).

[0333] In pursuit of optimal expression and purification of the NKCEs bearing multiple functional domains, the engager was designed and assembled in various modular formats (Figures 4A and 4B) to enhance protein expression and stability. The antigen-binding component of the NKCE was assembled in modular variants consisting of a scFv, a Fab, or a Fab with cross-mAb. In certain compositions, an IgGl-like Fc domain was also included in the overall structure to promote expression, folding, and stability. These building blocks were then used to assemble the various NKCE formats. (Figure 4B). All of these NKCEs were successfully expressed in a CHO-S expression system with varying yields and binding capacities.

[0334] The binding ability of these NKCEs to their target cells was evaluated using a flow cytometry -based cell binding assay in vitro and it was found that NKCE Format 3, termed NKCE- 3, exhibited superior binding to its target cells with an ECso at 7.9 nM for NK cells, 0.445 nM for U87MG cells, and 106.2 nM for GBM43 cells (Figures 4C and 4D). This was significantly higher than the binding of the other NKCE formats to the same target cells. In light of these results, NKCE-3 was selected for further characterization and functional studies.

[0335] Tetraval ent NKCEs can promote the trafficking of NK cells to GBM via the triggered release of CXCL10

[0336] The effect of NKCE-3 on NK cell proliferation and survival was measured using freshly isolated peripheral NK cells from healthy donors (Figure 4E and 4F). As predicted, NKCE-3 induced robust proliferation of NK cells from multiple donors (Figure 4E). In addition, the presence of NKCE-3 maintained NK cell viability, comparable to that induced by IL- 15 alone (53.70% vs 49.5%), while NK cells in the control group without any cytokine yielded only 9.38% viable cells (Figure 4F). Taken together, these data indicate that NKCE-3 incorporating the IL15N72D domain can induce NK cell proliferation and survival in vitro.

[0337] Within the structure of NKCE-3, a CXCL10 element with a cleavable linker was incorporated to induce the recruitment of NK cells by releasing CXCL10 locally in the TME. The linker is protease-sensitive and is cleaved in settings of upregulated TME protease activity. Consistent with the previous data, the ability of NKCE-3 to release CXCL10 induced potent migration of NK cells (Figure 4H) Compared to the control group without chemokine, a relative 7.96-fold increase in NK cells migrating toward NKCE-3 was observed. In addition, the release of CXCL10 via the cleavable linker was validated by Western Blot. After incubation with the protease, the released CXCL10 was detected successfully (Figure 4G), confirming the functionality of the NKCE’s ability to controllably release the chemokine ligand.

[0338] The NKCE-3 molecule containing the NKp46 moiety is endowed with the ability to activate NK cells. To assess whether NKCE-3 was able to prime NK cells, naive NK cells isolated from healthy donors were pretreated with NKCE-3 for 24 hours and then co-cultured with target tumor cells (Figures 41 and 4J, Figure 12A). Cytotoxicity assays showed that NKCE-3 pretreatment dramatically activates the killing capacity of NK cells against GBM cells, which were highly resistant to the killing of control NK cells in the resting state even at high E:T ratios. In addition to the direct cytotoxicity, IFN-y release from these NK cells in the presence of GBM cells was also measured. While resting NK cells showed minimal spontaneous IFN-g release, NKCE-3 pretreatment induced the activation of NK cells, resulting in a significant increase in IFN-y production. These NKCE-3 -activated NK cells were able to exert enhanced ant-tumor activity when cocultured with GBM target cells and release higher amounts of IFN-g. Both the higher killing ability and the higher production of IFN-g demonstrated that NKCE-3 exhibited a superior capacity to activate NK cells and thereby enhance their anti -tumor activity against GBM. NKCE-3 enhances NK cell anti-tumor activity in vitro

[0339] NKCE-3 binds to NK and GBM cells and induces co-engagement

[0340] Beyond the ability to bind to target cells alone, NKCE-3 was able to bind to NK cells and tumor cells simultaneously to tether them together, leading to the formation of immune synapses. To assess the co-engagement capacity of NKCE-3 upon co-culture of NK cells and GBM cells, double-positive populations of NK and GBM cells were measured after coculture of pre-labeled cells in the presence or absence of NKCE-3. The presence of NKCE-3 significantly increased the frequency of engaged NK cells and tumor cells (GBM43 cells, from 7.64% to 12.28% and U87MG cells from 8.42% to 10.05%; Figure 5A, Figure 12B), suggesting more co-engagement between NK cells and GBM tumor cells to be facilitated by NKCE-3.

[0341] NKCE-3 induces NK cell cytotoxicity against GBM

[0342] The effect of NKCE-3 on the enhancement of NK cell cytotoxicity against GBM cells was determined (Figure 5B). Strong tumor cell killing was induced by NKCE-3 both against GBM43 and U87MG cells at various E:T ratios (Figure 5B). Consistent with the IL-13Ra2 expression profile and the cell binding results, U87MG cells exhibited more potent cell death in the presence of NKCE-3 than patient-derived GBM43 cells.

[0343] NKCE-3 increases IFN-g release from NK cell against GBM more strongly than GBM exposure alone

[0344] IFN-g release was also measured from NK cells induced by NKCE-3 either alone or after exposure to target cells in a GBM co-culture system. Through its capacity to activate NK cells, NKCE-3 alone was able to stimulate NK cells to produce high amounts of IFN-g (Figure 5C) to a greater extent than NK cells in the presence of GBM cells but without engager. More strikingly, when NK cells were cocultured with both target tumor cells and NKCE-3, the release of IFN-g was even greater. The observed ability of NKCE-3 alone to stimulate production of IFN-g by NK cells was even more potent than exposure to GBM tumor cells, suggesting that NKCE-3 enhances NK cell activity substantively via the release of IFN-g.

[0345] NKCE-3 enhances NK cell lytic degranulation

[0346] Upon activation, the lytic degranulation of NK cells was also measured by CD 107a expression (Figure 5D). Target cells themselves, in the absence of any NKCE, induced significantly elevated degranulation of NK cells (CD107a+cells: GBM43 from 8.34% to 46.76%; U87MG from 8.35% to 26.50%). NKCE-3 alone showed a similar extent of induction of lytic degranulation on NK cells as did target cells. However, in the presence tumor cells, the addition of NKCE-3 in the co-culture system further enhanced the percentage of CD107a+NK cells (GBM43: 46.76% vs 58.97%, U87MG: 26.50% vs 43.07%). NKCE-3 improves the cytotoxicity ofNK cells in a 3D GBM tumor spheroid model

[0347] The cytotoxicity of NK cells in the presence of NKCE-3 was also evaluated using a 3D spheroid model (Figures 5E and 5F). mCherry-engineered GBM43 cells were used to establish 3D spheroids. After spheroid establishment, ex vzvo-expanded human NK cells were added to the wells containing the mCherry-GBM43 spheroids in the presence of NKCE-3. The killing of GBM by NK cells resulted in substantial tumor cell lysis, which was detected as a decrease in mCherry fluorescence. NKCE-3 significantly enhanced the anti-tumor activity of NK cells against GBM in the 3D spheroid model (Figure 5E). After 3 -days of coculture, the structure of the tumor spheroids collapsed, and the GBM cells were fully eliminated by NK cells in the groups treated with NKCE- 3 (both at low and high concentrations).

[0348] NKCE-3 induces NK cell anti-tumor activity against GBM in vivo

[0349] To evaluate the anti-tumor activity of the novel NKCE-3 in vivo, a xenograft orthotopic intracranial model of GBM in NSG mice was established. In detail, patient-derived GBM43 cells were genetically engineered to express a firefly luciferase gene to enable active monitoring of tumor progression throughout the study. To establish the orthotopic mouse model, NSG mice were first implanted intracranially with 1 * 105GBM43 -luciferase cells, then subsequently received 3 weekly injections of 2 * 106ex vivo expanded human NK cells, also intracranially (Figure 6A). Meanwhile, NKCE-3 was administrated to mice either via intracranial injection once a week via pre-mixing with NK cells (i.t.) or via intravenous injection (i.v.) 3 times a week. Tumor size and body weight were monitored throughout the study (Figures 6B-6E). Mice receiving expanded NK cells alone did not show significant tumor suppression, which highlights the deficiency in NK cells’ ability to control GBM growth alone. On the other hand, local NK cell treatment in combination with NKCE-3, wherein NKCE-3 was pre-mixed with NK cells, and both were injected intracranially into the mice, was able to reduce the tumor growth after repeated intracranial injections. Surprisingly, mice receiving NK cells locally alongside NKCE-3 intravenously recorded a drastic reduction in tumor growth, when compared with the PBS control group or mice receiving NK cells alone (Figures 6C and 6D). A survival benefit was also observed for mice treated with local NK cell-NKCE-3 combination immunotherapy or systemic NKCE-3 with local NK cell infusions. To further explore the potential mechanism of action, whole brain samples from mice were isolated at the endpoint and stained for human CXCL10 and granzyme B. Mice receiving NK cell injections, regardless of NKCE-3 treatment or not, were found to have increased levels of CXCL10 within the tumor (Figure 6F), which aligns with the data showing positive cooperation between NK cell trafficking and CXCL10. In addition, granzyme B staining showed functional activation of trafficked NK cells. In that respect, it was found that mice receiving NKCE-3 treatment, both intracranially and intravenously, showed the highest levels of granzyme B, indicating that in both treatment scenarios, NKCE-3 could reach the brain via systemic routes and, when administered alongside NK cells, maintain their functional activity and, in turn, enhance the efficacy of NK cell-based therapy against GBM. Overall, these data show that NKCE-3 can induce NK cell anti-tumor activity against GBM in vivo both locally and systemically and provides a therapeutic strategy for GBM immunotherapy via NK cell recruitment.

[0350] Discussion

[0351] An inherent dilemma constraining the efficacy of NK cell therapy against solid tumors is the extremely low infiltration of NK cells after adoptive transfer, especially in brain tumors like GBM, despite their presence associating with improved overall survival.15Clinical studies have shown promising anti-tumor activity of NK cell therapy in GBM patients; however, minimal NK cells are recruited to tumor sites after transplantation.16,17Indeed, NK cell recruitment to GBM is low compared to that of other immune cells.18Although chemokine receptor-ligand gradients can support immune recruitment, aberrant expression of one or both results in disrupted migratory capacity of immune cells in GBM.19By analyzing GBM patient tumor samples, it was found that high expression of CXCR3 on virtually all activated NK cells, including those in tumors, was matched by low levels of cognate CXC chemokine ligand CXCL10. The contribution of CXCL10 to the trafficking of NK cells into GBM was highlighted in previous work.14These analyses point to the fact that the mismatch between chemokine receptors and chemokine ligands in tumors, rather than the lack of their expression, may partly explain the low infiltration of NK cells into tumors. Given the high expression of CXCR3 on ex vivo expanded NK cells, but a low expression of CXCL10 in GBM itself, a feasible strategy to enhance NK cell recruitment would be to increase CXCL10 concentration locally at tumor sites which otherwise lack this ligand. This strategy not only enhances the chemokine gradient favorable to NK cell recruitment but also avoids potential side effects associated with systemic CXCL10 administration.

[0352] However, of relevance to the ability of infiltrative NK cells to sustain therapeutic activity in GBM, increased NK cell infiltration alone did not translate into improved overall NK antitumor activity. This aligns with notions of a highly immunosuppressive tumor microenvironment, which can contribute to NK cell dysfunction even when more NK cells reach the tumor.20The GBM TME is characterized by the presence of multiple immunosuppressive elements within a hypoxic / acidic physical environment replete with secreted metabolites (TGF-P, PGE2, adenosine) and immune suppressor cells, such as regulatory T cells and MDSCs.21These factors induce functional impairment of NK cells, leading to downregulation of activating receptors, up- regulation of inhibitory receptors, and alterations in immune cell metabolism.22Increasing the number of infiltrated cells is essential but insufficient to enhance the efficacy of NK cell-based immunotherapy against GBM. Targeting multiple mechanisms to improve both NK cell migration and resistance to the TME may be a more effective approach.

[0353] NKCEs can redirect NK cells to tumors by simultaneously binding to NK activating receptors on NK cells and tumor-associated antigens on tumor cells, serving as a bridge to tether them together to form immune synapses.23,24Such local tethering can stimulate NK cells and maintain their activation status in the tumor through functional domains engineered within the engager structure. To date, the majority of the NKCEs under development have been designed with 2 or 3 functional domains with the aim of activating NK cells and recognizing tumor cells in the form of linked scFvs, VHH or antibodies with or without cytokine, as well as Fabs, nanoengagers and linked peptides.9 25 27Immune recruitment and retention in GBM can be achieved by restoring CXCL10 levels in the tumor and thus the balance between CXC3- CXCL10.28The tetra-domain NKCE-3 with an IgG-like bispecific antibody targeting NKp46 and IL-13Ra2 with a modified IL15N72D29described in this study recruits NK cells via a locally cleavable CXCL10. Upon binding to tumors, the NKCE undergoes cleavage of the linker between CXCL10 and the other domains by a protease highly expressed in the TME, resulting in the release of CXCL10 at the tumor site.14The increased concentration of CXCL10 in the tumor is then sensed by peripheral NK cells — and potentially other immune cells — expressing high amounts of its receptor, CXCR3, thereby inducing their trafficking and recruitment to the tumor. In comparison to other methods that could specifically deliver CXCL10 locally to the tumor, NKCEs are capable of redirecting NK cells and sustaining their activation through the targeting of activating receptors in the immunosuppressive TME, thus avoiding recruitment-associated dysfunction seen with tumor-infiltrating NK cells. Moreover, this is the first multi-functional NKCE with a chemokine domain designed to address the challenge of insufficient NK and immune cell infiltration into solid tumors.

[0354] In addition to facilitating the expression and productivity of the NKCE, the IgG-like scaffold within the NKCE structure offers several advantages compared to other classic BiKE or TriKE with scFv domains. NK cells can induce target cell lysis through ADCC, a process in which NK cells recognize the Fc domain via CD16 expression, boosting their activation and cytotoxicity.30The incorporation of the Fc domain in our NKCE empowers NK cells with an additional activating signal beyond NKp46 engagement, allowing for co-targeting of NKp46 and CD16.31,32This equips NK cells with multiple and robust activation signals in the TME, which in turn, sustains the anti-tumor activity of NK cells against the tumors. Moreover, the IgG-like scaffold provides the NKCE with a bigger molecular weight, potentially enhancing its pharmacologic properties, such as an extended half-life in vivo.

[0355] Given the high expression of CXCR3 on activated NK cells but a lack of CXCL10 in GBM, increasing CXCL10 at tumor sites facilitated NK cell recruitment to GBM. Such local production of chemokine restored favorable chemokine levels and thus re-enabled disrupted interaction with cognate receptors on immune cells to occur. This multifunctional NKCE redirected NK cells, as well as other immune cells in the tumor’s proximity, toward GBM cells, allowing for immune and effector engagement, while the modified IL- 15 mediated the sustained survival and proliferation of NK cells. Evidence was provided that it is feasible and practical to target multiple signals simultaneously via the novel NKCE with various functional components to significantly improve the efficacy of NK cell-based immunotherapies. Interestingly, anti-tumor effects were observed both upon intracranial and systemic administration of the NKCE, suggesting that migration to the brain via systemic routes is feasible.

[0356] The present study reports the design, generation, and functional analysis of a novel CXCLIO-releasing NKCE with multi-functional domains for the treatment of GBM. The data showed that this novel NKCE facilitates NK cell recruitment via the locally increased CXCL10 concentration in the tumor, sustains NK cell proliferation and persistence, and directs NK cell- mediated cytotoxicity toward IL-13Ra2-positive GBM tumor cells, and consequently, augments the anti-tumor activity of NK cell-based immunotherapy against solid tumors. The study serves to illustrate that the delivery of CXCL10 in GBM tumors by a functional NKCE has the potential to restore local chemokine levels in the TME, and, in turn, increase NK cell recruitment and, ultimately, enhance resistance to the immunosuppressive TME through the targeting of multiple signals. The findings highlight the use of this novel NKCE with a CXCLIO-releasing domain and cytokine, as a potent anti-tumor therapeutic strategy for the treatment of cancer.

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[0372] 18. Kmiecik, J., Zimmer, J. & Chekenya, M. Natural killer cells in intracranial neoplasms: presence and therapeutic efficacy against brain tumours. J Neurooncol 116, 1-9 (2014). 19. Lachota, M. et al. Mapping the chemotactic landscape in NK cells reveals subset-specific synergistic migratory responses to dual chemokine receptor ligation. eBioMedicine 96, (2023).

[0373] 20. Zhang, W., Zhao, Z. & Li, F. Natural killer cell dysfunction in cancer and new strategies to utilize NK cell potential for cancer immunotherapy. Molecular Immunology 144, 58-70 (2022).

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[0375] 22. Shaim, H. etal. Targeting the av integrin / TGF-P axis improves natural killer cell function against glioblastoma stem cells. J Clin Invest 131, (2021).

[0376] 23. Pinto, S., Pahl, J., Schottelius, A., Carter, P. J. & Koch, J. Reimagining antibody-dependent

[0377] 10 cellular cytotoxicity in cancer: the potential of natural killer cell engagers. Trends in Immunology 43, 932-946 (2022).

[0378] 24. Demaria, O., Gauthier, L., Debroas, G. & Vivier, E. Natural killer cell engagers in cancer immunotherapy: Next generation of immuno-oncology treatments. European Journal of Immunology 51, 1934-1942 (2021).

[0379] 1525. Gleason, M. K. et al. CD16xCD33 bispecific killer cell engager (BiKE) activates NK cells against primary MDS and MDSC CD33+ targets. Blood 123, 3016-3026 (2014).

[0380] 26. Peipp, M. et al. HER2-specific immunoligands engaging NKp30 or NKp80 trigger NK-cell- mediated lysis of tumor cells and enhance antibody-dependent cell-mediated cytotoxicity. Oncotarget 6, 32075-32088 (2015).

[0381] 2027. Au, K. M., Park, S. I. & Wang, A. Z. Trispecific natural killer cell nanoengagers for targeted chemoimmunotherapy. Sci Adv 6, eaba8564 (2020).

[0382] 28. Wennerberg, E., Kremer, V., Childs, R. & Lundqvist, A. CXCLIO-induced migration of adoptively transferred human natural killer cells toward solid tumors causes regression of tumor growth in vivo. Cancer Immunol Immunother 64, 225-235 (2015).

[0383] 2529. Fousek, K. et al. An Interleukin- 15 Superagonist Enables Antitumor Efficacy of Natural Killer Cells Against All Molecular Variants of SCLC. Journal of Thoracic Oncology 18, 350-368 (2023).

[0384] 30. Nigro, C. Lo et al. NK-mediated antibody-dependent cell-mediated cytotoxicity in solid tumors: biological evidence and clinical perspectives. Annals of Translational Medicine 7, 105-105

[0385] 30 (2019).

[0386] 31. Gauthier, L. et al. Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity. Cell 177, 1701-1713. el6 (2019). Nikkhoi, S. K. et al. Bispecific immune cell engager enhances the anticancer activity of CD16+ NK cells and macrophages in vitro, and eliminates cancer metastasis in NK humanized NOG mice. J Immunother Cancer 12, e008295 (2024). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention.

[0387] All publications, patents, and patent applications, Genbank / accession numbers sequences, websites and other published materials referred to throughout the disclosure herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application, sequences, websites and other published materials was specifically and individually indicated to be incorporated by reference. In the event that the definition of a term incorporated by reference conflicts with a term defined herein, this specification shall control.

Claims

WHAT IS CLAIMED IS:

1. A multispecific polypeptide comprising i) an immune cell targeting moiety; ii) a tumor targeting moiety; iii) a cytokine molecule; and iv) a chemokine.

2. The polypeptide of claim 1, wherein the targeting moiety is an antibody, a ligand, or a receptor that binds to i) or ii).

3. The polypeptide of claim 1 or 2, wherein the immune cell targeting moiety is selected from a Natural Killer (NK) cell targeting moiety, a T cell targeting moiety, a B cell targeting moiety, a dendritic cell targeting moiety, or a macrophage cell targeting moiety.

4. The polypeptide of claim 2 or 3, wherein the NK cell targeting moiety targets one or more of NKp30, NKp40, NKp44, NKp46, KLRK1 / CD214 / NKG2D, DNAM1, DAP10, CD16a / FCGR3A, CD16b, CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160.

5. The polypeptide of any one of claims 2 to 4, wherein the NK cell targeting moiety targets NKp46.

6. The polypeptide of any one of claims 2 to 4, wherein the NK cell targeting moiety targets KLRK 1 / CD214 / NKG2D .

7. The polypeptide of any one of claims 2 to 4, wherein the NK cell targeting moiety targets CD16a / FCGR3A.

8. The polypeptide of claim 2 or 3, wherein the T cell targeting moiety targets one or more of CD3, TCRa, TCRP, TCRy, TCR^, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226.

9. The polypeptide of any one of claims 1-8, wherein the tumor targeting moiety binds to a cancer antigen present on a hematological cancer, a solid tumor, a metastatic cancer, soft tissue tumor, metastatic lesion, or a combination thereof.

10. The polypeptide of any one of claims 1-8, wherein the tumor targeting moiety binds to a cancer antigen on pancreatic, breast, colorectal, lung, skin, ovarian, glioblastoma, or liver cancer cells.

11. The polypeptide of any one of claims 1 to 10, wherein the tumor targeting moiety targets one or more of PDL1, CD47, mesothelin, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium- activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, IL-13Ra2, Telomerase, SAP-1, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, TRP-1 / -2, MC1R, P-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, P- catenin, CDK4, CDC27, CD47, a actinin-4, TRPl / gp75, TRP2, gplOO, Melan-A / MARTl, gangliosides, WT1, EphA3, Epidermal growth factor receptor (EGFR), CD20, MART -2, MART- 1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUI2, SAGE, TRG, TRP1, TSTA, Folate receptor alpha, LI -CAM, CAIX, EGFRvIII, gpA33, GD3, GM2, VEGFR, Intergrins (Integrin alphaVbeta3, Integrin alpha5Betal), Carbohydrates (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, carbonic anhydrase IX (CA9), carbonic anhydrase 12 (CA12), glucose transporter type 1 (GLUTl / solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)) or RANKL.

12. The polypeptide of any one of claims 1 to 11, wherein the tumor targeting moiety targets IL-13Ra2.

13. The polypeptide of any one of claims 1 to 11, wherein the tumor targeting moiety targets CA9.

14. The polypeptide of any one of claims 1 to 11, wherein the tumor targeting moiety targets15. The polypeptide of any one of claims 1 to 11, wherein the tumor targeting moiety targets glucose transporter type 1 (GLUTl / solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)).

16. The polypeptide of any one of claims 1 to 15, wherein the cytokine is selected from GM- CSF, IL-la, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-a, IFN-0, IFN-y, MIP-la, MIP-ip, TGF-P, TNF-a, TNFP or a mutation thereof.

17. The polypeptide of any one of claims 1 to 16, wherein the cytokine is interleukin- 15 (IL- 15) or mutation thereof.

18. The polypeptide of any one of claims 1 to 17, wherein the chemokine is selected from CCL1, CCL2, CCL3 (MIP-la), CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, XCL1, XCL2, or C S3 CL 1.

19. The polypeptide of any one of claims 1 to 18, wherein the chemokine is CXCL10.

20. The polypeptide of any one of claims 1 to 19, wherein the chemokine is joined by a tumor protease-sensitive linker.

21. The polypeptide of claim 20, wherein the linker is a urokinase plasminogen activator (uPA) or a matrix metalloproteinase (MMP).

22. The polypeptide of claim 21, wherein the MMP is selected from MMP-1, MMP-2, MMP- 3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-21, MMP-23, MMP-23A, MMP-23B, MMP-24, MMP-25, MMP -26, MMP-27 orMMP-28.

23. The polypeptide of any one of claims 1 to 22 containing any combination of at least one of each: i) an immune cell targeting moiety targeting KLRK1 / CD314 or CD16 / FCGR3A and ii) a tumor cell targeting moiety targeting CA9, CA12, or GLUT1 / SLC2A1.

24. The polypeptide of claim 23, wherein the immune cell targeting moiety binds KLRK1 / CD314 and the tumor targeting moiety binds CA9.

25. The polypeptide of claim 23, wherein the immune cell targeting moiety binds KLRK1 / CD314 and the tumor targeting moiety binds CA12.

26. The polypeptide of claim 23, wherein the immune cell targeting moiety binds KLRK1 / CD314 and the tumor targeting moiety binds GLUT1 / SLC2A1.

27. The polypeptide of claim 23, wherein the immune cell targeting moiety binds CD16 / FCGR3A and the tumor targeting moiety binds CA9.

28. The polypeptide of claim 23, wherein the immune cell targeting moiety binds CD16 / FCGR3A and the tumor targeting moiety binds CA12.

29. The polypeptide of claim 23, wherein the immune cell targeting moiety binds CD16 / FCGR3A and the tumor targeting moiety binds GLUT1 / SLC2A1.

30. The polypeptide of any one of claims 1-29 comprising(i) an IgG heavy chain (CH1-CH2-CH3) with a VH domain derived from an antibody targeting an immune cell and a linker with a sequence coding for a cytokine;(ii) an IgG light chain with a VL domain derived from an antibody targeting the immune cell;(iii) an IgG heavy chain (CH1-CH2-CH3) with a VH domain derived from an antibody targeting a cancer cell and a cleavable linker followed by a chemokine; and(iv) an IgG light chain with a VL domain derived from an antibody targeting the cancer cell.

31. The polypeptide of any one of claims 1 to 30, which comprises at least two non-contiguous polypeptide chains.

32. The polypeptide of any one of claims 1 to 31, wherein i), ii), iii) and / or iv) are linked to each other by a peptide linker.

33. The polypeptide of any one of claims 1 to 32, wherein one or more of the moieties have an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-13.

34. A host cell that express the polypeptide of any one of claim 1 to 33.

35. The host cell of claim 34, wherein i), ii), iii) and / or iv) are coded for by one or more polynucleotide sequences.

36. A pharmaceutical composition comprising the polypeptide of any one of claim 1 to 33 and a carrier.

37. A method to treat cancer comprising administering to a subject in need thereof an effective amount of the polypeptide of any one of claims 1-33.

38. A method to increase immune cell infiltration in cancer comprising administering to a subject in need thereof an effective amount of the polypeptide of any one of claims 1-37.

39. The method of claim 37 or 38, wherein the cancer is a solid tumor cancer, or a metastatic lesion.

40. The method of claim 39, wherein the solid tumor cancer is one or more of pancreatic, breast, colorectal, lung, skin, ovarian, glioblastoma or liver cancer.

41. The method of claim 37 or 38, wherein the cancer is a hematological cancer.

42. The method of any one of claims 37 to 41, wherein the peptide is administered by intratumoral or intravenous injection.

43. The method of any one of claims 37 to 42, further comprising administering a second therapeutic treatment.

44. The method of claim 43, wherein the second therapeutic treatment comprises a therapeutic agent, radiation, surgery or a combination thereof.

45. The method of claim 44, wherein the therapeutic agent is selected from: a chemotherapeutic agent, biological agent, hormonal therapy or combination thereof.

46. A method to increase immune cell infiltration in cancer comprising administering to a subject in need thereof an effective amount of the polypeptide of any one of claims 1-37.

47. A method to tether immune cells to cancer cells comprising contacting a immune cells and cancer cells with the polypeptide of any one of claims 1-37.

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