CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from and the benefit of United States Provisional Patent Application No. 63/237,634 filed on August 27, 2021 , the specification of which is hereby incorporated by reference in its entirety.

BACKGROUND

(a) Field

[0002] The subject matter disclosed generally relates to compositions comprising isolated natural killer (NK) cell, and more particularly to compositions comprising a complex formed between an isolated natural killer (NK) cell and a constant region of an IgE monoclonal antibody specific for a tumor-associated antigen, and a pharmaceutically acceptable carrier. The subject matter disclosed generally relates to methods of using the same.

(b) Related Prior Art

[0003] Natural Killer (NK) cells along with various monocytes and macrophage lineages that can be found in a tumor microenvironment. NK cells mediate a fraction of the therapeutic activity observed in tumor model, as ablation of that cell population reduces survival of mice inoculated with MUC1 positive tumors. Monoclonal IgE antibodies bind to cells in the tumor microenvironment and provide them antigen specificity as they undertake their tumor fighting activity therein. However, the biology remains incompletely understood, and the role of NK cells and tumor specific antigens directed monoclonal IgE antibodies remains to be teased apart and better understood. Ongoing experimentation is further characterizing the precise mechanism of therapeutic effect and evaluating IgE binding and phenotypic profiles of human NK cells.

[0004] Receptors to IgE constant domains in the forms of FcsR1 , which is a trimeric or tetrameric receptor, and CD23 the low affinity type II receptor are well known. Human and rodent forms of CD23 are known and expressed in low concentrations on various myeloid cells. However, their expression on NK cells is not well known, nor is the potential role of NK cells combined with IgE monoclonal antibodies in cancer therapy. Therefore, compositions and methods that leverage NK cells and IgE monoclonal antibodies as an anti-cancer treatment regimen are still needed.

SUMMARY

[0005] According to an embodiment, there is provided a composition comprising a complex formed between an isolated natural killer (NK) cell and a constant region of an IgE monoclonal antibody specific for a tumor-associated antigen bound on the isolated NK cell, and a pharmaceutically acceptable carrier.

[0006] The isolated NK cell may have been exposed to the tumor-associated antigen.

[0007] The IgE monoclonal antibody specific for a tumor-associated antigen may form an antibody immune complex with the tumor-associated antigen, or a fragment thereof, or a peptide thereof, or combinations thereof.

[0008] The IgE monoclonal antibody specific for a tumor-associated antigen may be bound to an FcsRI receptor.

[0009] The FcsRI receptor may be a trimeric FCsRIA receptor.

[0010] The IgE monoclonal antibody specific for a tumor-associated antigen may be an antibody specific to CA125, folate binding protein (FBP), HER2/neu, MUC1 or PSA.

[0011] The IgE monoclonal antibody specific for a tumor-associated antigen may be a monoclonal antibody specific to MUC1 .

[0012] The antibody specific to MUC1 may bind an epitope of MUC1 selected from SEQ ID NO: 5.

[0013] The heavy chain variable region (VH) of the antibody specific to MUC1 may be encoded by a nucleic acid comprising SEQ ID NO: 1 and a light chain variable region (VL) of the antibody specific to MUC1 may be encoded by a nucleic acid comprising SEQ ID NO: 2.

[0014] The heavy chain variable region (VH) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 6 and a light chain variable region ( L) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 7.

[0015] The heavy chain variable region (VH) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 10 and a light chain variable region (VL) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 18.

[0016] The heavy chain variable region (VH) and a light chain variable region (VL) of the antibody specific to MUC1 each may comprise three complementarity determining regions (CDR1 , CDR2 and CDR3), wherein the heavy chain (VH) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising DAWMD (SEQ ID NO:15), EIRSKANNHATYYAESVKG (SEQ ID NO:16), and GGYGFDY (SEQ ID NO:17), respectively; and the light chain (VL) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising RSSQSIVHSNGNTYLE (SEQ ID NO:23), KVSNRFS (SEQ ID NO:24), and FQGSHVPLT (SEQ ID NO:25), respectively.

[0017] The heavy chain variable region (VH) of the antibody specific to MUC1 may be encoded by a nucleic acid comprising SEQ ID NO: 3 and a light chain variable region (VL) of the antibody specific to MUC1 may be encoded by a nucleic acid comprising SEQ ID NO: 4.

[0018] The heavy chain variable region (VH) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 8 and a light chain variable region ( L) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 9.

[0019] The heavy chain variable region (VH) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 26 and a light chain variable region (VL) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 34.

[0020] The heavy chain variable region (VH) and a light chain variable region (VL) of the antibody specific to MUC1 each may comprise three complementarity determining regions (CDR1 , CDR2 and CDR3), wherein the heavy chain (VH) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising SYYMY (SEQ ID NO:31), EINPSNGGTDFNEKFKS (SEQ ID NO:32), and GGDYPWFAY (SEQ ID NO:33), respectively; and the light chain (VL) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising KSSQSLLYSSNQKNYLA (SEQ ID NO:39), WASTRES (SEQ ID NO:40), and QQYYSYPLT (SEQ ID NO:41), respectively.

[0021] The IgE monoclonal antibody specific for a tumor-associated antigen may be a monoclonal antibody specific to HER2/A/eu.

[0022] The IgE monoclonal antibody specific for a tumor-associated antigen may be a monoclonal antibody specific to PSA.

[0023] The heavy chain variable region (VH) of the antibody specific to PSA may be encoded by a nucleic acid comprising SEQ ID NO: 42 and a light chain variable region (VL) of the antibody specific to PSA may be encoded by a nucleic acid comprising SEQ ID NO: 52.

[0024] The heavy chain variable region (VH) of the antibody specific to PSA may comprise an amino acid sequence comprising SEQ ID NO: 43 and a light chain variable region (VL) of the antibody specific to PSA may comprise an amino acid sequence comprising SEQ ID NO: 53.

[0025] The heavy chain variable region (VH) of the antibody specific to PSA may comprise an amino acid sequence comprising SEQ ID NO: 44 and a light chain variable region (VL) of the antibody specific to PSA may comprise an amino acid sequence comprising SEQ ID NO: 53. [0026] The heavy chain variable region (VH) and a light chain variable region (VL) of the antibody specific to PSA each may comprise three complementarity determining regions (CDR1 , CDR2 and CDR3), wherein the heavy chain (VH) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising SYWMH (SEQ ID NO:49), AFHPENSDTNYNQKFKG (SEQ ID NO:50), and QTTRAEY (SEQ ID NO:51), respectively; and the light chain ( L) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising RSSQSLVHSNGDTYLH (SEQ ID NO:58), KVSNRFS (SEQ ID NO:59), and SQSAHVPLT (SEQ ID NQ:60), respectively.

[0027] The IgE monoclonal antibody specific for a tumor-associated antigen may be a murine monoclonal antibody (xenotypic), a chimeric monoclonal antibody, a humanized monoclonal antibody or a fully human monoclonal antibody.

[0028] The IgE monoclonal antibody specific for a tumor-associated antigen has a constant region that may be of human origin.

[0029] The IgE monoclonal antibody specific for a tumor-associated antigen has variable regions that are of human origin, non-human origin or any combination thereof.

[0030] The IgE monoclonal antibody specific for a tumor-associated antigen has a constant region that may be of human origin, and variable regions that are of murine origin.

[0031] The antibody specific to MUC1 may be mAb 3C6.hlgE, mAb 4H5.hlgE ora combination thereof.

[0032] The isolated natural killer (NK) cell may be isolated from a subject.

[0033] The subject may be a human subject.

[0034] The isolated natural killer (NK) cell may further comprise a Chimeric Antigen Receptor (CAR).

[0035] According to another embodiment, there is provided a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a composition according to the present invention.

[0036] The method may further comprise administering a therapeutically effective amount of an anti-cancer agent.

[0037] The method may further comprise administering a therapeutically effective amount of an anti-cancer agent after the composition.

[0038] The composition may be administered first and the anti-cancer agent may be administered 30 mins to 2 weeks after the composition. [0039] The method may further comprise administering an NK cell potentiating compound.

[0040] The NK cell potentiating compound may be interleukin (IL)-2, IL-12, IL-15, IL-18F, a matrix metalloproteinase (MMP) inhibitor, an inhibitor of an inhibitory NK receptor, and combinations thereof.

[0041] The anti-cancer agent may be selected from an anti-neoplastic agent, an immunotherapeutic agent, a photosensitizer, an immunostimulatory compound, an immune homeostatic checkpoint inhibitor, and combinations thereof.

[0042] The immunostimulatory compound may be a TLR3 agonist or a TLR4 agonist.

[0043] The TLR3 agonist may be polylC, polylCLC (Hiltonol®).

[0044] The immune homeostatic checkpoint inhibitor may be an anti-PD-1 antibody, an anti- PD-L1 antibody, an anti-CTLA-4 antibody, or molecular inhibitors of these receptors.

[0045] The anti-PD-1 antibody may be selected from the group consisting of nivolumab antibody, pembrolizumab antibody, pidilizumab antibody or combinations thereof.

[0046] The anti-PDL-1 antibody may be selected from the group consisting of B7-H1 antibody, BMS-936559 antibody, MPDL3280A (atezolizumab) antibody, MEDI-4736 antibody, MSB0010718C antibody, 10F-9G2 antibody or combinations thereof.

[0047] The anti-CTLA-4 antibody may be selected from the group consisting of ipilimumab or tremelimumab or combinations thereof.

[0048] The anti-cancer agent may comprise administering a combination of polylC, polylCLC (Hiltonol®) and an anti-PD-L1 antibody.

[0049] The polylC, polylCLC (Hiltonol®) may be administered about 1 day after the composition.

[0050] The polylC, polylCLC (Hiltonol®) may be administered as a first administration at about 1 day after the composition, and every 4 to 5 days after the first administration.

[0051] The anti-PD-L1 antibody may be administered about 1 day after administration of the polylC, polylCLC (Hiltonol®).

[0052] The anti-PD-L1 antibody may be administered about 1 day and about 3 days after administration of the polylC, polylCLC (Hiltonol®).

[0053] According to another embodiment, there is provided a method of killing a target cell comprising contacting the target cell with a composition according to the present invention. [0054] According to another embodiment, there is provided a method of treating cancer comprising administering therapeutically effective amounts of an isolated natural killer (NK) cell and an IgE monoclonal antibody specific for a tumor-associated antigen to a subject in need thereof.

[0055] The isolated NK cell may have been exposed to the tumor-associated antigen.

[0056] The IgE monoclonal antibody specific for a tumor-associated antigen may form an antibody immune complex with the tumor-associated antigen, or a fragment thereof, or a peptide thereof, or combinations thereof.

[0057] The FcsRI receptor may be a trimeric FCsRIA receptor.

[0058] The IgE monoclonal antibody specific for a tumor-associated antigen may be an antibody specific to CA125, folate binding protein (FBP), HER2/neu, MUC1 or PSA.

[0059] The IgE monoclonal antibody specific for a tumor-associated antigen may be a monoclonal antibody specific to MUC1 .

[0060] The antibody specific to MUC1 may bind an epitope of MUC1 selected from SEQ ID NO: 5.

[0061] The heavy chain variable region (VH) of the antibody specific to MUC1 may be encoded by a nucleic acid comprising SEQ I D NO: 1 and wherein a light chain variable region (VL) of the antibody specific to MUC1 is encoded by a nucleic acid comprising SEQ ID NO: 2.

[0062] The heavy chain variable region (VH) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 6 and wherein a light chain variable region ( L) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 7.

[0063] The heavy chain variable region (VH) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 10 and wherein a light chain variable region (VL) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 18.

[0064] The heavy chain variable region (VH) and light chain variable region (VL) of the antibody specific to MUC1 each may comprise three complementarity determining regions (CDR1 , CDR2 and CDR3), wherein the heavy chain (VH) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising DAWMD (SEQ ID NO:15), EIRSKANNHATYYAESVKG (SEQ ID NO:16), and GGYGFDY (SEQ ID NO:17), respectively; and the light chain (VL) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising RSSQSIVHSNGNTYLE (SEQ ID NO:23), KVSNRFS (SEQ ID NO:24), and FQGSHVPLT (SEQ ID NO:25), respectively. [0065] The heavy chain variable region (VH) of the antibody specific to MUC1 may be encoded by a nucleic acid comprising SEQ ID NO: 3 and a light chain variable region (VL) of the antibody specific to MUC1 is encoded by a nucleic acid comprising SEQ ID NO: 4.

[0066] The heavy chain variable region (VH) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 8 and wherein a light chain variable region ( L) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 9.

[0067] The heavy chain variable region (VH) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 26 and wherein a light chain variable region (VL) of the antibody specific to MUC1 may comprise an amino acid sequence comprising SEQ ID NO: 34.

[0068] The heavy chain variable region (VH) and light chain variable region (VL) of the antibody specific to MUC1 each may comprise three complementarity determining regions (CDR1 , CDR2 and CDR3), wherein the heavy chain (VH) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising SYYMY (SEQ ID NO:31), EINPSNGGTDFNEKFKS (SEQ ID NO:32), and GGDYPWFAY (SEQ ID NO:33), respectively; and the light chain (VL) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising KSSQSLLYSSNQKNYLA (SEQ ID NO:39), WASTRES (SEQ ID NO:40), and QQYYSYPLT (SEQ ID NO:41), respectively.

[0069] The IgE monoclonal antibody specific for a tumor-associated antigen may be a monoclonal antibody specific to HER2/A/eu.

[0070] The IgE monoclonal antibody specific for a tumor-associated antigen may be a monoclonal antibody specific to PSA.

[0071] The heavy chain variable region (VH) of the antibody specific to PSA may be encoded by a nucleic acid comprising SEQ ID NO: 42 and a light chain variable region (VL) of the antibody specific to PSA is encoded by a nucleic acid comprising SEQ ID NO: 52.

[0072] The heavy chain variable region (VH) of the antibody specific to PSA may comprise an amino acid sequence comprising SEQ ID NO: 43 and wherein a light chain variable region (VL) of the antibody specific to PSA may comprise an amino acid sequence comprising SEQ ID NO: 53.

[0073] The heavy chain variable region (VH) of the antibody specific to PSA may comprise an amino acid sequence comprising SEQ ID NO: 44 and wherein a light chain variable region (VL) of the antibody specific to PSA may comprise an amino acid sequence comprising SEQ ID NO: 53.

[0074] The heavy chain variable region (VH) and light chain variable region (VL) of the antibody specific to PSA each may comprise three complementarity determining regions (CDR1 , CDR2 and CDR3), wherein the heavy chain (VH) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising SYWMH (SEQ ID NO:49), AFHPENSDTNYNQKFKG (SEQ ID NO:50), and QTTRAEY (SEQ ID NO:51), respectively; and the light chain (VL) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising RSSQSLVHSNGDTYLH (SEQ ID NO:58), KVSNRFS (SEQ ID NO:59), and SQSAHVPLT (SEQ ID NQ:60), respectively.

[0075] The IgE monoclonal antibody specific for a tumor-associated antigen may be a murine monoclonal antibody (xenotypic), a chimeric monoclonal antibody, a humanized monoclonal antibody or a fully human monoclonal antibody.

[0076] The IgE monoclonal antibody specific for a tumor-associated antigen may have a constant region that is of human origin.

[0077] The IgE monoclonal antibody specific for a tumor-associated antigen may have variable regions that are of human origin, non-human origin or any combination thereof.

[0078] The IgE monoclonal antibody specific for a tumor-associated antigen may have a constant region that is of human origin, and variable regions that are of murine origin.

[0079] The antibody specific to MUC1 may be mAb 3C6.hlgE, mAb 4H5.hlgE or a combination thereof.

[0080] The isolated natural killer (NK) cell may be isolated from a subject.

[0081] The subject may be a human subject.

[0082] The isolated natural killer (NK) cell may further comprise a Chimeric Antigen Receptor (CAR).

[0083] The method may further comprise administering a therapeutically effective amount of an anti-cancer agent.

[0084] The method may further comprise administering a therapeutically effective amount of an anti-cancer agent after the composition.

[0085] The isolated natural killer (NK) cell and IgE monoclonal antibody specific for a tumor- associated antigen may be administered first and the anti-cancer agent is administered 30 mins to 2 weeks after the composition.

[0086] The method may further comprise administering an NK cell potentiating compound.

[0087] The NK cell potentiating compound may be interleukin (IL)-2, IL-12, IL-15, IL-18F, a matrix metalloproteinase (MMP) inhibitor, an inhibitor of an inhibitory NK receptor, and combinations thereof. [0088] The anti-cancer agent may be selected from an anti-neoplastic agent, an immunotherapeutic agent, a photosensitizer, an immunostimulatory compound, an immune homeostatic checkpoint inhibitor, and combinations thereof.

[0089] The immunostimulatory compound may be a TLR3 agonist or a TLR4 agonist.

[0090] The TLR3 agonist may be polylC, polylCLC (Hiltonol®).

[0091] The immune homeostatic checkpoint inhibitor may be an anti-PD-1 antibody, an anti- PD-L1 antibody, an anti-CTLA-4 antibody, or molecular inhibitors of these receptors.

[0092] The anti-PD-1 antibody may be selected from the group consisting of nivolumab antibody, pembrolizumab antibody, pidilizumab antibody or combinations thereof.

[0093] The anti-PDL-1 antibody may be selected from the group consisting of B7-H1 antibody, BMS-936559 antibody, MPDL3280A (atezolizumab) antibody, MEDI-4736 antibody, MSB0010718C antibody, 10F-9G2 antibody or combinations thereof.

[0094] The anti-CTLA-4 antibody may be selected from the group consisting of ipilimumab or tremelimumab or combinations thereof.

[0095] The anti-cancer agent may comprise administering a combination of polylC, polylCLC (Hiltonol®) and an anti-PD-L1 antibody.

[0096] The polylC, polylCLC (Hiltonol®) may be administered about 1 day after the composition.

[0097] The polylC, polylCLC (Hiltonol®) may be administered as a first administration at about 1 day after the composition, and every 4 to 5 days after the first administration.

[0098] The anti-PD-L1 antibody may be administered about 1 day after administration of the polylC, polylCLC (Hiltonol®).

[0099] The anti-PD-L1 antibody may be administered about 1 day and about 3 days after administration of the polylC, polylCLC (Hiltonol®).

[00100] According to another embodiment, there is provided a method of killing a target cell comprising contacting the target cell with therapeutically effective amounts of an isolated natural killer (NK) cell and an IgE monoclonal antibody specific for a tumor-associated antigen to a subject in need thereof.

[00101] According to another embodiment, there is provided a use of a composition according to the present invention for the treatment of cancer in a subject in need thereof. [00102] According to another embodiment, there is provided a use of therapeutically effective amounts of an isolated natural killer (NK) cell and an IgE monoclonal antibody specific for a tumor- associated antigen for the treatment of cancer in a subject in need thereof.

[00103] According to another embodiment, there is provided a composition according to the present invention for use in the treatment of cancer in a subject in need thereof.

[00104] According to another embodiment, there is provided a therapeutically effective amounts of an isolated natural killer (NK) cell and an IgE monoclonal antibody specific for a tumor- associated antigen for use in the treatment of cancer in a subject in need thereof.

[00105] According to another embodiment, there is provided a use of a composition according to any one of claims 1 to 30 in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

[00106] According to another embodiment, there is provided a use of an isolated natural killer (NK) cell and an IgE monoclonal antibody specific for a tumor-associated antigen in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

[00107] The following terms are defined below.

[00108] The term “antibody”, which is also referred to in the art as “immunoglobulin” (Ig), as used herein refers to a protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA, Ig D, IgE, IgG, and IgM. When an antibody is correctly folded, each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences. For example, the immunoglobulin light chain folds into a variable (Vi_) and a constant (CL) domain, while the heavy chain folds into a variable (VH) and multiple constant (eg CHI, CH2, CHS) domains. Interaction of the heavy and light chain variable domains (VH and L) results in the formation of an antigen binding region (Fv). Each domain has a well-established structure familiar to those of skill in the art.

[00109] The present invention is particularly concerned with Immunoglobulin E (IgE) antibodies. Monomers of IgE consist of two constant heavy chains (E chain) and two light chains, with the £ chain containing 4 Ig-like constant domains (Cs1-Cs4). IgE's main function is immunity to parasites such as helminths like Schistosoma mansoni, Trichinella spiralis, and Fasciola hepatica. IgE is utilized during immune defense against certain protozoan parasites such as Plasmodium falciparum. The IgE also has an essential role in type I hypersensitivity, which manifests in various allergic diseases, such as allergic asthma, most types of sinusitis, allergic rhinitis, food allergies, and specific types of chronic urticaria and atopic dermatitis. IgE also plays a pivotal role in responses to allergens, such as: anaphylactic reactions to drugs, bee stings, and antigen preparations used in desensitization immunotherapy.

[00110] The light and heavy chain variable regions are responsible for binding the target antigen and can therefore have unique variable region sequences for each clone. The constant regions show less sequence diversity and are responsible for interacting with a number of natural proteins on cell surfaces or in circulation to elicit important immune response activities. The variable region of an antibody contains the antigen-binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The majority of sequence variability occurs in six hypervariable regions, three each per variable heavy (VH) and light (VL) chain; the hypervariable regions combine to form the antigen-binding site, and contribute to binding and recognition of an antigenic determinant. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as their size, shape, and chemistry of the surface they present to the antigen. Various schemes exist for identification of the regions of hypervariability, the two most common being those of Kabat and of Chothia and Lesk. Kabat et al (1991) define the “complementaritydetermining regions” (CDRs) based on sequence variability at the antigen-binding regions of the H and VL domains. Chothia and Lesk (1987) define the “hypervariable loops” (H or L) based on the location of the structural loop regions in the VH and VL domains. These individual schemes define CDR and hypervariable loop regions that are adjacent or overlapping. Those of skill in the antibody art often utilize the terms “CDR” and “hypervariable loop” interchangeably, and they may be so used herein. The CDR/loops are identified herein according to the Kabat scheme (i.e., CDR1 , 2 and 3, for each variable region), as identified in the sequence table below. The CDRs/loops are also identified herein according to the Chothia, AbM (Whitelegg, N. R., and Rees, A. R. 2000), Contact (MacCallum, R. M. et al. 1996), or IMGT (Lefranc, M. P. 1997 and Lefranc, M. P. et al. 2003) schemes (i.e., CDR1 , 2 and 3, for each variable region), as identified in the sequence table below.

[00111] An “antibody fragment”, “antigen-binding fragment”, and “antigen-binding fragment thereof” as referred to herein may include any suitable antigen-binding antibody fragment known in the art. The antibody fragment may be a naturally-occurring antibody fragment, or it may be a non-naturally occurring antibody fragment obtained, for example, by manipulation of a naturally-occurring antibody or by using recombinant methods. For example, an antibody fragment may include, but is not limited to, a Fv, a single-chain Fv (scFv; a molecule consisting of VL and VH connected with a peptide linker), a Fab, a F(ab’)2, single-domain antibody (sdAb; a fragment composed of a single VL or VH), or a multivalent presentation of any of these. Antibody fragments such as those just described may require one or more linker sequences, disulfide bonds, or other type of covalent bond to link different portions of the fragments; those of skill in the art will be familiar with the requirements of the different types of fragments for their construction.

[00112] In a non-limiting example, the antibody fragment may be a sdAb derived from a naturally-occurring source. Heavy chain antibodies of camelid origin (Hamers-Casterman et al, 1993) lack light chains and thus their antigen binding sites consist of one domain, termed VHH . sdAb have also been observed in shark and are termed VNAR (Nuttall et al, 2003). Other sdAb may be engineered based on human Ig heavy and light chain sequences (Jespers et al, 2004; To et al, 2005). As used herein, the term “sdAb” includes an sdAb directly isolated from a VH, VHH , L, or NAR reservoir of any origin through phage display or other technology, an sdAb derived from the aforementioned sdAb, a recombinantly produced sdAb, as well as an sdAb generated through further modification of such sdAb by humanization, affinity maturation, stabilization, solubilization, camelization, or other methods of antibody engineering. Also encompassed by the present invention are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the sdAb.

[00113] SdAb possess desirable properties for antibody molecules, such as high thermostability, high detergent resistance, relatively high resistance to proteases (Dumoulin et al, 2002) and high production yield (Arbabi-Ghahroudi et al, 1997). They can also be engineered to have very high affinity by isolation from an immune library (Li et al, 2009) or by in vitro affinity maturation (Davies & Riechmann, 1996). Further modifications to increase stability, such as the introduction of one or more non-canonical disulfide bonds (Hussack et al, 2011a, b; Kim et al, 2012), may also be brought to the sdAb.

[00114] A person of skill in the art would be well-acquainted with the structure of a singledomain antibody (see, for example, 3DWT, 2P42 in Protein Data Bank). An sdAb comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR/hypervariable loops form the antigen-binding site. However, and as would be understood by those of skill in the art, not all CDRs may be required for binding the antigen. For example, and without wishing to be limiting, one, two, or three of the CDRs may contribute to binding and recognition of the antigen by the sdAb of the present invention. The CDR of the sdAb or variable domain are referred to herein as CDR1 , CDR2, and CDR3.

[00115] The term “scFv” is intended to refer to single-chain variable fragment, although an scFv is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This scFv protein retains the specificity of the original immunoglobulin, despite removal of the constant Fc regions and the introduction of the linker. ScFv molecules were created to facilitate phage display, where it is highly convenient to express the antigen-binding domain as a single peptide. As an alternative, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma.

[00116] Divalent (or bivalent) scFvs (di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. Another possibility is the creation of scFvs with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. For example, a diabody drugs could be dosed much lower than other therapeutic antibodies and are capable of highly specific targeting of tumors in vivo. Still shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

[00117] All of these formats can be composed from variable fragments with specificity for two different antigens, in which case they are types of bispecific antibodies. The furthest developed of these are bispecific tandem di-scFvs, known as bi-specific T-cell engagers (BiTE antibody constructs).

[00118] The present invention further encompasses an antibody or an antigen-binding fragment that is “humanized” using any suitable method known in the art, such as, but not limited to CDR grafting or veneering. Humanization of an antibody or antibody fragment comprises replacing an amino acid in the antibody or antigen-binding fragment sequence with its human counterpart, as found in the human consensus sequence, without substantial loss of antigen-binding ability or specificity; this approach reduces immunogenicity of the antibody or fragment thereof when introduced into human subjects. In the process of CDR grafting, one or more than one of the CDR defined herein may be fused or grafted to a human variable region (VH, or L), to other human antibody (IgA, IgD, IgE, IgG, and IgM), to a human antibody fragment framework region (Fv, scFv, Fab) or to another protein of similar size and nature onto which a CDR can be grafted (Nicaise et al, 2004). In such a case, the conformation of the one or more than one hypervariable loop is likely preserved, and the affinity and specificity of the sdAb for its target (i.e., MUC16) is likely minimally affected. CDR grafting is known in the art and is described in at least the following: US Patent No. 6180370, US Patent No. 5693761 , US Patent No. 6054297, US Patent No. 5859205, and European Patent No. 626390. Veneering, also referred to in the art as “variable region resurfacing”, involves humanizing solvent-exposed positions of an antibody or antigenbinding fragment; thus, preserving buried non-humanized residues, which may be important for CDR conformation, are preserved while minimizing the potential for immunological reaction against solvent- exposed regions. Veneering is known in the art and is described in at least the following: US Patent No. 5869619, US Patent No. 5766886, US Patent No. 5821123, and European Patent No. 519596. Persons of skill in the art would also be amply familiar with methods of preparing such humanized antibody fragments and humanizing amino acid positions.

[00119] The antibody or antigen-binding fragment thereof according to the present invention may comprise an additional sequence to aid in expression, detection, localization or purification of the antibody or antigen-binding fragment. Any such sequence or tag known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody or antigen-binding fragment thereof may comprise a targeting or signal sequence [such as, but not limited to an endoplasmic reticulum retention signal (KDEL), ompA or pelB, a detection/purification tag (such as, but not limited to c-Myc, His5, or His6), or a combination of any two or more thereof. In another example, the additional sequence may be a biotin recognition site such as that described by Cronan et al in WO 95/04069 or by Voges et al. in WO/2004/076670. As is also known to those of skill in the art, a linker sequence may be used in conjunction with the additional sequences or tag, or may serve as a detection/purification tag.

[00120] The present invention further encompasses “monoclonal antibodies” (mAb). Monoclonal antibodies are antibodies that are made by identical immune cells which are all clones belonging to a unique parent cell. Monoclonal antibodies can have monovalent affinity, in that they bind to the same epitope (the part of an antigen that is recognized by the antibody).

[00121] As used herein, the term “chimeric antibody” refers to an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[00122] As used herein, the term “NK cells” refers to a sub-population of lymphocytes that is involved in non-conventional immunity. NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD16, CD56 and/or CD57, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express “self’ MHC/HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art.

[00123] Within the context of this invention, “potentiated”, “active,” or “activated” NK cells designate biologically active NK cells, more particularly NK cells having the capacity of lysing target cells. For instance, an “active” NK cell is able to kill cells that express an NK activating receptor-ligand and fails to express “self’ MHC/HLA antigens (KIR-incompatible cells). “Potentiated”, “active,” or “activated” cells can also be identified by any other property or activity known in the art as associated with NK activity, such as cytokine (e.g., IFN-y and TNF-a) production of increases in free intracellular calcium levels. For the purposes of the present invention, “potentiated”, “active,” or “activated” NK cells refer particularly to NK cells in vivo that are not inhibited via stimulation of an inhibitory receptor, or in which such inhibition has been overcome, e.g., via stimulation of an activating receptor.

[00124] As used herein, the term “activating NK receptor” refers to any molecule on the surface of NK cells that, when stimulated, causes a measurable increase in any property or activity known in the art as associated with NK activity, such as cytokine (for example IFN-y and TNF-a) production, increases in intracellular free calcium levels, the ability to target cells in a redirected killing assay as described, e.g., elsewhere in the present specification, or the ability to stimulate NK cell proliferation. The term “activating KIR receptor” includes but is not limited to NKp30, NKp44, NKp46, NKG2D, IL- 12R, IL-15R, IL-18R and IL-21 R. The term “activating NK receptor” as used herein excludes the IL-2 receptor (IL-2R). Methods of determining whether an NK cell is active or proliferating or not are described in more detail below and are well known to those of skill in the art.

[00125] As used herein, the term “inhibiting” or “inhibitory” NK receptor refers to any molecule on the surface of NK cells that, when stimulated, causes a measurable decrease in any property or activity known in the art as associated with NK activity, such as cytokine (e.g., IFN-y and TNF-a) production, increases in intracellular free calcium levels, or the ability to lyse target cells in a redirected killing assay as described, e.g., elsewhere in the present specification. Examples of such receptors include KIR2DL1 , KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1 , KIR3DL2, KIR3DL3, LILRB1 , NKG2A, NKG2C NKG2E and LILRB5. Methods of determining whether an NK cell is active or not are described in more detail below and are well known to those of skill in the art.

[00126] In the present invention, the term “block an inhibitory receptor or stimulates an activating receptor of an NK cell” refers to the ability of certain compounds, preferably antibodies, fragments or derivatives thereof; to preferably directly interact with at least one inhibitory or activating NK cell receptor, e.g., KIR, NKG2A/C, NKp30, NKp44, NKp46 and others listed herein, and either neutralizing inhibitory signals of the receptor (in the case of inhibitory receptors) or stimulate signaling from the receptor (in the case of activating receptors). With inhibitory receptors, preferably the compound, preferably an antibody or a fragment thereof, is able to block the interaction between HLA and the receptor. When the compound is an antibody, the antibodies may be polyclonal or, preferably, monoclonal. They may be produced by hybridomas or by recombinant cells engineered to express the desired variable and constant domains. The antibodies may be single chain antibodies or other antibody derivatives retaining the antigen specificity and the lower hinge region or a variant thereof such as a Fab fragment, a Fab'2 fragment, a CDR and a ScFv. These may be polyfunctional antibodies, recombinant antibodies, humanized antibodies, or variants thereof.

[00127] The term “subject” as used herein, is a human patient or other animal such as another mammal with functional mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, dendritic cells, and Langerhans cells. In humans, the appropriate cells express the high affinity receptor for IgE (FcsRI) for the administered composition of the invention.

[00128] The term “tumor-associated antigen” (TAA) as used herein can be any type of cancer antigen that may be associated with a tumor as is known in the art and includes antigens found on the cell surface, including tumor cells, as well as soluble cancer antigens. Several cell surface antigens on tumors and normal cells have soluble counterparts. Such antigens include, but are not limited to those found on cancer-associated fibroblasts (CAFs), tumor endothelial cells (TEC) and tumor-associated macrophages (TAM). Examples of cancer-associated fibroblasts (CAFs) target antigens include but are not limited to: carbonic anhydrase IX (CAIX); fibroblast activation protein alpha (FAPa); and matrix metalloproteinases (MMPs) including MMP-2 and MMP-9. Examples of Tumor endothelial cell (TECs) target antigens include, but are not limited to vascular endothelial growth factor (VEGF) including VEGFR-1 , 2, and 3; CD-105 (endoglin), tumor endothelia markers (TEMs) including TEM1 and TEM8; MMP-2; Survivin; and prostate-specific membrane antigen (PMSA). Examples of tumor associated macrophage antigens include but are not limited to: CD105; MMP-9; VEGFR-1 , 2, 3 and TEM8. According to some embodiments, the tumor associated antigen may be CA125, folate binding protein (FBP), HER2/neu, MUC1 or PSA.

[00129] Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[00130] It is noted that terms like “preferably”, “commonly”, and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[00131] For the purposes of describing and defining the present invention it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[00132] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[00133] Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:

[00134] Fig. 1A illustrates the expression of IgE receptor (FcsRIa) in pancreatic adenocarcinoma. Shown is an immunostaining for FcsRIa and FCyRIII in Pancreatic ductal adenocarcinoma (PDAC) tumor and adjacent normal tissues.

[00135] Fig. 1 B illustrates boxplot of FCER1A, FCER1G, FCGR3A, and FCGR1A expression levels in different subtypes of PDAC.

[00136] Fig. 1C illustrates immunofluorescence staining for mast cells markers (Tryptase) and FcsRIa in primary pancreatic tumor tissue.

[00137] Fig. 1 D shows a histogram presenting the mean fluorescence intensity (MFI) for the immunofluorescence staining of Fig. 1 C in corresponding PDAC sample.

[00138] Fig. 1 E shows a histogram plot which displays histological score for the quantitation of FcsRIa positive cells in the normal pancreas (n=3), primary pancreatic tumor and matched metastatic liver samples (n=8). Scoring of FcsRIa positive cells in IHC were assessed using a formula as described in the method section of Example 1. IHC data and boxplots from Figs. 1A to 1 D were compared using One-way ANOVA, p values: *<0.05, **<0.005, for multiple comparison. [00139] Fig. 1 F shows a schematic representation of flow cytometer gating followed for FcsRIa positive immune cells in the pancreatic tumor. Human pancreatic tumor samples were acquired through RAPID autopsy program and processed for single cell suspension. Subsequently, cells were stained for cell surface markers using fluorescent labeled antibodies.

[00140] Fig. 2A illustrates anti-MUC1 .IgE-based immunotherapy relieved tumor burden and prolonged survival in subcutaneous pancreatic tumor-bearing mice. The cartoon depicts humanized anti-MUC1.lgE antibody that recognizes the ‘PDTRPAP’ region in the N-terminal domain of MUC1 on tumor cells.

[00141] Fig. 2B shows FACS plot which demonstrates the binding capacity of anti-MUC1 . IgE (3C6.hlgE) to the MUC1 expressing S2-013 tumor cells. Binding was determined by immunofluorescence staining of epsilon and kappa chains of anti-MUC1 .IgE bound to S2-013.MUC1 cells.

[00142] Fig. 2C shows histogram which represents median fluorescence intensity (MFI) of Epsilon (E) and Kappa (K) chains on S2-013.MUC1 cells.

[00143] Fig. 2D shows FACS plots with corresponding MFI which demonstrate FcsRIa expression by T cells, mast cells, and dendritic cells in the blood of mice from different genetic backgrounds. Cells are gated on live cells and fluorescence minus one (FMO) control for FcsRIa was also utilized for the study.

[00144] Fig. 2E illustrates a diagrammatic depiction of the subcutaneous tumor model followed for dTg mice. The picture represents the day of tumor implantation and treatment regimen with anti- MUCl .lgE (25 p.g/ml, /.v.), anti-PD-L1 (200 pig/injection , /.v.) and PolylCLC (200 pig /injection, /.v.). As show, treatment began with anti-MUC1.lgE post 7 days of tumor implantation and followed for every 10 days afterwards. PolylCLC treatment began a day after anti-MCU1.lgE and was followed every 5 days, Anti-PD-L1 was given every 1 ^st and 3 ^rd day after PolylCLC.

[00145] Fig. 2F shows Kaplan-Meier plots which represents time-to-tumor progression (TTP) in PancO2.MUC1 subcutaneous tumor bearing dTg mice in different treatment groups.

[00146] Fig. 2G illustrates histogram which represents tumor growth curve for indicated treatment groups in subcutaneous PancO2.MUC1 tumor-bearing mice (n=10).

[00147] Fig. 2H shows a cartoon which depicts the re-challenge experiments followed for anti- MUCl .lgE + anti-PD-L1 + PolylCLC treated tumor-free mice. Tumor free and naive mice were challenged with PancO2.MUC1 and Panc02.Neo cells on the opposite flanks. [00148] Fig. 2I shows a histogram which represents tumor growth curve of PancO2.MUC1and Panc02.Neo tumors in re-challenged dTg mice (n=4). In Fig. 2: Log-rank test for TTP curves; two-way ANOVA with Bonferroni’s test for tumor volume, p values: *<0.05, **<0.005, ***<0.0005. Values are presented as average ± SEM, unpaired t-test for MFI plots, p value: ***<0.0005.

[00149] Fig. 3A shows anti-MUC1 .IgE-based immunotherapy relieved tumor burden and prolonged survival of orthotropic pancreatic tumor-bearing mice. Shown is a Kaplan-Meier plot which represents survival of KPC.MUC1 orthotopic tumor bearing mice in different treatment groups (n=10).

[00150] Fig. 3B shows tumor growth curves for anti-PSA.IgE + RatlgG2a + saline and anti- MUCl .lgE + anti-PD-L1 + PolylCLC in orthotopic tumor bearing dTg mice. Treatment with anti- MUCl .lgE (25 pig) begin at day 5 and was administered every 5 days after. Treatment dose and schedule for anti-PD-L1 and PolylCLC is the same as mentioned for the subcutaneous model above.

[00151] Fig. 3C shows a Kaplan-Meier plot which represents survival of KPC.MUC1 orthotopic tumor bearing dTg mice in different treatment groups (n=10).

[00152] Fig. 3D shows tumor growth curves for anti-PSA.IgE + RatlgG2a + saline and anti- MUC1 . IgE + anti-PD-L1 + PolylCLC in orthotopic tumor bearing dTg mice.

[00153] Fig. 3E shows Kaplan-Meier plot and tumor growth curves which represents survival and tumor volumes for h FcsRIa single Tg mice in different treatment groups (n=8).

[00154] Fig. 3F showsKaplan-Meier plot and tumor growth curves which represent survival and tumor volumes for h MUC1 single Tg mice in different treatment groups (n=8).

[00155] Fig. 3G shows IHC analysis for Ki67 (proliferation marker) and cleaved Caspase-3 in the tumor section from different treatment groups.

[00156] Fig. 3H shows quantification of Ki67 and cleaved Caspase-3 positive cells as percentage of total cells in each tumor section (n=4 mice/group). In Fig. 3: Kaplan-Meier curves were compared using Log-rank test; tumor volumes were compared using two-way ANOVA with Bonferroni’s post-test, p value: *<0.05, ***<0.0005, ***<0.0005. IHC data represents analysis of 4 mice/group, oneway ANOVA, p value: *<0.05.

[00157] Fig. 4A illustrates that Ova-induced IgE did not attenuate pancreatic tumor growth in early and late tumor model. (A) Shown is a schematic presentation of early OVA-tumor model where KPC tumor cells (5x10 ³ ) were implanted orthotopically in mice before the beginning of OVA aerosol challenges in mice. Mice sensitized and challenged with saline served as control. Mice that were sensitized and challenged with OVA but with no tumor implantation served as another control to account for the effect of tumor cells on serum IgE levels. [00158] Fig. 4B shows a histogram which presents serum IgE levels in different groups.

[00159] Fig. 4C shows a histogram which presents differential count of eosinophils in the bronchoalveolar lavage (BAL) fluid from mice in different groups.

[00160] Fig. 4D shows a Histogram which presents tumor volume in tumor-bearing mice in early-OVA tumor model.

[00161] Fig. 4E shows a Histogram which presents tumor weight in tumor-bearing mice in early-

OVA tumor model.

[00162] Fig. 4F shows tumor growth curves for OVA and saline challenged mice. Tumor volume was measured using ultrasonography every week.

[00163] Fig. 4G shows schematic presentation of late ova-tumor model where KPC tumor cells were implanted orthotopically in mice after 1 cycle of OVA aerosol challenges. Mice sensitized and challenged with saline served as control. Mice sensitized and challenged with OVA but with no tumor implantation served as another control to account for the effect of tumor cells on serum IgE levels.

[00164] Fig. 4H shows a histogram which presents serum IgE levels in different groups.

[00165] Fig. 4I shows a histogram which presents differential count of eosinophils in the BAL fluid from mice in different groups.

[00166] Fig. 4J shows a histogram which presents tumor volume in tumor-bearing mice in late- OVA-tumor model.

[00167] Fig. 4K shows a histogram which present tumor weight in tumor-bearing mice in late- OVA-tumor model.

[00168] Fig. 4L shows tumor growth curves for OVA and saline challenged mice. T umor volume was measured using ultrasonography every week. In Figs. 4A - L: IgE levels, tumor growth, and tumor volume were compared between groups using unpaired t-test. Values are presented as average ± SEM. Tumor growth curve was analyzed using two-way ANOVA with Bonferroni’s test for tumor volume, p values: *<0.05, **<0.005, ***<0.0005.

[00169] Fig. 5A shows that NK and CD8 T cell abrogated efficacy of anti-MUC1 .IgE-based combination therapy. Shown is a Kaplan-Meier plot which represents survival of PancO2.MUC1 subcutaneous tumor in the presence and absence of CD4, CD8, and NK cells in different treatment groups (n=6)

[00170] Fig. 5B shows a Kaplan-Meier plot which represents survival of KPC.MUC1 orthotopic tumor-bearing dTg mice in the presence and absence of CD4, CD8, and NK cells in different treatment groups (n=6). Depletion of NKs, CD4, and CD8 T cells was followed by treatment with anti-NK1.1 (100 pg), anti-CD8 (200 pg), and anti-CD4 (200 pg) at days -6, -2, 0, +2, +6, +15, and +25; (day 0 is tumor implantation). Treatment with antibodies for subcutaneous and orthotopic study is mentioned in previous examples.

[00171] Fig. 5C shows Kaplan-Meier plot which displays survival of KPC.MUC1 orthotopic tumor-bearing mice treated with anti-MUC1.lgE-based and anti-MUC1.lgG-based combination with or without NK cell depletion (n=7/group).

[00172] Fig. 5D shows flow cytometer-based quantitation for CD103 ⁺ DC (of total DCs), macrophages, GR1 ⁺ (of CD11 b ⁺ cells), NK (of total live cells), CD8 T cells (of total live cells), and PD- 1 ⁺ TIGIT ⁺ NK (of total NKs) in the tumors from different treatment groups (n=5).

[00173] Fig. 5E shows enzyme-linked immune absorbent spot (ELISPOT) images and quantitation for the activity of splenic CD8 T cells from treated mice against KPC.MUC1 tumor cells (n=3).

[00174] Fig. 5F shows flow cytometer-based analysis of MUC1-FITC peptide-anti-MUC1 . IgE uptake (antigen-antibody complex) by spleen-derived DCs from anti-MUC1.lgE-based combination treated dTg mice. Mouse IgE was used as control for the assay.

[00175] Fig. 5G shows CD8 T cell proliferation assay using carboxyfluorescein succinimidyl ester (CFSE)-based CD8 T cell proliferation in the presence and absence of antigen-antibody loaded DCs post 3-day co-culture at different ratio.

[00176] Fig. 5H shows FACS plot which represents CD107a positive NKs from treated tumorbearing mice in ex vivo assay. Splenic NKs were harvested from treated tumor-bearing mice, cultured in the presence of S2-013.MUC1 (10:1 co-culture ratio), and treated with the respective combination.

[00177] Fig. 5I shows a histogram which presents quantitation of percent de-granulated NKs of total NK in CD107a-based degranulation assay. In Figs. 5A-I: the Kaplan-Meier curves were compared using Log-rank test; frequency of different immune subsets in treated tumor was compared using oneway ANOVA. Values are presented as average ± SEM, unpaired t-test for ELISPOT and degranulation assay, p values: *<0.05, **<0.005.

[00178] Fig. 6A shows that anti-MUC1 .IgE + anti-PD-L1 + PolylCLC treatment enhanced NK cell function. It shows a scatter plot which presents percentages of CD107a positive NKs upon coculture with human spleen-derived NKs with MUC1 expressing S2-013 cell line in different treatment groups. NK cells were isolated from the spleen of healthy donors. NK-tumor cell co-culture were treated with anti-MUC1.lgE (20 pg), anti-PSA.IgE (20 pg), RatlgG2a (2 pg), anti-PD-L1 (2 pg), and PolylCLC (10 pg) for 4 hr.

[00179] Fig. 6B shows a scatter plots present percentages of CD107a positive NKs upon coculture with human spleen derived NKs with the Panel (B) cell line in different treatment groups. NK cells were isolated from the spleen of healthy donors. NK-tumor cell co-culture were treated with anti- MUd .lgE (20 pg), anti-PSA.IgE (20 pg), RatlgG2a (2 pg), anti-PD-L1 (2 pg), and PolylCLC (10 pg) for 4 hr.

[00180] Fig. 6C shows a histogram plot which represents the percentages of lysed S2013.MUC1 upon co-culture with human spleen derived NKs in the different treatment groups (ex vivo ADCC assay). For ADCC assay, tumor cells were labeled with CFSE dye. NK-Tumor cell cocultures were treated with anti-MUC1 . IgE (20 pg), anti-PSA.IgE (20 pg), RatlgG2a (2 pg), anti-PD-L1 (2 pg), and PolylCLC (10 pg) for 4 hr. Post incubation, tumor cell lysis was determined by propidium iodide staining.

[00181] Fig. 6D shows a histogram plot which represents the percentages of lysed Panel upon co-culture with human spleen derived NKs in the different treatment groups (ex vivo ADCC assay). For ADCC assay, tumor cells were labeled with CFSE dye. NK-Tumor cell co-cultures were treated with anti-MUC1.lgE (20 pg), anti-PSA.IgE (20 pg), RatlgG2a (2 pg), anti-PD-L1 (2 pg), and PolylCLC (10 pg) for 4 hr. Post incubation, tumor cell lysis was determined by propidium iodide staining.

[00182] Fig. 6E shows flow cytometer quantitation of granzyme B and IFN-y production by human splenic NKs upon co-culture with S2-103.MUC1 in different treatment groups. NK-tumor cell co-culture was treated with anti-MUC1.lgE (20 pg), RatlgG2a (2 pg), anti-PSA.IgE (20 pg), anti-PD-L1 (2 pg), and PolylCLC (10 pg) for 6 hr. Values are presented as average ± SEM. Levels of granzyme B and IFN-y were determined using intracellular cytokine staining.

[00183] Fig. 6F shows flow cytometer quantitation of granzyme B and IFN-y production by human splenic NKs upon co-culture with Panel (F) in different treatment groups. NK-tumor cell coculture was treated with anti-MUC1.lgE (20 pg), RatlgG2a (2 pg), anti-PSA.IgE (20 pg), anti-PD-L1 (2 pg), and PolylCLC (10 pg) for 6 hr. Values are presented as average ± SEM. Levels of granzyme B and IFN-y were determined using intracellular cytokine staining. In Fig. 6: Percent CD107a positive NKs and percent tumor cell lysis were compared in different treatment groups using one-way ANOVA, intracellular cytokine quantitation utilized unpaired t-test, p values: *<0.05, **<0.005, ***<0.0005.

[00184] Fig. 7A illustrates that anti-MUC1 .IgE-combination treatment did not alter GZMB expression in NK cells. Shown is relative mRNA expression of GZMB in anti-MUC1.lgE + anti-PD-L1 + PolylCLC treated NK cells upon co-culture with S2-013.MUC1 . Unpaired t-test, p values: *<0.05. [00185] Fig. 7B illustrates that anti-MUC1 .IgE-combination treatment did not alter PRF1 expression in NK cells. Shown is relative mRNA expression of PRF1 in anti-MUC1 . IgE + anti-PD-L1 + PolylCLC treated NK cells upon co-culture with S2-013.MUC1. Unpaired t-test, p values: *<0.05.

[00186] Fig. 8A illustrates that the anti-MUC1. IgE-combination induced SMAD1 phosphorylation in NKs. Shown is a tabular chart which presents the fold change of top 10 phosphorylated proteins enriched in anti-MUC1 .IgE + anti-PD-L1 + PolylCLC treated NK cells upon co-culture with Panel cells. NKs were co-cultured with Panel cells in the presence and absence of anti-MUC1.lgE (20 pg), anti-PD-L1 (2 pg), and PolylCLC (10 pg) for 30 mins. Post treatment, cells were harvested and processed for phosphoarray analysis using a phosphoarray as per manufactures protocol.

[00187] Fig. 8B shows a tabular chart which depicts the fold change of all the phosphorylated SMAD proteins enriched in in anti-MUC1 .IgE + anti-PD-L1 + PolylCLC treated NK cells upon co-culture with Panel cells. NKs were co-cultured as in Fig. 8A above.

[00188] Fig. 8C shows histogram which presents absorbance (O.D) at 598 for MTT assay to monitor survival of NK cells following treatment with anti-MUC1.lgE and anti-PSA.IgE-based combination therapy NK cells were treated with anti-MUC1 .IgE (20 pg), anti-PSA.IgE (20 pg), anti-PD- L1 (2 pg), and PolylCLC (10 pg) for 12 hr.

[00189] Fig. 8D shows an immunoblot which presents phosphorylated SMAD1/SMAD2 and total SMAD1/SMAD2 in the anti-MUC1 .IgE + anti-PD-L1 + PolylCLC treated NK cells upon co-culture with S2-013.MUC1 for 30 and 120 mins.

[00190] Fig. 8E shows the relative mRNA expression oi TGFB2, ID1, ID2, and ID3 in anti- MUCl .lgE + anti-PD-L1 + PolylCLC treated NK cells upon co-culture with S2-013.MUC1.

[00191] Fig. 8F shows a flow cytometer plot which displays intracellular phosphorylated SMAD1 in the NK cells post treatment with anti-MUC1 .IgE-based combination in the presence and absence of Dorsomorphin (20 pm) for 5 hours.

[00192] Fig. 8G shows a histogram plot which represents the percentages of lysed S2013.MUC1 upon co-culture with human spleen-derived NKs in the different treatment groups (ex vivo ADCC assay) in the presence and absence of Dorsomorphin (BMP-SMAD1 inhibitor). NK-tumor cell co-cultures were treated with anti-MUC1.lgE (20 pg), anti-PSA.IgE (20 pg), rat lgG2a (2 pg), anti- PD-L1 (2 pg), and PolylCLC (10 pg). Dorsomorphin (20 pm) was added to the co-culture at the mentioned dose for 5 hr. [00193] Fig. 8H shows a flow cytometer plot which displays intracellular IFN- y in the NK cells post treatment with anti-MUC1.lgE-based combination in the presence and absence of Dorsomorphin (20 pm) and SJB-043 (USPI inhibitor that degrade ID1) for 5 hours.

[00194] Fig. 8I illustrates a flow cytometer plot which displays phosphorylated SMAD1 in the NKs in the tumors of treated mice. It is important to note that given the low abundance of NKs in the tumor, we secured phosphorylation data by flow cytometer using 2000 events from 2 mice in each group.

[00195] Fig. 8J shows a schematic representation of mechanism of action that underlies anti- MUC1.lgE-based combination induced NK cell activation. In Figs. 8A-J: values are presented as average ± SEM, relative expression of mRNA was compared using unpaired t-test, percent tumor lysis was compared in different treatment groups using one-way ANOVA, unpaired t-test, p values: *<0.05, **<0.005, ***<0.0005.

[00196] It will be noted that throughout the appended drawings, like features are identified by like reference numerals.

DETAILED DESCRIPTION

[00197] In embodiments there are disclosed compositions comprising a complex formed between an isolated natural killer (NK) cell and a constant region of an IgE monoclonal antibody specific for a tumor-associated antigen, and a pharmaceutically acceptable carrier. The present invention stems in part from the novel observation that NK cells express the high affinity receptor for IgE antibodies.

[00198] Natural killer cells, also known as NK cells or large granular lymphocytes (LGL), are a type of cytotoxic lymphocyte critical to the innate immune system. The role of NK cells is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cells, acting at around 3 days after infection, and respond to tumor formation. Typically, immune cells detect the major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing the death of the infected cell by lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named “natural killers” because they do not require activation to kill cells that are missing “self’ markers of MHC class 1 . This role is especially important because harmful cells that are missing MHC I markers cannot be detected and destroyed by other immune cells, such as T lymphocyte cells.

[00199] According to an embodiment, the isolated NK cell of the present invention may have been exposed to the tumor-associated antigen. [00200] According to an embodiment, the isolated natural killer (NK) cell of the present invention may be isolated from the subject in which the composition will be used, so as to avoid unwanted immune rejection of the NK cells present in the composition. In a preferred embodiment, the subject is a human subject.

[00201] According to an embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen is bound to an FcsRI receptor. According to an embodiment, the FcsRI receptor may be an FcsRIa receptor. According to another embodiment, the IgE monoclonal antibody specific for a tumor-associated antigen is bound by another means than an FcsRI receptor.

[00202] According to another embodiment, the isolated natural killer (NK) cell may further comprise a Chimeric Antigen Receptor (CAR).

[00203] According to another embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen may form an antibody immune complex with the tumor-associated antigen, or a fragment thereof, or a peptide thereof, or combinations thereof.

[00204] According to another embodiment, the NK cell may have further been treated with an NK cell potentiating compound. NK cell activity is regulated by a complex mechanism that involves both stimulating and inhibitory signals. Accordingly, effective NK cell-mediated therapy can be achieved both by a stimulation of these cells or a neutralization of inhibitory signals. It will be appreciated that any compound that has the effect of blocking, inhibiting, or otherwise downregulating an inhibitory receptor of an NK cell, or of activating, stimulating, or otherwise promoting the activity or expression of an activating receptor of an NK cell, can be used. This includes compounds such as cytokines, as well as small molecules, polypeptides, and antibodies that can bind to NK cell receptors and directly inhibit or stimulate them. It will also be appreciated that the mechanism by which the receptors are blocked or stimulated is not critical to the advantages provided by the invention. For example, the compounds can increase the expression of an activating receptor, or inhibit the expression of an inhibitory receptor, the compounds can prevent the interaction between a ligand and an inhibitory receptor or enhance the interaction between a ligand and an activating receptor, or the compounds can bind directly to the receptors and inhibit them (in the case of inhibitory receptors) or activate them (in the case of activating receptors). The critical parameter is the effect that the compounds have on the ability of therapeutic complex the composition of the present invention to target tumor cells in vivo.

[00205] Any inhibitory receptor on the surface of an NK cell can be targeted by the present NK cell potentiating compounds. NK cells are negatively regulated by major histocompatibility complex (MHC) class l-specific inhibitory receptors. These specific receptors bind to polymorphic determinants of major histocompatibility complex (MHC) class I molecules or HLA and inhibit natural killer (NK) cell lysis. In humans, a family of receptors termed killer Ig-like receptors (KIRs) recognize groups of HLA class I alleles.

[00206] There are several groups of KIR receptors, including KIR2DL, KIR2DS, KIR3DL and KIR3DS. KIR receptors having two Ig domains (KIR2D) identify HLA-C allotypes: KIR2DL2 (formerly designated p58.1) or the closely related gene product KIR2DL3 recognizes an epitope shared by group 2 HLA-C allotypes (Cw1 , 3, 7, and 8), whereas KIR2DL1 (p58.2) recognizes an epitope shared by the reciprocal group 1 HLA-C allotypes (Cw2, 4, 5, and 6). The recognition by KIR2DL1 is dictated by the presence of a Lys residue at position 80 of HLA-C alleles. KIR2DL2 and KIR2DL3 recognition is dictated by the presence of an Asn residue at position 80. Importantly the great majority of HLA-C alleles have either an Asn or a Lys residue at position 80. One KIR with three Ig domains, KIR3DL1 (p70), recognizes an epitope shared by HLA-Bw4 alleles. Finally, a homodimer of molecules with three Ig domains KIR3DL2 (p140) recognizes HLA-A3 and -A11.

[00207] Although KIRs and other class-l inhibitory receptors may be co-expressed by NK cells, in any given individual's NK repertoire, there are cells that express a single KIR and thus, the corresponding NK cells are blocked only by cells expressing a specific class I allele group. Accordingly, when inhibitory receptors are targeted, the present methods will often involve the administration of compounds that target multiple inhibitory receptors, thereby ensuring a broad-based effect that reaches a maximum range of NK cells.

[00208] In certain embodiments, the compound, preferably an antibody or a fragment thereof, blocks an inhibitory receptor of an NK cell, neutralizing the inhibitory signal of at least one inhibitory receptor selected from the group consisting of KIR2DL2, KIR2DL3, KIR2DL1 , KIR3DL1 , KIR3DL2, NKG2A and NKG2C. More preferably, the compound, preferably an antibody or a fragment thereof, that blocks the inhibitory receptor of an NK cell, is a compound, preferably an antibody or a fragment thereof that neutralizes the inhibitory signal of KIR2DL2, KIR2DL 3 and/or KIR2DL1 . Such compounds may also be termed inhibitors of an inhibitory NK receptor.

[00209] The invention also contemplates the use of a combination of several compounds, preferably antibodies or a fragment thereof, that block different inhibitory receptors of NK cells. The compounds, preferably antibodies or a fragment thereof, that block inhibitory receptors of NK cells are specific of an inhibitory receptor selected from KIR2DL1 , KIR2DL2, KIR2DL3, KIR3DL1 , KIR3DL2, NKG2A and NKG2C and are able to inhibit the related KIR- or NKG2A/C-mediated inhibition of NK cell cytotoxicity. For example, the compounds that block inhibitory receptors of NK cells can comprise an antibody having a specificity for KIR2DL1 and another having a specificity for KIR2DL2 and/or KIR2DL3. The combination of compounds that block inhibitory receptors of NK cells may able to inhibit the KIR2DL1-, KIR2DL2-, and KIR2DL3-mediated inhibition of NK cell cytotoxicity. In other embodiments, a cocktail of one or more compounds targeting one or more inhibitory receptors, as well as one or more compounds targeting one or more activating receptors, may be administered.

[00210] For example, monoclonal antibodies specific for KIR2DL1 have been shown to block the KIR2DL1 Cw4 (or the like) alleles. In an other example, monoclonal antibodies against KIR2DL2/3 have also been described that block the KIR2DL2/3 HLACw3 (or the like) alleles. Anti NKG2A antibodies have been shown to block the inhibitory interaction between NKG2A and HLA-E.

[00211] Optionally, the antibody can be selected from the group consisting of GL183 (KIR2DL2, L3, available from Immunotech, France and Beckton Dickinson, USA); EB6 (KIR2DL1 , available from Immunotech, France and Beckton Dickinson, USA); AZ138 (KIR3DL1 , available from Moretta et al, Univ. Genova, Italy); Q66 (KIR3DL2, available from Immunotech, France); Z270 (NKG2A, available from Immunotech, France); P25 (NKG2A/C, available from Moretta et al., Univ. Genova, Italy); and DX9, Z27 (KIR3DL1 , available from Immunotech, France and Beckton Dickinson, USA).

[00212] In embodiments, the invention uses monoclonal antibodies, as well as fragments and derivatives thereof, wherein the antibody, fragment or derivative cross reacts with several KIR or NKG2A/C receptors at the surface of NK cells and neutralizes their inhibitory signals.

[00213] In one embodiment, the invention may use a monoclonal antibody that binds a common determinant of human KIR2DL receptors and inhibit the corresponding inhibitory pathway. In embodiments, the invention may use a monoclonal antibody that binds KIR2DL1 and KIR2DL2/3 receptors at the surface of human NK cells and inhibits KIR2DL1- and KIR2DL2/3-mediated inhibition of NK cell cytotoxicity. The antibody specifically inhibits binding of HLA-c molecules to KIR2DL1 and KIR2DL2/3 receptors. More preferably, the antibody facilitates NK cell activity in vivo. Because KIR2DL1 and KID2DL3 (or KIR2DL2) are sufficient for covering most of the HLA-C allotypes, respectively group 1 HLA-C allotypes and group 2 HLA-C allotypes, such antibodies may be used to increase the efficiency of a therapeutic antibody in most human individuals, typically in about 90% of human individuals or more. In such an embodiment, any of the antibodies described in PCT Patent Application no. PCT/FR 04/01702 filed Jul. 1 , 2004 can be used in accordance with the invention.

[00214] In a particular object of this invention, the antibody that blocks the inhibitory receptor of an NK cell is a monoclonal antibody, wherein the antibody binds a common determinant of KIR2DL human receptors and inhibits KIR2DL-mediated inhibition of NK cell cytotoxicity. The antibody more specifically binds to the same epitope as monoclonal antibody DF200 or NKVSF1 produced by hybridoma DF200 and NKVSF1 respectively and/or competes with monoclonal antibody DF200 or NKVSF1 produced by hybridoma DF200 and NKVSF1 respectively, for binding to a KIR receptor at the surface of a human NK cell. As discussed, examples of antibodies, functional assays and assays to determine whether antibodies compete for binding with the antibodies are described in PCT Patent Application no. PCT/FR 04/01702.

[00215] In another embodiment, the monoclonal antibody may be monoclonal antibody DF200 produced by hybridoma DF200. In another embodiment, the monoclonal antibody is EB6, or the antibody binds to the same epitope as monoclonal antibody EB6, or competes for binding with monoclonal antibody EB6. In other embodiments, the antibody may be a fragment or derivative of either of antibodies DF200 or EB6. The hybridoma producing antibody DF200 has been deposited at the CNCM culture collection, as Identification no. “DF200”, registration no. CNCM I-3224, registered 10 Jun. 2004, Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25, Rue du Docteur Roux, F-75724 Paris Cedex 15, France. The antibody NKVSF1 is available from Serotec (Cergy Sainte-Christophe, France), Catalog ref no. MCA2243.

[00216] In another embodiment of the present invention, the NK cell potentiating compound used to enhance the efficacy of therapeutic antibodies stimulates an activating receptor of an NK cell. Any activating receptor can be used, e.g., NKp30 (see, e.g., PCT WO 01/36630), NKp44 (see, e.g., Vitale et al. (1998) J. Exp. Med. 187:2065-2072), NKp46 (see, e.g., Sivori et al. (1997) J. Exp. Med. 186:1129-1136; Pessino et al. (1998) J. Exp. Med. 188:953-960), NKG2D (see, e.g., OMIM 602893), IL-12R, IL-15R, IL-18R, IL-21 R, or an activatory KIR receptor, for example a KIR2DS4 receptor, or any other receptor present on a substantial fraction of NK cells, and whose activation leads to the activation or proliferation of the cell, preferably even if the cell had previously been inhibited via an inhibitory receptor such as an inhibitory KIR receptor. The compound can be any molecular entity, including polypeptides, small molecules, and antibodies. Exemplary compounds include any ligands, including natural, recombinant or synthetic ligands, which interact with activating receptors. For example, a compound which stimulates an activating receptor of an NK cell may be a cytokine such as IL-2 which interacts with the IL-2 receptor (IL-2R), IL-12 which interacts with the IL-12 receptor (IL-12R), IL-15 which interacts with the IL-15 receptor (IL-15R), IL-18 which interacts with the IL-18 receptor (IL-18R), IL-21 which interacts with the IL-21 receptor (IL-21 R). Such compounds are known from e.g., IL-12 (Research Diagnostics, NJ, DI-212), IL-15 (Research Diagnostics, NJ, RDI-215), IL-21 (Asano et al, FEBS Lett. 2002; 528:70-6). Preferably, a compound which stimulates an activating receptor of an NK cell is a compound other than IL-2. Other exemplary compounds which stimulate an activating receptor of an NK cell include antibodies which bind an NK cell receptor selected from the group consisting of NKp30, NKp44, NKp46, NKG2D, KIR2DS4 and other activatory KIR receptors. [00217] In certain embodiments, the activatory receptor may be a Natural Cytotoxicity Receptor (NCR) found on NK cells, preferably the NCR selected from the group consisting of NKp30, NKp44 or NKp46, and the compound that stimulates an activating receptor is, binds to the same epitope as, or competes for binding with any of the monoclonal antibodies selected from the group consisting of AZ20, A76, Z25, Z231 , and BAB281.

[00218] It will also be appreciated that when the compound that blocks the inhibitory receptor of an NK cell or stimulates an activatory receptor of an NK cell is an antibody, such antibody may by polyclonal or, preferably, monoclonal. The antibody may be produced by a hybridoma or by a recombinant cell engineered to express the desired variable and constant domains. The antibody may be a single chain antibody or other antibody derivative retaining the antigen specificity and the lower hinge region or a variant thereof. The antibody may be a polyfunctional antibody, recombinant antibody, humanized antibody, or a fragment or derivative thereof. The fragment or a derivative thereof is preferably selected from a Fab fragment, a Fab'2 fragment, a CDR and a ScFv. Preferably a fragment is an antigen-binding fragment. An antibody that comprises an antibody fragment may also include but are not limited to bispecific antibodies.

[00219] The binding of any compound to any of the herein-described NK cell receptors can be detected using any of a variety of standard methods. For example, colorimetric ELISA-type assays can be used, as can immunoprecipitation and radioimmunoassays. Competition assays may be employed, e.g., to compare the binding of a test compound to a compound known to bind to an NK cell receptor, in which the control (e.g., BAB281 , which specifically binds to NKp46) and test compounds are admixed (or pre-adsorbed) and applied to a sample containing the epitope-containing protein, e.g., NKp46 in the case of BAB281 . Protocols based upon ELISAs, radioimmunoassays, Western blotting and the use of BIACORE are suitable for use in such simple competition studies and are well known in the art.

[00220] The NK cell potentiating compounds of this invention, preferably antibodies, may exhibit partial inhibitory or stimulating activity, e.g., partially reduce the KIR2DL-mediated inhibition of NK cell cytotoxicity, or partially activate an NK cell through any level of stimulation of NCRs or other receptors. Most NK cell potentiating compounds are able to inhibit (or stimulate, in the case of activating receptors) at least 20%, at least 30%, 40% or 50% or more of the activity of the NK cell, e.g., as measured in a cell toxicity assay, in comparison to cells in the absence of the compound. Also preferred, the compound can provide an increase of depletion of target cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 1000%, or more relative to the depletion level in the absence of the compound. Alternatively, preferred compounds of this invention, preferably antibodies, are able to induce the lysis of matched or HLA compatible or autologous target cell population, i.e., cell population that would not be effectively lysed by NK cells in the absence of the antibody. Accordingly, compounds of this invention may also be defined as facilitating NK cell activity in vivo.

[00221] The invention also contemplates embodiments in which compounds that stimulate activating receptors, or, preferably, block the inhibitory receptor of an NK cell, are fragments of such a monoclonal antibody having substantially the same antigen specificity, including, without limitation, a Fab fragment, a Fab'2 fragment, a CDR and a ScFv. Furthermore, the monoclonal antibody may be humanized, human, or chimeric (e.g., a bispecific or functionalised antibody). While antibodies stimulating activating receptors can also be fragments, they are preferably full length. Derivatives, e.g., with modified sequences or with conjugated heterologous functional groups or other compounds, can be used for any of the antibodies described herein.

[00222] According to embodiments, the composition of the present invention comprises IgE monoclonal antibody specific for a tumor-associated antigen. As used herein, tumor-associated antigen (TAA) can be any type of cancer antigen that may be associated with a tumor as is known in the art and includes antigens found on the cell surface, including tumor cells, as well as soluble cancer antigens. Several cell surface antigens on tumors and normal cells have soluble counterparts. For example, the tumor associated antigen may be CA125, folate binding protein (FBP), HER2/neu, MUC1 or Prostate-Specific Antigen (PSA). Preferably, the tumor associated antigen is MUC1.

[00223] In one embodiment, the antibody of the invention is an IgE monoclonal antibody specific for an epitope of MUC1 . In one embodiment, the antibody of the invention is specific for the epitope of MUC1 comprising amino acids STAPPAHGVTSAPDTRPAPG [SEQ ID NO: 5] of MUC1. The exact epitope lies in one of the 20 amino acid repeats that characterize the external domain of MUC1. In one embodiment, the antibody of the invention is capable of binding MUC1 at the epitope defined at STAPPAHGVTSAPDTRPAPG [SEQ ID NO: 5],

[00224] Any of the herein-described antibodies can be genetically modified or engineered to be human-suitable, e.g., humanized, chimeric, or human antibodies. Methods for humanizing antibodies are well known in the art. Generally, a humanized antibody according to the present invention has one or more amino acid residues introduced into it from the original antibody. These murine or other nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. (1986) Nature 321 :522; Riechmann et al. (1988) Nature 332:323; Verhoeyen et al. (1988) Science 239:1534 (1988)). In some cases, such “humanized” antibodies are chimeric antibodies (Cabilly et al., U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from the original antibody. In practice, humanized antibodies according to this invention are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in the original antibody.

[00225] Another method of making “humanized” monoclonal antibodies is to use a XenoMouse® (Amgen, Thousand Oaks, Calif.) as the mouse used for immunization. A XenoMouse is a murine host that has had its immunoglobulin genes replaced by functional human immunoglobulin genes. Thus, antibodies produced by this mouse or in hybridomas made from the B cells of this mouse, are already humanized. The XenoMouse is described in U.S. Pat. No. 6,162,963. An analogous method can be achieved using a HuMAb-Mouse™ (Medarex).

[00226] Human antibodies may also be produced according to various other techniques, such as by using, for immunization, othertransgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et al., Nature 362 (1993) 255), or by selection of antibody repertoires using phage display methods. Such techniques are known to the skilled person and can be implemented starting from monoclonal antibodies as disclosed in the present application.

[00227] The IgE antibodies of the present invention may also be derivatized to “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in the original antibody, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (Cabilly et al., supra; Morrison et al. (1984) Proc. Natl. Acad. Sci. 81 :6851).

[00228] According to an embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen of the present invention may be a monoclonal antibody specific to MUC1 in which a heavy chain variable region (VH) of the antibody specific to MUC1 is encoded by a nucleic acid comprising SEQ ID NO: 1 and wherein a light chain variable region (VL) of the antibody specific to MUC1 is encoded by a nucleic acid comprising SEQ ID NO: 2.

[00229] According to an embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen of the present invention may be a monoclonal antibody specific to MUC1 in which a heavy chain variable region (VH) of the antibody specific to MUC1 comprises an amino acid sequence comprising SEQ ID NO: 6 and wherein a light chain variable region ( L) of the antibody specific to MUC1 comprises an amino acid sequence comprising SEQ ID NO: 7. According to another embodiment, the IgE monoclonal antibody specific for a tumor-associated antigen of the present invention may be a monoclonal antibody specific to MUC1 in which a heavy chain variable region (VH) of the antibody specific to MUC1 comprises an amino acid sequence comprising SEQ ID NO: 10 and wherein a light chain variable region (VL) of the antibody specific to MUC1 comprises an amino acid sequence comprising SEQ ID NO: 18.

[00230] According to another embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen of the present invention may be a monoclonal antibody specific to MUC1 in which a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody specific to MUC1 each comprise three complementarity determining regions (CDR1 , CDR2 and CDR3), wherein the heavy chain ( H) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising DAWMD (SEQ ID NO:15), EIRSKANNHATYYAESVKG (SEQ ID NO:16), and GGYGFDY (SEQ ID NO:17), respectively; and the light chain (VL) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising RSSQSIVHSNGNTYLE (SEQ ID NO:23), KVSNRFS (SEQ ID NO:24), and FQGSHVPLT (SEQ ID NO:25), respectively.

[00231] The above-described antibody may be for example the mAb 3C6.hlgE antibody described in PCT/US2014/042065.

[00232] According to an embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen of the present invention may be a monoclonal antibody specific to MUC1 in which a heavy chain variable region (VH) of the antibody specific to MUC1 is encoded by a nucleic acid comprising SEQ ID NO: 3 and a light chain variable region (VL) of the antibody specific to MUC1 is encoded by a nucleic acid comprising SEQ ID NO: 4.

[00233] According to an embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen of the present invention may be a monoclonal antibody specific to MUC1 in which a heavy chain variable region (VH) of the antibody specific to MUC1 comprises an amino acid sequence comprising SEQ ID NO: 8 and wherein a light chain variable region (VL) of the antibody specific to MUC1 comprises an amino acid sequence comprising SEQ ID NO: 9. According to another embodiment, the IgE monoclonal antibody specific for a tumor-associated antigen of the present invention may be a monoclonal antibody specific to MUC1 in which a heavy chain variable region (VH) of the antibody specific to MUC1 comprises an amino acid sequence comprising SEQ ID NO: 26 and wherein a light chain variable region (VL) of the antibody specific to MUC1 comprises an amino acid sequence comprising SEQ ID NO: 34.

[00234] According to another embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen of the present invention may be a monoclonal antibody specific to MUC1 in which a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody specific to MUC1 each comprise three complementarity determining regions (CDR1 , CDR2 and CDR3), wherein the heavy chain (VH) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising SYYMY (SEQ ID NO:31), EINPSNGGTDFNEKFKS (SEQ ID NO:32), and GGDYPWFAY (SEQ ID NO:33), respectively; and the light chain (VL) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising KSSQSLLYSSNQKNYLA (SEQ ID NO:39), WASTRES (SEQ ID NQ:40), and QQYYSYPLT (SEQ ID NO:41), respectively.

[00235] The above-described antibody may be for example the mAb 4H5.hlgE antibody described in PCT/US2014/042065.

[00236] According to an embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen of the present invention may be a monoclonal antibody specific to HER2/A/eu such as, for example, as described in T. R. Daniels, et al., Cancer Immunol Immunother, DOI 10.1007/S00262-011-1150-z. .

[00237] According to an embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen of the present invention may be a monoclonal antibody specific to PSA in which a heavy chain variable region (VH) of the antibody specific to PSA is encoded by a nucleic acid comprising SEQ ID NO: 42 and a light chain variable region ( L) of the antibody specific to PSA is encoded by a nucleic acid comprising SEQ ID NO: 52.

[00238] According to an embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen of the present invention may be a monoclonal antibody specific to PSA in which a heavy chain variable region (VH) of the antibody specific to PSA comprises an amino acid sequence comprising SEQ ID NO: 43 and wherein a light chain variable region (VL) of the antibody specific to PSA comprises an amino acid sequence comprising SEQ ID NO: 53. According to another embodiment, the IgE monoclonal antibody specific for a tumor-associated antigen of the present invention may be a monoclonal antibody specific to PSA in which a heavy chain variable region (VH) of the antibody specific to PSA comprises an amino acid sequence comprising SEQ ID NO: 44 and wherein a light chain variable region (VL) of the antibody specific to PSA comprises an amino acid sequence comprising SEQ ID NO: 53.

[00239] According to another embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen of the present invention may be a monoclonal antibody specific to PSA in which a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody specific to PSA each comprise three complementarity determining regions (CDR1 , CDR2 and CDR3), wherein the heavy chain (VH) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising SYWMH (SEQ ID NO: 49), AFHPENSDTNYNQKFKG (SEQ ID NO: 50), and QTTRAEY (SEQ ID NO: 51), respectively; and the light chain (VL) CDR1 , CDR2 and CDR3 comprise an amino acid sequence comprising RSSQSLVHSNGDTYLH (SEQ ID NO: 58), KVSNRFS (SEQ ID NO: 59), and SQSAHVPLT (SEQ ID NO: 60), respectively.

[00240] According to other embodiments, the IgE monoclonal antibody specific for a tumor- associated antigen may be a murine monoclonal antibody (xenotypic), a chimeric monoclonal antibody, a humanized monoclonal antibody or a fully human monoclonal antibody.

[00241] According to another embodiment, the IgE monoclonal antibody specific for a tumor- associated antigen may have a constant region that is of human origin.

[00242] According to other embodiments, the IgE monoclonal antibody specific for a tumor- associated antigen may have variable regions that are of human origin, non-human origin or any combination thereof.

[00243] According to other embodiments, the IgE monoclonal antibody specific for a tumor- associated antigen may have a constant region that is of human origin, and variable regions that are of murine origin.

[00244] In embodiments, the compositions of the present invention comprise pharmaceutically acceptable diluent, carrier or excipient. The composition may comprise a single antibody of the present invention as described above, or may be a mixture of antibody of the present invention. Furthermore, in a composition comprising a mixture of antibody of the present invention, the antibody or antigenbinding fragment thereof may have the same specificity, or may differ in their specificities; for example, and without wishing to be limiting in any manner, the composition may comprise antibody specific to MUC1 (same or different epitope).

[00245] The composition may also comprise a pharmaceutically acceptable diluent, excipient, or carrier. The diluent, excipient, or carrier may be any suitable diluent, excipient, or carrier known in the art, and must be compatible with other ingredients in the composition, with the method of delivery of the composition, and is not deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in suspension form. For example, and without wishing to be limiting, when the composition is provided in suspension form, the carrier may comprise saline, a suitable buffer, or additives to improve solubility, stability or viability of the composition; In a specific, non-limiting example, the composition may be so formulated as to deliver the antibody or antigen-binding fragment to the gastrointestinal tract of the subject. Thus, the composition may comprise suitable technologies for delivery of the NK cell and antibody complex of the present invention. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the antibody and the isolated NK cell. [00246] In another embodiment, there is disclosed a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a composition according to the present invention.

[00247] In another embodiment, there is disclosed a method of treating cancer comprising administering therapeutically effective amounts of an isolated natural killer (NK) cell and an IgE monoclonal antibody specific for a tumor-associated antigen to a subject in need thereof.

[00248] According to another embodiment, there is disclosed a method of killing a target cell comprising contacting the target cell with therapeutically effective amounts of an isolated natural killer (NK) cell and an IgE monoclonal antibody specific for a tumor-associated antigen to a subject in need thereof.

[00249] According to another embodiment, there is disclosed a use of a composition according to the present invention for the treatment of cancer in a subject in need thereof.

[00250] According to another embodiment, there is disclosed a use of therapeutically effective amounts of an isolated natural killer (NK) cell and an IgE monoclonal antibody specific for a tumor- associated antigen for the treatment of cancer in a subject in need thereof.

[00251] According to another embodiment, there is disclosed a composition according to the present invention for use in the treatment of cancer in a subject in need thereof.

[00252] According to another embodiment, there is disclosed a therapeutically effective amounts of an isolated natural killer (NK) cell and an IgE monoclonal antibody specific for a tumor- associated antigen for use in the treatment of cancer in a subject in need thereof.

[00253] According to another embodiment, there is disclosed a use of a composition according to any one of claims 1 to 30 in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

[00254] According to another embodiment, there is disclosed a use of an isolated natural killer (NK) cell and an IgE monoclonal antibody specific for a tumor-associated antigen in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

[00255] According to another embodiment, the methods of the present invention may further comprise administering an anti-cancer agent. The anti-cancer agent may be chosen from an anti- neoplastic agent, an immunotherapeutic agent, a photosensitizer, an immunostimulatory compound, an immune homeostatic checkpoint inhibitor, and combinations thereof.

[00256] Indeed, it is contemplated that the present methods and therapeutic strategies may be used alone or in combination with anti-cancer agents to increase overall patient survival. The anti- neoplastic agents/cytotoxic therapeutic agents include, but are not limited to, angiogenesis inhibitors, antiproliferative agents, kinase inhibitors, receptor tyrosine kinase inhibitors, aurora kinase inhibitors, polo-like kinase inhibitors, bcr-abl kinase inhibitors, growth factor inhibitors, COX-2 inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDS), antimitotic agents, alkylating agents, antimetabolites, intercalating antibiotics, platinum containing agents, growth factor inhibitors, ionizing radiation, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biologic response modifiers, immunologicals, antibodies, hormonal therapies, retinoids/deltoids plant alkaloids, proteasome inhibitors, HSP-90 inhibitors, histone deacetylase inhibitors (HDAC) inhibitors, purine analogs, pyrimidine analogs, MEK inhibitors, CDK inhibitors, ErbB (such as ErbB2) receptor inhibitors, phosphoinositide 3-kinases (PI3Ks) I Akt signaling inhibitors, mTOR inhibitors and combinations thereof as well as other antitumor agents.

[00257] Angiogenesis inhibitors include, but are not limited to, EGFR inhibitors, PDGFR inhibitors, VEGFR inhibitors, TTE2 inhibitors, IGFIR inhibitors, matrix metalloproteinase 2 (MMP-2) inhibitors, matrix metalloproteinase 9 (MMP-9) inhibitors, thrombospondin analogs such as thrombospondin- 1 and N-Ac-Sar-Gly-Val-D-allolle-Thr-Nva-He-Arg-Pro- NHCH2CH3 or a salt thereof and analogues of N-Ac-Sar-Gly-Val-D-allolle-Thr-Nva-lle-Arg- PrO-NHCFhCHs such as N-Ac-GlyVal- D-alle-Ser-Gln-lle-Arg-ProNHCH2CH3 or a salt thereof.

[00258] Examples of EGFR inhibitors include, but are not limited to, Iressa (gefitinib), Tarceva (erlotinib or OSI-774), Icotinib, Erbitux (cetuximab), EMD-7200, ABX-EGF, HR3, IgA antibodies, TP- 38 (IVAX), EGFR fusion protein, EGF- vaccine, anti-EGFr immunoliposomes, Tykerb (lapatinib) and AZD-8931 (sapitinib).

[00259] Examples of PDGFR inhibitors include, but are not limited to, CP-673,451 and CP- 868596.

[00260] Examples of VEGFR inhibitors include, but are not limited to, Avastin (bevacizumab), Sutent (sunitinib, SUI 1248), Nexavar (sorafenib, BAY43-9006), CP-547,632, axitinib (AG13736), Apatinib, cabozantinib, Zactima (vandetanib, ZD-6474), AEE788, AZD-2171 , VEGF trap, Vatalanib (PTK-787, ZK-222584), Macugen, M862, Pazopanib (GW786034), ABT-869 and angiozyme.

[00261] Examples of thrombospondin analogs include, but are not limited to, TSP-I and ABT- 510.

[00262] Examples of aurora kinase inhibitors include, but are not limited to, VX-680, AZD-1152 and MLN-8054. Example of polo-like kinase inhibitors include, but are not limited to, BI-2536. [00263] Examples of bcr-abl kinase inhibitors include, but are not limited to, Gleevec (imatinib) and Dasatinib (BMS354825).

[00264] Examples of platinum containing agents includes, but are not limited to, cisplatin, Paraplatin (carboplatin), eptaplatin, lobaplatin, nedaplatin, Eloxatin (oxaliplatin) or satraplatin.

[00265] Examples of mTOR inhibitors includes, but are not limited to, CCI-779, rapamycin, temsirolimus, everolimus, RAD001 , INK-128 and ridaforolimus.

[00266] Examples of HSP-90 inhibitors includes, but are not limited to, geldanamycin, radicicol, 17-AAG, KOS-953, 17-DMAG, CNF-101 , CNF-1010, 17-AAG-nab, NCS-683664, Mycograb, CNF- 2024, PU3, PU24FC1 , VER49009, IPI-504, SNX-2112 and STA-9090.

[00267] Examples of histone deacetylase inhibitors (HDAC) includes, but are not limited to, Suberoylanilide hydroxamic acid (SAHA), MS-275, valproic acid, TSA, LAQ-824, Trapoxin, tubacin, tubastatin, ACY-1215 and Depsipeptide.

[00268] Examples of MEK inhibitors include, but are not limited to, PD325901 , ARRY-142886, ARRY-438162 and PD98059.

[00269] Examples of CDK inhibitors include, but are not limited to, flavopyridol, MCS-5A, CVT- 2584, seliciclib (CYC-202, R-roscovitine), ZK-304709, PHA-690509, BMI-1040, GPC-286199, BMS- 387,032, PD0332991 and AZD-5438.

[00270] Examples of COX-2 inhibitors include, but are not limited to, CELEBREX™ (celecoxib), parecoxib, deracoxib, ABT-963, MK-663 (etoricoxib), COX-189 Lumiracoxib), BMS347070, RS 57067, NS-398, Bextra (valdecoxib), paracoxib, Vioxx (rofecoxib), SD- 8381 , 4-Methyl-2-(3,4-dimethylphenyl)- l-(4-sulfamoyl-phenyl-IH-pyrrole, T-614, JTE-522, S-2474, SVT-2016, CT-3, SC-58125 and Arcoxia (etoricoxib).

[00271] Examples of non-steroidal anti-inflammatory drugs (NSAIDs) include, but are not limited to, Salsalate (Amigesic), Diflunisal (Dolobid), Ibuprofen (Motrin), Ketoprofen (Orudis), Nabumetone (Relafen), Piroxicam (Feldene), Naproxen (Aleve, Naprosyn), Diclofenac (Voltaren), Indomethacin (Indocin), Sulindac (Clinoril), Tolmetin (Tolectin), Etodolac (Lodine), Ketorolac (Toradol) and Oxaprozin (Daypro).

[00272] Exambles of ErbB (e.g. ErbB2) receptor inhibitors include, but are not limited to, CP- 724-714, CI-1033, (canertinib), Herceptin (trastuzumab), Omitarg (2C4, petuzumab), TAK-165, GW- 572016 (lonafarnib), GW-282974, EKB-569, PI-166, AZD-8931 (sapitinib), dHER2 (HER2 Vaccine), APC8024 (HER2 Vaccine), anti-HER/2neu bispecific antibody, B7.her2lgG3, AS HER2 trifunctional bispecific antibodies, mAB AR-209 and mAB 2B-1 . [00273] Exambles of Phosphoinositide 3-kinase inhibitor include, but are not limited to, Wortmannin, LY294002, hibiscone C, Idelalisib, Copanlisib, Duvelisib, Taselisib, Perifosine, Idelalisib, Buparlisib, Duvelisib, Alpelisib, Umbralisib, Copanlisib, PX-866, Dactolisib, CUDC-907, Voxtalisib (also known as SAR245409, XL765), CUDC-907, ME-401 , IPI-549, SF1126, RP6530, INK1117, pictilisib, XL147 (also known as SAR245408), Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, RP6503, PI-103, GNE-477, and AEZS-136.

[00274] Examples of alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, trofosfamide, Chlorambucil, melphalan, busulfan, mitobronitol, carboquone, thiotepa, ranimustine, nimustine, temozolomide, AMD-473, altretamine, AP-5280, apaziquone, brostallicin, bendamustine, carmustine, estramustine, fotemustine, glufosfamide, KW- 2170, mafosfamide, and mitolactol, carmustine (BCNU), lomustine (CCNU), Busulfan, Treosulfan, Decarbazine and Temozolomide.

[00275] Examples of antimetabolites include but are not limited to, methotrexate, 6- mercaptopurine riboside, mercaptopurine, uracil analogues such as 5-fluorouracil (5-FU) alone or in combination with leucovorin, tegafur, UFT, doxifluridine, carmofur, cytarabine, cytarabine ocfosfate, enocitabine, S-l, Alimta (premetrexed disodium, LY231514, MTA), Gemzar (gemcitabine), fludarabine, 5-azacitidine, capecitabine, cladribine, clofarabine, decitabine, eflornithine, ethnylcytidine, cytosine arabinoside, hydroxyurea, TS-I, melphalan, nelarabine, nolatrexed, ocfosate, disodium premetrexed, pentostatin, pelitrexol, raltitrexed, triapine, trimetrexate, vidarabine, vincristine, vinorelbine, mycophenolic acid, tiazofurin, Ribavirin, EICAR, hydroxyurea and deferoxamine.

[00276] Examples of antibiotics include intercalating antibiotics but are not limited to, aclarubicin, actinomycins such as actinomycin D, amrubicin, annamycin, adriamycin, bleomycin a, bleomycin b, daunorubicin, doxorubicin, elsamitrucin, epirbucin, glarbuicin, idarubicin, mitomycin C, nemorubicin, neocarzinostatin, peplomycin, pirarubicin, rebeccamycin, stimalamer, streptozocin, valrubicin, zinostatin and combinations thereof.

[00277] Examples of topoisomerase inhibiting agents include, but are not limited to, one or more agents selected from the group consisting of aclarubicin, amonafide, belotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, irinotecan HCL (Camptosar), edotecarin, epirubicin (Ellence), etoposide, exatecan, gimatecan, lurtotecan, orathecin (Supergen), BN-80915, mitoxantrone, pirarbucin, pixantrone, rubitecan, sobuzoxane, SN-38, tafluposide and topotecan.

[00278] Examples of antibodies include, but are not limited to, Rituximab, Cetuximab, Bevacizumab, Trastuzumab, specific CD40 antibodies and specific IGFIR antibodies, [00279] Examples of hormonal therapies include, but are not limited to, exemestane (Aromasin), leuprolide acetate, anastrozole (Arimidex), fosrelin (Zoladex), goserelin, doxercalciferol, fadrozole, formestane, tamoxifen citrate (tamoxifen), Casodex, Abarelix, Trelstar, finasteride, fulvestrant, toremifene, raloxifene, lasofoxifene, letrozole, flutamide, bicalutamide, megesterol, mifepristone, nilutamide, dexamethasone, predisone and other glucocorticoids.

[00280] Examples of retinoids/deltoids include, but are not limited to, seocalcitol (EB 1089, CB 1093), lexacalcitrol (KH 1060), fenretinide, Aliretinoin, Bexarotene and LGD-1550.

[00281] Examples of plant alkaloids include, but are not limited to, vincristine, vinblastine, vindesine and vinorelbine.

[00282] Examples of proteasome inhibitors include, but are not limited to, bortezomib (Velcade), MGI 32, NPI-0052 and PR-171.

[00283] Examples of immunologicals include, but are not limited to, interferons and numerous other immune enhancing agents. Interferons include interferon alpha, interferon alpha-2a, interferon, alpha-2b, interferon beta, interferon gamma- 1a, interferon gamma- 1 b (Actimmune), or interferon gamma-nl and combinations thereof. Other agents include filgrastim, lentinan, sizofilan, TheraCys, ubenimex, WF-10, aldesleukin, alemtuzumab, BAM-002, decarbazine, daclizumab, denileukin, gemtuzumab ozogamicin, ibritumomab, imiquimod, lenograstim, lentinan, melanoma vaccine (Corixa), molgramostim, OncoVAC- CL, sargaramostim, tasonermin, tecleukin, thymalasin, tositumomab, Virulizin, Z-100, epratuzumab, mitumomab, oregovomab, pemtumomab (Y-muHMFGI), Provenge (Dendreon), CTLA4 (cytotoxic lymphocyte antigen 4) antibodies and agents capable of blocking CTLA4 such as MDX-010.

[00284] Examples of biological response modifiers are agents that modify defense mechanisms of living organisms or biological responses, such as survival, growth, or differentiation of tissue cells to direct them to have anti-tumor activity. Such agents include krestin, lentinan, sizofrran, picibanil and ubenimex.

[00285] Examples of pyrimidine analogs include, but are not limited to, 5-Fluorouracil, Floxuridine, Doxifluridine, Ratitrexed, cytarabine (ara C), Cytosine arabinoside, Fludarabine, and Gemcitabine.

[00286] Examples of purine analogs include but are not limited to, Mercaptopurine and thioguanine.

[00287] Examples of antimitotic agents include, but are not limited to, ABT-751 , paclitaxel, docetaxel, epothilone D (KOS-862) and ZK-EPO. [00288] The antibodies or antigen binding fragments thereof of the present invention are also intended to be used as a radiosensitizer that enhances the efficacy of radiotherapy. Examples of radiotherapy include but are not limited to, external beam radiotherapy (XBRT), or teletherapy, brachtherapy or sealed source radiotherapy, unsealed source radiotherapy.

[00289] The antibodies or antigen binding fragments thereof of the present invention can also be used in combination with a different class of Bcl-2 inhibitors, such as ABT263 or ABT737.

[00290] According to some embodiments, the cytotoxic agent may be at least one of gemcitabine and abraxane.

[00291] According to yet another embodiment, the additional antibody or therapeutically fragment thereof may be oregovomab antibody B43.13, AR9.6 antibody, or combinations thereof.

[00292] According to yet another embodiment, the immunostimulatory compound may be a TLR3 agonist or a TLR4 agonist.

[00293] According to another embodiment, the TLR3 agonist may be polylC, polylCLC (Hiltonol®).

[00294] According to another embodiment, the immune homeostatic checkpoint inhibitor may be an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or molecular inhibitors of these receptors.

[00295] According to another embodiment, the anti-PD-1 antibody may be selected from the group consisting of nivolumab antibody, pembrolizumab antibody, pidilizumab antibody or combinations thereof.

[00296] According to another embodiment, the anti-PDL-1 antibody may be selected from the group consisting of B7-H1 antibody, BMS-936559 antibody, MPDL3280A (atezolizumab) antibody, MEDI-4736 antibody, MSB0010718C antibody, 10F-9G2 antibody or combinations thereof.

[00297] According to another embodiment, the anti-CTLA-4 antibody may be selected from the group consisting of ipilimumab or tremelimumab or combinations thereof.

[00298] According to an embodiment, the chemotherapy regimen may be Folfirinox.

[00299] In embodiments, the methods may further comprise administering a therapeutically effective amount of an anti-cancer agent after the composition of the present invention. For example, the composition may be administered first and the anti-cancer agent may be administered 30 mins to 2 weeks, or from about 30 mins to 1 week, or from about 30 mins to 10 days, or from about 30 mins to 9 days, or from about 30 mins to 8 days, or from about 30 mins to 7 days, or from about 30 mins to 6 days, or from about 30 mins to 5 days, or from about 30 mins to 4 days, or from about 30 mins to 3 days, or from about 30 mins to 2 days, or from about 30 mins to 1 day, or from about 30 mins to 24 hours, or from about 30 mins to 22 hours, or from about 30 mins to 20 hours, or from about 30 mins to 18 hours, or from about 30 mins to 16 hours, or from about 30 mins to 14 hours, or from about 30 mins to 12 hours, or from about 30 mins to 12 hours, or from about 30 mins to 10 hours, or from about 30 mins to 9 hours, or from about 30 mins to 8 hours, or from about 30 mins to 7 hours, or from about 30 mins to 6 hours, or from about 30 mins to 5 hours, or from about 30 mins to 4 hours, or from about 30 mins to 3 hours, or from about 30 mins to 2 hours, or from about 30 mins to 1 hour after the composition.

[00300] According to an embodiment, the anti-cancer agent may comprise administering a combination of polylC, polylCLC (Hiltonol®) and an anti-PD-L1 antibody. The polylC, polylCLC (Hiltonol®) may be administered about 1 day after the composition. According to another embodiment, the polylC, polylCLC (Hiltonol®) may be administered as a first administration at about 1 day after the composition, and every 4 to 5 days after the first administration.

[00301] According to an embodiment, the anti-PD-L1 antibody may administered about 1 day after administration of the polylC, polylCLC (Hiltonol®). According to another embodiment, the anti- PD-L1 antibody may be administered about 1 day and about 3 days after administration of the polylC, polylCLC (Hiltonol®).

[00302] According to another embodiment, the method may further comprise administering an NK cell potentiating compound. The NK cell potentiating compound is as defined above.

[00303] In embodiments of the present invention, the methods of the present invention may be for the treatment of cancer from which the tumor may be chosen from a pancreatic tumor, a gall bladder tumor, a gastric tumor, a colon tumor, an ovarian tumor, a breast tumor, and a liver tumor.

[00304] According to yet another embodiment, the present invention also encompasses use of the composition of the present invention for treating cancer in a patient in need thereof. According to yet another embodiment, the present invention also encompasses use of the composition of the present invention in method for the treatment of cancer in a patient in need thereof.

[00305] According to yet another embodiment, the present invention also encompasses kits for use for inhibiting cancer tumor growth in a patient in need thereof. The kits may comprise a therapeutic monoclonal antibody specific for a tumor associated antigen, at least one immunostimulatory compound, at least one immune homeostatic checkpoint inhibitor, and instructions on how to use the kit. [00306] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE 1

PANCREATIC DUCTAL ADENOCARCINOMA MODEL

MATERIALS AND METHODS

[00307] Pancreatic ductal adenocarcinoma (PDAC) is currently the most lethal carcinoma. As per estimation, approximately .47,050 people will die of PDAC in 2020. Late diagnosis and chemoresistance are the significant factors that underlie the reduced survival rate of 10%. Unlike other solid tumors, PDAC has proven to be refractory, primarily to single-agent immunotherapeutic approaches. This suggests that more profound immunosuppression exists in pancreatic cancer compared to other solid tumors. Thus, multi-pronged therapeutic approaches may be required to overcome the immunosuppressed microenvironment and provide superior survival benefits for pancreatic cancer patients.

[00308] The current immunotherapeutic strategies against PDAC primarily focus on activating CD8 T cells. Such approaches have largely ignored immunosuppressed innate immune players, such as natural killer cells (NKs), that respond to tumor insults by generating IFN-y for T cell activation. To date, only a few NK cell-targeted strategies have been employed in the preclinical models for solid tumors, and they have faltered in activating intra-tumoral NKs activity in clinical settings (NCT01885897). Hence, a therapeutic combination that boosts both cytotoxic T cells (CTLs) and NKs mediated anti-tumor response would provide long-lasting therapeutic benefits in PDACs.

[00309] The present example investigates MUC1 targeted IgE antibody in combination with toll like receptor three (TLR3) agonist (PolylCLC, Hiltonol™) and anti-PD-L1 against pancreatic tumors in double transgenic (dTg) mice that express human MUC1 and human IgE antibody receptor (FcsRI a). MUC1 is a critical therapeutic target in pancreatic cancer, whose N and C-terminal domain contributes to immune evasion in the pancreatic tumor microenvironment. To date, successful MUC1-based immunotherapies have remained elusive. Two isotypes of humanized MUC1 antibodies (anti-human MUd .lgE and anti-human MUd .IgG antibody) are tested. The study demonstrated that anti- MUd .lgE + anti-PD-L1 + PolylCLC produces NK and CD8 T cell-mediated antigen-specific cellular immune responses that restrict pancreatic tumor growth in an otherwise immunosuppressed preclinical model of pancreatic cancer. Interestingly, protective benefits of anti-MUCI .IgG + anti-PD-L1 + PolylCLC were dependent on CD8 T cells only. Also, the anti-MUCI . IgE-based unique therapeutic combination attenuated the proportion of PD-1 ⁺ TIM3 ⁺ NK cells and increased the numbers of CD103 ⁺ dendritic cells inside the tumor. It was also noted that this combination promoted MUC1 antigen cross- presentation to CD8 T cells and NK cell-mediated tumor killing in the ex vivo assay. Furthermore, the data demonstrated that anti-MUC1.lgE-based combination increased NK cell activity via increased SMAD1/5 phosphorylation and the ID1 (inhibitor of differentiation 1) axis. Moreover, blockade of the BMP-2/SMAD1-ID1 axis using dorsomorphin (BMP-2 receptor inhibitor) and SJB-043 (A deubiquitinating enzyme inhibitor that degrades ID1) inhibited the degranulation and IFN-y producing capabilities of activity of anti-MUC1 . IgE + anti-PD-L1 + PolylCLC treated NK cells. Together, the study demonstrated that tumor targeted IgE antibodies can open new avenues for IgE-based therapeutics, which has not been explored for pancreatic cancer yet.

[00310] The following example provides the material and methods for all the examples 2 to 7 presented below.

Mice

[00311] hMUCI/hFcsRIa dTg mice were developed by crossing MUCl .Tg and hFcsRIa.Tg mice. These mice are immunologically tolerant to human MUC1 and human FcsRIa. While MUCl .Tg is described previously (K. Mehla et al., Cancer Immunol Immunother 67, 445-457 (2018), hFcsRIa Tg were purchased from Jackson Laboratories and bred for our study. Age-matched hMUCI/hFcsRIa dTg, MUCl .Tg, and hFcsRIa.Tg mice were obtained from the breeding colony at the University of Nebraska Medical Center. Mice were genotyped for FcsRIa and MUC1 expression using primers; hFcsRIoc-FP (5'-AGT CAG TCT TGA ATG GCT TCC TG-3'; SEQ ID NO: 134), hFcsRIa-RP (5 -TCT TCG TCC CAT CAC TTC TGC TT-3'; SEQ ID NO: 135), hMUC1-FP (5'-GTA TCG GCC TTT CCT TCC CCA T-3'; SEQ ID NO: 136), hMUC1-RP (5 -ACC TTA AGT GCA CCA GTC CCT C-3'; SEQ ID NO:

137), internal control primers, IC-FP (5'-CTA GGC CAC AGA ATT GAA AGA TCT-3'; SEQ ID NO:

138), IC-RP (5 -GTA GGT GGA AAT TCT AGC ATC ATC C-3'; SEQ ID NO: 139). All animal studies were performed under the Institutional Animal Care and Use Committee guidelines.

Cell lines

[00312] PancO2.MUC1 (expressing human MUC1), Panc02.Neo, and KPC.MUC1 (expressing human MUC1) are described earlier [K. Mehla et al., Cancer Immunol Immunother67, 445-457 (2018)] and [K. Mirikane, Int Immunol. 2001 Feb;13(2):233-40], These cells were maintained in DMEM supplemented with 10% FBS (Bio Whittaker, Walkersville, MD) and G418 sulfate (Mediatech, Herndon, VA).

Human MUCl.lgE and MUCl.IgG antibodies

[00313] The adherent CHO-K1-3C6 3F3 4C5 3D9-H2L2 chimeric human IgE anti-MUC1 monoclonal antibody (also named 3C6.hlgE) producing cell line and the anti-human MUCl .IgG secreting mouse hybridoma line, AR20.5 [K. Mehla et al., Cancer Immunol Immunother 67, 445-457 (2018)] were provided by OncoQuest Pharmaceuticals Inc.. These two cell lines were cultured in DMEM/F12 media supplemented with 10% FBS and weaned to DMEM with 5% FBS. The MABB (SP2/0 Ag 14) mouse hybridoma cell line producing anti-human PSA IgE was a gift of OncoQuest Pharmaceuticals Inc. and was cultured in IMDM with 5% FBS. All antibody conditioned culture supernatants were centrifuged and filtered through 0.25 pm PMSF filters.

[00314] The monoclonal IgE antibody 3C6.hlgE is used in all these examples unless otherwise indicated. It comprises a heavy chain variable region encoded by a nucleic acid sequence comprising SEQ ID NO: 1 ; a light chain variable region encoded by a nucleic acid sequence comprising SEQ ID NO: 2. Put another way, this antibody has a heavy chain variable region (VH) which comprises an amino acid sequence comprising SEQ ID NO: 6 and a light chain variable region (VL) which comprises an amino acid sequence comprising SEQ ID NO: 7.

[00315] The IgG monoclonal antibody specific for an epitope of MUC1 AR20.5 (Mab AR20.5), is disclosed in Qi, W, et al.; Hybrid Hybridomics. 2001 ; 20(5-6):313-24. MAb AR20.5 reacts strongly with either the soluble form or the cell surface epitope of MUC1 on many human cancer cell lines. In one embodiment, the antibody of the invention is specific for the epitope of MUC1 comprising amino acids STAPPAHGVTSAPDTRPAPG [SEQ ID NO: 5] of MUC1 . The exact epitope lies in one of the 20 amino acid repeats that characterize the external domain of MUC1 . In one embodiment, the antibody of the invention is capable of binding MUC1 at the epitope defined at STAPPAHGVTSAPDTRPAPG [SEQ ID NO: 5],

[00316] MUd .IgG (AR20.5) conditioned media was eluted from a Pierce Protein L agarose capture liquid chromatography column (Pierce 20510), whereas the MUCl .lgE (3C6.hlgE) and PSA.IgE conditioned media was eluted from a Human IgE capture liquid chromatography column prepared with bound Omalizumab (Xolair; Novartis Pharmaceuticals Ltd/Genentech, South San Francisco, CA). Subsequently, all Ig eluate peaks were dialyzed against 300 buffer changes of PBS in 10k MWCO Slide-A-Lyzer G2 dialysis cassettes (ThermoFisher 87732). Dialysates were quantified by 280 nm absorbance via a NanoDrop One spectrophotometer (ThermoFisher). Western blot as well as ELISA confirmed antigenicity. Purified antibody lots were documented and lyophilized for long-term storage. Dialysate antibodies were reconstituted in pure water to 1X PBS and sterile filtered prior to in vivo use.

[00317] In flow cytometer studies, fluorescence conjugated antibodies for CD56, CD3, CD107a, IFN-y, Granzyme B, TNF-a, IL6, CD49b, CD11 b, CD11c, F4/80, MHCII, Gr-1 , PD-1 , TIGIT, and CD103 were purchased from Biolegend Inc. BMP inhibitors Dorsomorphin (cat no: 3093) was purchased from Tocris (Bristol, UK).

Tumor model and antibody treatment

[00318] PancO2.MUC1 cells (1x10 ⁶ cells/1 OOptl) were challenged subcutaneously between the scapulae in hMUCI/hFcsRIa dTg mice. Post challenge mice were randomized into different experimental groups (Saline control; Anti-PD-L1 ; PolylCLC; Anti-MUC1.lgE; Anti-PD-L1 + PolylCLC; Anti-MUC1 . IgE + Anti-PD-L1 ; Anti-MUC1 .gE + PolylCLC; and Anti-MUC1 . IgE + PolylCLC + Anti-PD- L1 , n=8 mice per group), and were treated with their respective antibodies. Anti-PD-L1 was purchased from BioXcell (Cat: BE0101 , BioXcell Inc., New Hampshire, USA), and is the 10F.9G2 monoclonal antibody which reacts with mouse PD-L1 (programmed death ligand 1) also known as B7-H1 orCD274. Unless otherwise indicated, this is the anti-PD-L1 antibody used in all examples. Post-treatment, mice were monitored for time-to-tumor progression (TTP) using established protocols (K. Mehla et al., Cancer Immunol Immunother67, 445-457 (2018)). Tumor diameters (2/tumor) were measured weekly, and tumor volume was calculated using formula volume = ^{[length x wldth} < ² )] Mi _{ce were} euthanized when tumors reached 1.2 cm in diameter by following IACUC requirements. For orthotopic studies, KPC.MUC1 tumor cells (3x10 ⁴ cells/30 pl) were implanted in the pancreas of eight- to nine-weeks-old female immunocompetent hMUCI/hFcsRIa dTg mice using established protocols. Post-implantation mice were randomized into different experimental groups and treated with the respective antibodies. Subsequently, mice were monitored for tumor growth using ultrasonography and euthanized when reaching the euthanasia criteria (tumor volume=1500-2000mm ³ ) as per the institutional IACUC guidelines. Tumor volume was measured and calculated as described previously in (K. Mehla et al., Cancer Immunol Immunother 67, 445-457 (2018)).

Immunohistochemistry staining and immunological assays

[00319] Spleen and primary tumors were harvested from tumor-bearing mice at the time of necropsies. Harvested tissues were subsequently processed forTIL (tumor-infiltrating lymphocyte) and IHC (formalin-fixed) analyses. Using established protocols, we processed tumor tissues for single cells and then stained for flow cytometer analysis. FACS samples were acquired by the BD LSRII flow cytometer, and data were analyzed by FlowJo software TreeStar Version 8.8.7. Formalin-fixed tissue sections were embedded in paraffin, and a 5 pm section was stained for Ki67 (cat no: ThermoFisher, clone SolA15) and Caspase 3 (cat no: CST, D175) markers. Post staining, slides were imaged using a Leica microscope equipped with a LAS-AF processing system. For IHC analyses, positively stained cells were measured in 3-4 non-overlapping 20x fields by using Imaged software. Expression of SMAD1 , SMAD2, phosphoSMADI , and phosphoSMAD2 was determined using western blot analysis. SMAD1 and SMAD2 antibodies were obtained from Santa Cruz, CA, USA. PhosphoSMADI (cat no:13820S, CST, MA, USA) and phospho-SMAD2 (cat no:3108S, CST) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). An ECL chemiluminescence detection kit and a Bio-Rad ChemiDoc MP imaging system were used to obtain images. For immunohistochemistry, pancreatic tumor arrays were obtained through the RAPID autopsy program. Samples were stained for FcsRIoc antibody (LS-B3150-50) and quantitated using a histological scoring method as described previously (Hirsch FR et al., J clin Oncol, 21 (20) 3798-807 (2003)). Briefly, FcsRIoc staining was quantitated using a formula, histological score = {1 x (% Cells 1+) +2 x (% Cells 2+) + 3x (% Cells 3)}, where 1 , 2, and 3 corresponds to weak, moderate, and strong staining respectively.

O A challenges in pancreatic tumor model

[00320] C57BL/6J mice were sensitized and challenged to ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO) as previously described with modifications (K. J. Warren et al., Respir Res 20, 51 (2019)). For sensitization, 20 pg OVA was mixed with 2 mg of AI(OH)3 (Thermo Fischer Scientific, Waltham, MA) in 0.2 ml saline and administered intraperitoneally (OVA sensitization) on days 1 , 4, and 7 in mice. Non-sensitized animals received regular saline injections. On days 17 through 20, mice were challenged with nebulized 1.5% OVA in saline or saline alone (OVA and saline challenged groups, respectively) for 20 minutes. Mice were subsequently implanted with the orthotopic tumor on day 0. Post implantation mice were challenged with nebulized 1.5% OVA in saline or saline alone (OVA and saline challenged groups, respectively) for 20 minutes twice a week until the end of the study. In the early time point study, KPC tumor cells (3x10 ⁴ cells/30 pl) were implanted on day 12, whereas in the late time point study, tumors cells were implanted on day 23. To validate the asthmatic phenotype, we collected blood from the submandibular vein. We analyzed for IgE level by utilizing ELISA assay kits (cat no: 501128838, eBioscience Inc, CA, USA) as per the manufacturer’s protocol.

Expression of Fc-Receptors in PDAC subtypes

[00321] PDAC normalized expression data from Bailey et al. was collected and boxplots plotted for each pancreatic subtype using Matlab (P. Bailey et al., Nature 531 , 47-52 (2016)). Outliers are plotted as data points that reside approximately 2.7 standard deviations beyond the mean expression. Statistical analysis comparing PDAC subtypes was performed using one-way ANOVA for multiple comparisons.

Statistical analyses

[00322] TTP curves for different treatment groups were assessed using Kaplan-Meyer plots and log-rank tests. Tumor volume measurement over time was calculated using two-way ANOVA analyses with repeated measurement (Bonferroni post-hoc test). The percentage of immune subsets in different mice subsets was measured using one-way ANOVA with a Bonferroni post-test adjustment for multiple measurements in Prism 6 software (GraphPad). All the p values < 0.05 were considered significant.

Anti-MUC1.lgE binding assay

[00323] MUC1 specific binding ability of anti-MUC1.lgE (3C6.hlgE) antibody was confirmed using human MUC1 expressing tumor (PancO2.MUC1) in the in vitro assay. Here, tumor cells were incubated with or without anti-MUC1.lgE antibody for 12 hr. Post incubation, cells were harvested and stained for epsilon (E) and kappa (K) chains on anti-MUC1.lgE antibody using fluorescent conjugated antibodies.

Immunological assays

[00324] Tumor cell killing activity of NK cells was determined using ADCC assays using the standard protocols. Briefly, human splenic NKs were harnessed using a magnetic isolation kit. Purified NKs were co-cultured with CFSE-labeled tumor cells in the different ratios (10:1 , 5:1 , 2.5:1) in the presence and absence of treatment. Post 4 hours of culture, cells were harvested and stained for propidium iodide stain (P1340MP, ThermoFisher, Inc) and acquired by BD LSRII flow cytometer and data was analyzed by FlowJo software TreeStar Version 8.8.7. CSFE-based proliferation assay was utilized to measure CD8 T cells proliferation upon co-culture with anti-antibody loaded DCs using established protocols. CD107a (cat no:328620, Biolegend, Inc) based degranulation assay was performed to determine the functional activity of NK cells, as described (S. Shabrish, et al. J Immunol Res 2016, 3769590 (2016)). Antigen peptide assay for DCs using FITC-MUC1 peptide was performed as previously described. FITC-MUC1 peptide (16-mer containing DTRPAP sequence (HiLyte Fluor™ 488 - GVTSAPDTRPAPGSTA-OH; SEQ ID NO: 61), utilized for uptake assay, was purchased from Anaspec Inc (Eurogenetec). Phosphoproteome analysis of treated NK cells was performed using the phosphoexplorer (Full Moon Biosystems, Inc, CA, USA). Full Moon Biosystems, Inc performed image quantitation and analysis of array slides. ELISPOT assay was performed using mouse IFN- y ELISpot kit (cat no: EL485, RnD Systmes, Inc.) as per manufacturer’s protocol.

Immunodepletion studies

[00325] CD8, CD4, and NK cells were depleted in subcutaneous and orthotopic tumor models using appropriate antibodies and methods, as described in (K. Mehla et al., Cancer Immunol Immunother 67, 445-457 (2018)). EXAMPLE 2

PANCREATIC TUMOR HARBORED FCERIO CHAIN EXPRESSING CELLS

[00326] The FCERI receptor is a high-affinity IgE receptor that consists of one a subunit, one p subunit, and two y subunits. While the extracellular domain of the a subunit binds to IgE heavy chain, the p and y subunit relays the signal to downstream pathways. The FcsRIa expression in pancreatic tumor tissues was first determined. In Fig. 1A, the normal tissue adjacent to the tumor displays a notable expression of FCyRI and FcsRIa. While FCyRIII, also known as CD16, is a receptor for monomeric IgG-type antibodies, FcsRIa binds to the Fc region of IgE antibody isotype with high affinity. Earlier, Bailey et al. (P. Bailey et al., Nature 531 , 47-52 (2016)) classified PDAC into different molecular subtypes that exhibit distinct histopathological characteristics and survival. Hence, the expression of IgG and IgE receptor genes (FCER1A, FCER1G, FCGR3A, and FCGR1A) was investigated in different subtypes of pancreatic cancer by using previously published expression data from P. Bailey et al., Nature 531 , 47-52 (2016). Herein, noted significant enrichment of the FCGR3A in the squamous subtype of PDAC was noted (Fig. 1 B). In contrast, uniform expression of FCER1A, albeit low as compared to FCGR3A, was observed in all the subtypes of PDAC. Surprisingly, significant expression of FCER1G in the squamous subtype as compared to other subtypes of PDAC was identified. Next, the cell types that express FcsRIa inside the pancreatic tumor was determined by using immunofluorescence staining. This study revealed the expression of FcsRIa on mast cells, monocytes, and a small fraction of eosinophils inside the pancreatic tumor (Figs. 1C and E). Subsequently the expression of FcsRIa was assessed in the primary tumor and matched liver metastatic tissue specimens. The data demonstrate a lack of significant difference in FcsRIa expression between the primary tumor and liver metastatic site (Fig. 1 D).

EXAMPLE 3

ANTI-MUC1.IGE + ANTI-PD-L1 + POLYICLC COMBINATION REJECTED THE GROWTH OF MUC1 -EXPRESSING SUBCUTANEOUS TUMOR CELLS IN DTG MICE

[00327] After confirming the expression FcsRIa positive cells in PDAC, the efficacy of mouse chimeric anti-human MUd .lgE (3C6.hlgE) antibody in the preclinical model of pancreatic cancer was investigated (P. Z. Teo et al., Cancer Immunol Immunother 61 , 2295-2309 (2012)). This antibody targets the DTRPAP sequence in the tandem repeat of the human MUC1 antigen (Fig. 2A). The MUC1 specific binding ability of anti-MUC1 .IgE antibody was confirmed using human PancO2.MUC1 in the in vitro assay (Figs. 2C-D). The data demonstrated increased mean fluorescence intensity for £ and K chains on anti-MUC1.lgE-treated PancO2.MUC1 cells compared to non-treated and fluorescence minus control (FMO) control sets. As mentioned earlier, dTg mice are employed for preclinical studies. Our data demonstrated FcsRIa expression on CD11 b ⁺ myeloid cells, DCs, and NKs, but not T cells in hMUCI/hFcsRIa dTg mice (Fig. 2D). Next, the therapeutic efficacy of anti-MUC1.lgE was investigated in combination with the TLR3 agonist (PolylCLC, Hiltonol™) and a checkpoint inhibitor (anti-PD-L1) against PancO2.MUC1 tumors in dTg mice. The subcutaneous tumor model studies were performed using tumor cells and antibodies as depicted in the schematic presentation (Fig. 2E). The dose of anti- MUC1 . IgE determined in pilot experiments. Anti-MUC1 . Ig E does not display any adverse effects, such as anaphylactic shock, even at a dose up to 40 pg in mice (data not shown). In this study, a significant proportion of anti-MUC1.lgE + PolylCLC + anti-PD-L1 treated mice rejected PancO2.MUC1 and remained tumor-free. Besides, the rest of the anti-MUC1.lgE-combination treated mice that failed to reject tumor had significantly attenuated pancreatic tumor growth compared to other treatment combination sets (Fig. 2F-G). Next, the MUC1 specific memory response in tumor-free mice was examined. To do this, a tumor re-challenge experiment was performed by implanting Panc02.Neo control and PancO2.MUC1 tumor cells on the opposite flanks of tumor-free mice. Herein, unchallenged (naive) dTg animals served as controls (Fig. 2H). Neo and PancO2.MUC1 cell lines exhibit indistinguishable growth rates in the in vitro system (K. Mehla et al., Cancer Immunol Immunother 67, 445-457 (2018). Importantly, no rejection of PancO2.MUC1 and Panc02.Neo tumors was observed upon re-challenge in previously treated mice. However anti-MUC1.lgE-combination treated mice demonstrated a significantly slower growth rate of PancO2.MUC1 compared to Panc02.Neo tumors (Fig. 2I). However, in naive mice, both the tumors grew at similar rates. Overall, the data demonstrated that anti-MUC1 .IgE + PolylCLC + anti-PD-L1 provides a MUC1 specific immune response that hinders tumor growth in heterotopic models.

EXAMPLE 4

ANTI-MUC1.IGE + ANTI-PD-L1 + POLYICLC COMBINATION RESTRICTED KPC.MUC1 ORTHOTOPIC TUMORS IN HMUCI/HFcsRIa DTG MICE BUT NOT IN SINGLE TRANSGENIC

MICE

[00328] Next, the therapeutic benefits of anti-MUC1 .IgE-based combination was assessed in the orthotopic murine model of pancreatic cancer. This model shows an aggressive phenotype and mimics the pathological features of human PDAC. To accomplish this, KPC.MUC1 tumor cells was utilized. Anti-PSA.IgE in combination with saline and rat lgG2a as a control set for tumor studies was also employed. Anti-PSA.IgE antibody targets prostate antigen, which is not present in pancreatic cancer. In this study, treatment with anti-MUC1 .IgE + anti-PD-L1 + PolylCLC significantly delayed tumor growth and prolonged survival of orthotopic tumor-bearing mice as compared to control mice (Fig. 3A-B). Furthermore, anti-MUC1.lgE + anti-PD-L1 + PolylCLC significantly reduced tumor burden and provided superior survival benefits in contrast to anti-PD-L1 + PolylCLC and anti-PSA.IgE + anti- PD-L1 + PolylCLC treatment (Fig. 3C-D). Also, anti-MUC1 . IgE + anti-PD-L1 + PolylCLC combination displayed an improved anti-tumor response as compared to anti-MUC1.lgG-based combination, however, no statistical significance was reached. The above data suggested that while anti-MUC1.lgG and anti-MUC1.lgE-combination triggers similar anti-tumor pathways, anti-MUC1.lgE activates a unique anti-tumor response that contributes to improved therapeutic benefits. To further dissect the critical role of FcsRIa-lgE in attributing the enhanced anti-tumor response of anti-MUC1.lgE, the therapeutic efficacy of anti-MUC1.lgE and anti-MUC1 .IgG-based combinations was investigated in different transgenic mice; hFcsRIa single transgenic (Tg) and hMUC1 single Tg mice. The study demonstrated that anti-MUC1 .IgE + anti-PD-L1 + PolylCLC exhibited tumor protective benefits only in hMUCI/hFcsRIa dTg mice. The absence of either hMUC1 or hFcsRIa attenuated the anti-tumor responses of anti-MUC1 .IgE-based combination treatment against the pancreatic tumor (Fig. 3E-F). In contrast, anti-MUC1. IgG-based combination requires hMUC1 expression for its anti-tumor response.

[00329] Overall, this data implied that the requirement of the FcsRIa-dependent pathway underlies the improved therapeutic efficacy of anti-MUC1 .IgE-based combination over anti-MUC1 .IgG- based therapy in pancreatic tumor-bearing mice. Next, the effect of anti-MUC1 .IgE-based combination on tumor proliferation and apoptosis in the treated mice was investigated (Fig. 3H). While no significant difference in apoptosis among different treatment cohorts was observed, anti-MUC1. IgE-based combination treated mice displayed a significant decrease in Ki67 staining, a marker for tumor proliferation, compared to other treatment sets. Overall, the data suggested that anti-MUC1.lgE + PolylCLC + anti-PD-L1 therapy significantly restricted tumor proliferation and prolonged the overall survival of pancreatic tumor-bearing mice.

[00330] Previous meta-analysis studies suggested an inverse relationship between allergy and the incidence of pancreatic cancer. However, it is not known if IgE antibodies generated in response to allergic reactions can mount an immune response against the pancreatic tumor antigens. To decipher this puzzle, a unique mouse model was developed to study tumor growth of KPC mice-derived tumor cell lines before and after the aerosol challenges with ovalbumin (OVA) in OVA-induced allergic model (Figs. 4A, 4C). OVA challenges significantly increased circulating IgE in both naive and pancreatic tumor-bearing mice compared to saline-challenged control mice (Figs. 4B, 4D). However, no impact of OVA-induced IgE on the growth of the pancreatic tumor was observed. There was no significant difference in the pancreatic tumor growth, weight, and volume between saline and OVA- challenged mice in both the early- and late-tumor models (Figs. 4C-E, H-l). Together, the data imply that allergen specific IgE is ineffective in limiting pancreatic tumor growth. This study presents a careful control for anti-MUC1.lgE therapy, where MUC1 -targeted IgE demonstrated a MUC1 specific anti- tumor response. The data underlies the significance of tumor-specificity of I g E antibodies in controlling pancreatic tumor growth.

EXAMPLE 5

NKS AND CD8 T CELL DEPLETION REDUCED THE EFFICACY OF ANTI-MUC1.IGE-BASED COMBINATION THERAPY IN TUMOR-BEARING MICE

[00331] Next, the key immune subsets that underlie the therapeutic efficacy of anti-MUC1 . IgE + PolylCLC + anti-PD-L1 was studied. Now referring to Fig. 5A, which shows that elimination of NKs and CD8, but not CD4, T cells reduced the survival of anti-MUC1.lgE-combination treated subcutaneous tumor-bearing mice. Similarly, NK and CD8 T cell depletion reduced survival despite treatment with anti-MUC1 .IgE + PolylCLC + anti-PD-L1 in orthotopic tumor-bearing mice (Fig. 5B). It was previously demonstrated that anti-MUC1.lgG + PolylCLC + anti-PD-L1 combination induced a CD8 T, but not NK cell-mediated, anti-tumor response against pancreatic cancer in MUC1 .Tg mice. To understand the distinction between anti-MUC1 .IgG and anti-MUC1.lgE-based combination therapy in the context of NK cells, the efficacy of these two therapies in the presence or absence of NKs in tumorbearing dTg mice was compared. As expected, NK cell depletion did not alter the anti-tumor response of anti-MUC1 .IgG + anti-PD-L1 + PolylCLC treatment, but significantly attenuated the prolonged survival benefits of anti-MUC1.lgE-based combination therapy in orthotopic tumor-bearing mice (Fig. 5C). Subsequently, the tumor-infiltrating lymphocytes (TILs) assessment showed that anti-MUC1.lgE + anti-PD-L1 + PolylCLC did not alter the total number of NKs but significantly reduced the proportion of PD-1 ⁺ TIGIT ⁺ NK cells in treated tumors compared to other control counterparts (Fig. 5D). PD-1 and TIGIT are checkpoint inhibitors that are common between CD8 and NK cells. TIGIT restricts NK cell function, and blockade of TIGIT increases the anti-tumor response of NK cells against trastuzumab- coated breast cancer cells. This data demonstrated that anti-MUC1 .IgE-based combination therapy is effective in restricting the functional exhaustion of NK cells. Additionally, our data showed a significant increase in CD103 ⁺ DCs, but no significant alteration in macrophages and MDSCs percentages in anti- MUd .lgE + anti-PD-L1 + PolylCLC treated tumors, as compared to other treatment groups. CD103 ⁺ DCs are conventional type cDC1s that cross-present tumor antigens to the CD8 T cells. Increased prevalence of CD103 ⁺ DCs indicates the boosting of CD8 T-mediated immune response in anti-MUC1 .IgE treated tumors. It is worth noting that anti-MUC1.lgE + anti-PD-L1 + PolylCLC treatment moderately increased the frequency of CD8 T cells.

[00332] With the goal of understanding CD8 and NK cell mediated anti-tumor response, the CD8 T and NK cell function from treated tumor-bearing mice was assessed. Splenic CD8 cells from anti-MUC1.lgE + anti-PD-L1 + PolylCLC mice demonstrated increased production of IFN-y upon incubation with KPC.MUC1 cells in the ex vivo ELISPOT assay (Fig. 5E). DCs express FcsRIa receptors and can cross-present antigen to CD8 T cells. Perhaps increased CD103 ⁺ DCs in anti- MUCl .lgE-combination treated mice indicate DC-mediated activation of CD8 T cells. To test this hypothesis, an antigen uptake assay was performed using FITC labeled MUC1 peptide (16 aa, sequence HiLyte Fluor™ 488 - GVTSAPDTRPAPGSTA -OH; SEQ ID NO: 61) - anti-MUC1 . IgE antibody immune complex and co-incubated the loaded DCs with CFSE-labeled autologous CD8 T cells. The data demonstrate increased proliferation (CFSE dilution) of CD8 T cells upon incubation with MUC1- peptide-loaded DCs compared to untreated DCs (Figs. 5F-G). Subsequently, the function of NK cells from the treated mice was investigated. CD107a, also known as LAMP1 , is an established marker of NK cell degranulation. The extracellular appearance of CD107a suggests the fusion of lysosome with the plasma membrane of NK cells, transporting lytic granule outside for cytotoxic killing of target cells (Blood. 2013 Jun 6;121 (23):4672-83.). Notably, NKs from anti-MUC1 .IgE + anti-PD-L1 + PolylCLC mice demonstrated increased degranulation as compared to control mice (Figs. 5H-I). Together, the data shows that while both anti-MUC1.lgG and anti-MUC1 .IgE promoted CD8 T cell-mediated efficacy, additional enhancement of NKs activity provided improved (but not significant) therapeutic efficiency of anti-MUC1 .IgE + PolylCLC + anti-PD-L1 against pancreatic cancer.

EXAMPLE 6

ANTI-MUC1. IGE-BASED COMBINATION INCREASED NK CELL FUNCTION.

[00333] Next, the effect of anti-MUC1 .IgE-based combination on the activity of human spleen- derived NK cells was investigated. Human splenic NKs were acquired from a healthy donor and NK cell functional assays were performed, such as ADCC and a degranulation assay. Herein, the S2013.MUC1 was utilized and Panel pancreatic cancer cells are used as target cell lines. The data demonstrated a significant NK cell degranulation following anti-MUC1 .IgE + anti-PD-L1 + PolylCLC treated NKs compared to other treatment groups (Fig. 6A). Besides, anti-MUC1. IgE-based combination enhanced NKs-mediated tumor killing, as evidenced in the increased proportion of propidium iodide (PI) positive tumor cells in the ADCC assay. To further decipher enhanced NK cell activity in anti-MUC1 .IgE + anti-PD-L1 + PolylCLC treated mice, the cytokine-producing abilities of NKs was examined. Surprisingly, anti-MUC1 .IgE + anti-PD-L1 + PolylCLC treatment moderately increased granzyme B production upon incubation with two different tumor cell lines. As mentioned earlier, the extracellular appearance of CD107a indicates the transport of lytic granules (granzyme B and perforin) to the target cells. The data suggested that while anti-MUC1 .IgE + anti-PD-L1 + PolylCLC did not alter the production of lytic granule (granzyme B) in NKs, it did increase the release of existing lytic granules. This observation was confirmed in the expression data where it was noted that anti-MUC1.lgE treatment did not induce GZMB and PRF1 (perforin) expression (Fig. 7A-B). However, a significantly increased production of IFN-y by NK cell post-anti-MUC1 .IgE-based combination treatment was noted. As IFN-y aids in helper T cell differentiation, the data suggested that anti-MUC1 . IgE + anti-PD-L1 + PolylCLC strongly triggered the anti-tumor capacity of NKs cells as well as enhanced adaptive immunity by NKs.

EXAMPLE 7

ANTI-MUC1.IGE + ANTI-PD-L1 + POLYICLC COMBINATION ACTIVATED NK CELLS BY TRIGGERING BMP-SMAD1 AXIS SIGNALING.

[00334] The observed distinction between anti-MUC1.lgG- and anti-MUC1.lgE-combination therapy regarding NK cell activity led to the exploration of the mechanism underlying NK cell activation. To elucidate this, a phospho-proteome analysis of splenic NK cells upon co-culture with Panel tumor cells in the presence and absence of anti-MUC1 .IgE + anti-PD-L1 + PolylCLC treatment for 30 mins was performed. The data showed an enrichment of phosphorylated proteins that are critical for NK cell activity, such as CAMK2A (Calcium/calmodulin-dependent protein kinase II alpha) and PLCG2 (Phospholipase C Gamma 2) (Fig. 8A). While CaMKII activation induces the IFN-y production by NK cells, PLC-y2 activation is critical for NK cell-mediated cytotoxicity. Surprisingly, anti-MUC1.lgE- combination treatment displayed significant upregulation in Caspase 9 (p-Tyr-153) and SMAD1 phosphorylation (p-Ser-465). Earlier studies report that NKs required caspase for cytokine release and proliferation. It was further confirmed that anti-MUC1 .IgE-based combination does not affect the viability of NKs (Fig. 8C). Next, the importance of SMAD1 phosphorylation in NKs following anti- MUCl .lgE + anti-PD-L1 + PolylCLC treatment was explored. SMAD pathways critically regulate NK cell activity. While activation of BMP-SMAD1 maintains the optimal effector function of NKs, TGF-P- SMAD2 signaling inhibits NKs activity. Phosphorylated (Ser 465) SMAD1/5/8 forms a complex with SMAD4 and translocates to the nucleus for the activation of downstream target genes, such as ID1 , ID2, and ID3. In contrast, TGF-p triggers phosphorylation of SMAD2/3, which translates into the complex formation with SMAD4 and activates downstream targets. Hence, the phosphorylation status of all the SMADs in our phospho-array data was determined. Herein, a significant up-regulation of SMAD1 (p-Ser 465) phosphorylation is observed. In contrast, the level of phosphorylated SMAD2 (p- Ser 467) and SMAD3 (p-Thr179) remain unaltered following anti-MUC1.lgE + anti-PD-L1 + PolylCLC treatment (Fig. 8B). The data was confirmed by immunoblot using S2013.MUC1 and Panel cells at different time points. Anti-MUC1.lgE + anti-PD-L1 + PolylCLC treatment significantly increased the levels of pSMADI , but not total SMAD1 , at 30 mins and 120 mins time points (Fig. 8D). As anticipated, pSMAD2 remains unaltered in anti-MUC1.lgE + anti-PD-L1 + PolylCLC treated NK cells. Subsequently, the impact of these treatments on the downstream target of BMP-SMAD1 and TGF-P- SMAD2 signaling was investigated. While anti-MUC1.lgE + anti-PD-L1 + PolylCLC treatment significantly increased ID1 expression, ID2 and ID3 expression displayed moderate increases (Fig. 8E). Furthermore, anti-MUC1.lgE + anti-PD-L1 + PolylCLC moderately downregulated TGFB2 expression. ID1 regulates NF-KB activity and, in turn, upregulates TNF-a and IFN-y production in T cells. It was also noted, in the phosphorylation data, an enrichment of phosphorylated NF-KB-p65 in anti-MUC1 . IgE + anti-PD-L1 + PolylCLC as compared to the control set. Furthermore, anti-MUC1 . IgE- combination treatment increases IFN-y production by NK cells. To further understand the importance of SMAD1 phosphorylation, the BMP-SMAD1 signaling pathway was blocked using Dorsomorphin and NK cell function was determined. Dorsomorphin treatment significantly attenuated SMAD1 phosphorylation and degranulation in anti-MUC1.lgE + anti-PD-L1 + PolylCLC treated NK cells (Fig. 8F-G). Subsequently inhibition of ID1 (SJB-043) in anti-MUC1 .IgE + anti-PD-L1 + PolylCLC treated NK by using SJB-043 attenuates IFN-y production was demonstrated. Finally, SMAD1 phosphorylation in intra-tumoral NK cells of anti-MUC1.lgE-based combination-treated mice was confirmed (Fig. 8I). Together, the data suggested that anti-MUC1.lgE-based combination regulated NK cell function via the SMAD1-ID1-IFN-y axis. Hence in this study a novel IgE-based immunotherapy that enhanced the function of both innate and adaptive immunity players inside the pancreatic tumor is demonstrated (Fig. 8J).

EXAMPLE 8

DISCUSSION OF RESULTS OF EXAMPLE 1 - 7

[00335] The above examples demonstrated that the high-affinity IgE receptor (FcsRIa) is uniformly present in primary pancreatic and metastatic tumors. Likewise, the lack of significant distinction in the expression of FcsRIa among different pancreatic cancer subtypes implies that IgE- based therapies can uniformly benefit all the patients, thus overcoming the limitation of the development of subtype-specific therapy. Furthermore, the examples show the expression of FcsRIa in the CD14 ⁺ monocytes, mast cells, neutrophils, and basophils in our primary pancreatic tumors. Anti- MUC1 .IgE (3C6.hlgE), utilized here, is a mouse chimeric humanized antibody that targets the variable tandem repeat domain of MUC1. This antibody recognizes human MUC1 and binds to the corresponding human FcsRIa subunit of the FcsRIa receptor. DTg mice that express human MUC1 and human FcsRIa were used - the former establishes immunological tolerance to MUC1 in this animal model, and the latter is required for the biological activity of this humanized antibody in mice. Bone marrow-derived mast cells, eosinophils, and DCs are known to express FcsRIa from hFcsRIa Tg mice. The present results showed for the first time the expression of FcsRIa on circulating CD11 b ⁺ myeloid cells and NKs, but not CD3 T cells in dTg mice. The results also demonstrated that anti-MUC1 .IgE + anti-PD-L1 + PolylCLC induced MUC1 -specific cellular immune responses that mediate rejection of PancO2.MUC1 tumors in a subcutaneous model of pancreatic cancer. Similarly, the results show that anti-MUC1.lgE + anti-PD-L1 + PolylCLC significantly restricted the growth of KPC.MUC1 orthotopic tumors as compared to anti-PD-L1 + PolylCLC and anti-PSA.IgE + anti-PD-L1 + PolylCLC. Anti- PSA.IgE, utilized in the current study, is a chimeric antibody against prostate-specific antigen (PSA). Hence, a lack of anti-tumor activity of anti-PSA.IgE reinforces the fact that absence of a specific target antigen prevents IgE-FcsRIa cross-linking and non-specific activation of immune players in our model. The results also emphasize the critical importance of tumor-targeted specificity of IgE-FcsRIa interaction by studying the efficacy of IgE-based therapy in mice with different genetic backgrounds. Here, the efficacy of anti-MUC1 .IgE-based combination against KPC.MUC1 tumors was compared in single Tg hMUC1 or hFcsRIa mice. As anticipated, the anti-tumor responses by anti-MUC1 . IgE + anti- PD-L1 + PolylCLC in single Tg mice were distinct from dTg mice: while anti-MUC1. IgE-based combination therapy prolonged the survival of tumor-bearing dTg mice, it failed to rescue hMUC1 and hFcsRIa dTg mice from pancreatic tumor burden and hence exhibited shortened survival. Therefore, tumor protective benefits of anti-MUC1.lgE requires both human MUC1 and IgE receptor in this preclinical model.

[00336] The present examples also demonstrated that anti-MUC1 .IgE + anti-PD-L1 + PolylCLC provided improved, but not significant, anti-tumor immunity in dTg mice as compared to anti-MUC1 .IgG + anti-PD-L1 + PolylCLC. Therefore, while anti-MUC1 .IgE and anti-MUC1.lgG-based combination employs common effector mediators, the slightly improved response with anti-MUC1 .IgE suggests the involvement of unique anti-tumor mechanisms. The data presented herein demonstrated that NK depletion abrogated the therapeutic efficacy of anti-MUC1 .IgE- but not anti-MUC1.lgG-based combination. Interestingly, both the isotypes of the MUC1 -targeted antibody required CD8 T cell effector mediators for their efficacy against pancreatic cancer, as CD8 depletion attenuated anti-tumor immunity. It is noteworthy that no significant difference in the intra-tumoral level of CD8 T cells between anti-MUC1.lgG and anti-MUC1. IgE-based combination treatment was observed, but CD103+ DCs were significantly elevated in anti-MUC1. IgE-based combination treated mice. Previously, DCs have been shown to express FcsRIa and cross-present antigens to CD8 T cells. Similarly, the results show that splenic DCs from dTg mice could take up anti-MUC1 .lgE-MUC1-FITC complex and stimulate CD8 T cell proliferation. Hence, the data suggested that CD103 ⁺ DC-CD8 T cell interactions underlied the cell-mediated immune response of anti-MUC1.lgE + anti-PD-L1 + PolylCLC.

[00337] The presented results demonstrate an additional component of NK cell activity utilizing anti-MUC1.lgE not seen with anti-MUC1.lgG administration. NK cell depletion abrogated the therapeutic efficacy of anti-MUC1 .IgE- but not anti-MUC1 .IgG-based combination. NK cells execute a rapid response without the need of priming. For NK-mediated cytotoxic killing, FcyRllla expressed on NK cells binds to the Fc regions of IgG antibodies. Interestingly, the expression of FcsRIa on NK cell was not known. Previously, a study showed that NK cells did not express the FcsR receptor; however, incubation of NK cells with IgE-anti-lgE immune complexes induced 10% of FcsR+ expression on NK cells [H. Kimata and A. Saxon, J Clin Invest 82, 160-167 (1988)]. Interestingly, a small proportion of FcsRIcCNK cells in dTg mice was observed herein, and they showed increased degranulation of NKs from anti-MUC1 .IgG + anti-PD-L1 + PolylCLC treated mice in an ex vivo assay. The present results suggest that increased NK cell activity in the present model could be both the direct and also indirect effect of anti-MUC1.lgE via other immune players. Perhaps CD103 ⁺ DCs in the anti-MUC1 .IgG + anti- PD-L1 + PolylCLC treated mice enhances NK activity. Furthermore, no significant impact on NK cell proportions inside the treated tumors was observed. In contrast, the data displayed a substantial reduction in TIGIT and PD-1 expressing NKs population in anti-MUC1.lgE + anti-PD-L1 + PolylCLC treated mice. Given the inhibitory role of TIGIT and PD-1 on NKs, the data demonstrated that anti- MUC1 .IgE-combination therapy improved NK-mediated immunity in tumor-bearing mice. This is further substantiated by the observed increased antibody-mediated killing, degranulation, and IFN-y production by human splenic NKs upon treatment with anti-MUC1 .IgE-based combination therapy. NK- mediated anti-tumor effects mainly centered on IFN-y production, through which it activates cytotoxic T cells, DCs, and macrophages. Given this, it can be concluded that anti-MUC1 . Ig E + PolylCLC + anti- PD-L1 trigger both an innate and adaptive immune player, making it distinct from anti-MUC1.lgG- combination therapy.

[00338] The data also demonstrated significant phosphorylation of SMAD1 , but not SMAD2, protein. Furthermore, blocking of SMAD1 significantly impaired NK cell activity. The TGF-P-SMAD2 pathway contributes to NK cell dysfunction. Activation of SMAD2 by TGF-p inhibits the production of IFN-y by NK cells. In contrast, the BMP-SMAD1 axis has been shown to maintain effector NK cell activity. Furthermore, it is interesting to note that IL-2 and PolylCLC combination has been shown to enhance NK cell activity via the BMP-SMAD1 axis. Of note, anti-MUC1.lgE, combined with PolylCLC + anti-PD-L1 , but not alone, significantly increased SMAD1 phosphorylation at 30- and 120-mins time points. Unexpectedly, no noticeable change in pSMADI following PolylCLC + anti-PD-L1 treatment was observed. Besides, the present results confirmed the presence of pSMADI , but not pSMAD2, in the intra-tumoral NK of anti-MUC1.lgE + PolylCLC + anti-PD-L1 treated mice. SMAD1/5/8 phosphorylation activates downstream target genes, including ID1/2/3. The present results demonstrated significantly increased induction of ID1 in anti-MUC1 .IgE-combination treated NKs. In contrast, while ID2 and ID3 demonstrated moderate induction, TGFB2 expression was downregulated following anti-MUC1 . IgE + PolylCLC + anti-PD-L1 treatment of NKs. NK cells can also produce TGF- P, and TGF-p ⁺ NK cells show a negative regulatory role in HIV infection. Importantly, ID1 regulated IFN-y expression in T cells. The intracellular cytokine results shown herein documented the increased expression of IFN-y, but not granzyme B, following anti-MUC1 .IgE-based combination treatment. Most importantly, the results demonstrated that blocking of BMP pathways via Dorsomorphin and ID1 inhibition attenuated intracellular levels of IFN-y in treated NK cells, further confirming that the pSMAD1-ID1 axis underlies anti-MUC1 . IgE + PolylCLC + anti-PD-L1 -mediated NK cell activation. It is worth noting that this is the first time that the role of NKs in IgE-mediated anti-tumor immunity is demonstrated. In the previous report, anti-folate receptor IgE was shown to control ovarian tumor growth via macrophage activation (D. H. Josephs et al., Cancer Res 77, 1127-1141 (2017)). The authors argued that anti-Folate receptor IgE can promote ADCP-mediated killing by recruiting macrophage through the TNF-oc/MCP-1 axis. Interestingly, D. H. Josephs et al. did not demonstrate NKs and CD8 T cells’ role in their tumor model.

[00339] Again, it is essential to reiterate that IgE’s antigen specificity plays a vital role in executing the anti-tumor response as non-specific IgE, such as OVA-induced IgE, failed to restrict tumor growth in C57BL/6J mice. Furthermore, the results of the acute OVA-induced asthma-PDAC model contradicts the previous notion that the allergic phenotype can reduce the risk of PDAC cases. Of note, the OVA-induced model displays the clinical feature of asthma, such as high serum levels of IgE and airway inflammation. The present study also demonstrated elevated IgE levels upon OVA installation irrespective of pancreatic tumor burden, validating the acute asthmatic phenotype. Nonetheless, the presented data suggested that tumor-targeted IgE, in combination with other immune players, provided robust anti-tumor immunity against pancreatic cancer and hence can help develop better IgE-based therapeutics for PDAC.

EXAMPLE 9

USE OF ANTI-MUC1.IGE + NK-92® CELLS + POLYICLC COMBINATION

[00340] The goal of the following experimental design is to examine if anti-MUC1 .IgE can enhance the therapeutic efficacy of administered isolated NK-92® cells in the preclinical model of pancreatic cancer in athymic nude mice.

[00341] In the studies presented above, anti-MUC1 .IgE based-therapeutic combination has shown tumor-protective benefits in a preclinical model of pancreatic tumors. Furthermore, these studies suggest NK and CD8 T cell-mediated anti-tumor activity of IgE based combination treatment. The major hurdle in PDAC therapies is a scarcity of NK cells in PDAC tumors. Therefore, a combination of anti-MUC1 .IgE with NK-based cellular therapy is believed to be advantageous to PDAC patients. This will be tested using athymic nude mice that do not harbor T and NK cells. These mice are the perfect model for studying immunotherapies against human xenografts. [00342] As a source of isolated NK cells for cell therapy, the NK-92® cell line (available from ATCC, with ATCC Number CRL-2407) is used. NK-92® is an immortal cell line that has features and characteristics of natural killer (NK) cells that every person has circulating in the blood. Blood NK cells and NK-92® cells recognize invaders such as viruses and fungi. NK-92® cells, like blood NK cells, can attack cancer cells. NK-92® cells can be expanded to larger numbers and retain the ability to consistently kill tumor cells. When NK-92® cells bind to a cancer or infected cell, they secrete perforin, which punches holes in target cells, followed by granzymes, which induce apoptosis in the target cells. NK-92® cells also attack cancer cells through the Fas-Fas ligand system and are capable of producing cytokines that by themselves can kill cancer cells (such as TNF-alpha) or stimulate and expand other immune cells such as interferon.

[00343] NK-92® cells also carry the FcyRIII receptor for ADCC assay. Previously, IgE antibodies have shown to initiate downstream signaling via FcyRIII receptor. Hence the model is suitable for examining NK-mediated tumor protective benefits of anti-MUC1.lgE in athymic nude (or NSG mice). In these mice, humanized anti-MUC1 .IgE will only act on NK-92® cells. Additionally, the requirement of polylCLC for inducing expression of the Fc8 receptor, and enhancing the anti-tumor action of anti-MUC1.lgE on NK-92® cells in the treated tumor-bearing mice will be examined.

Experimental Design

[00344] Athymic nude mice (n = 15 for each group) are implanted with MUC1 expressing human pancreatic tumor cell lines (1x 10 ⁶ cells/30 pl) in the pancreas using orthotopic injections. Following tumor implantation, the animals are segregated into the following groups:

1) Saline control;

2) NK-92® cells (adoptively transferred intravenously at days 7, 10, 14, 17, 21 after tumor cell inoculation);

3) Anti-MUC1.lgE (day 8 and every 7 days, 25 pg/1 OOpI, i.p injections);

4) NK-92® cells + Anti-MUC1 .IgE (25 pg/100 pl);

5) NK-92® cells + Anti-MUC1 .IgE (25 pg/ 100 pl) + PolylCLC (200 pg, ip, starting at day 8, every 5 days)+ Isotype control for macrophage depletion;

6) NK-92® cells +PolylCLC (200 pg, ip, starting at day 8, every 5 days);

7) NK-92® cells + Anti-MUC1 .IgE (25 pg/ 100 pl) +PolylCLC (200 pg, ip, starting at day 8, every 5 days) +Macrophage depletion (i.e. anti-CSF1 R treatment); 8) Anti-MUC1 . Ig E (25ug/ 10Oul) +PolylCLC (200ug, i.p, starting at day 8, every 5 days) + Isotype control for macrophage depletion;

9) Anti-MUC1.lgE (25 pg/ 100 pl) +PolylCLC (200 pg, i.p, starting at day 8, every 5 days) + Macrophage Depletion (i.e. anti-CSF1 R treatment);

10) Gemcitabine (40mg/kg, twice a week, i.p).

[00345] The mice are monitored for overall survival and tumor growth using ultrasonography. Animals are sacrificed when they appear moribund. Tumor and spleen are harvested from all treatment groups and MUC1 expression is evaluated in these organs (in time point studies, from 5 mice of each group). In parallel, the presence of NK-92® cells in the tumor and spleen is determined using the NK- 92® cell-specific marker with a flow cytometer. The NK-92® from the spleen or periphery are harvested and tested with ex vivo assays to determine their functionality in the control and all the treatment groups. The impact of macrophage depletion on NK-92® cell activity is examined in the anti-MUC1 . IgE + polylCLC treated mice. Also, tumor cell death I proliferation is evaluated using cleaved caspase-3 and Ki67 staining, respectively.

Outcomes

[00346] Saline control mice are expected to fail to restrict tumor growth and will die within a month of tumor implantation. Anti-MUC1.lgE treatment alone is expected to have a moderate effect on the tumor volume and overall survival of mice. Anti-MUC1 .IgE + NK-92® cells is expected to control tumor growth and prolong the overall survival of mice. Furthermore, the addition of PolylCLC is expected to further increase the anti-tumor response of anti-MUC1 .IgE + NK-92® combination in pancreatic tumor-bearing mice. Macrophage depletion is expected to diminish the therapeutic efficacy of NK-92® cells in anti-MUC1 .IgE-based combination treated mice.

EXAMPLE 10

USE OF ANTI-MUC1.IGE + POLYICLC COMBINATION TO MODULATE MACROPHAGE- MEDIATED ANTI-TUMOR RESPONSE

[00347] The goal of the following experimental design is to examine the efficacy of anti- MUd .lgE alone or in combination with polylCLC in increasing macrophage-mediated anti-tumor response in the preclinical model of pancreatic cancer in athymic nude mice.

[00348] Macrophages are critical players for phagocytosis and have been shown to express FcsRIa receptor. One object of this designs is to test the combination of anti-MUC1 .IgE and PolylCLC to determine if it will significantly modulate the function of macrophages and increase macrophage mediated anti-tumor response in the treated mice. Macrophage depletion is thought to be able to attenuate NK/CD8 T-cell mediated anti-tumor response in anti-MUC1.lgE-based combination treated mice.

Experimental Design

[00349] Double transgenic (dTg) mice described above (n =10 per group) are implanted with MUC1 expressing pancreatic tumor cell lines (1x 10 ⁶ cells/30 pl) in the pancreas using orthotopic injections. Macrophages are depleted using anti-CSF1 R antibody (Bio X Cell BE0213) as follow; (200 pg, 3 times a week until the completion of the experiment, start 2 days before tumor implantation). Following tumor implantation, the animals are segregated into the following groups:

1) Saline control

2) Anti-MUC1.lgE (day 8 and every 7 days, 25 pg/100 pl, i.p injections)

3) Anti-MUC1.lgE (25 pg/ 100 pl) +PolylCLC (200 pg, i.p, starting at day 8, every 5 days)+ isotype control antibody for anti-CSF1 R

4) PolylCLC (200 pg, i.p, starting at day 8, every 5 days)

5) Anti-MUC1 .1 gE (25 pg/ 100 pl) + PolylCLC (200 pg, i.p, starting at day 8, every 5 days) + Anti- CSF1 R (200 pg/100 pl)

6) Gemcitabine (40mg/kg, twice a week, i.p)

[00350] The mice are monitored for overall survival and tumor growth using ultrasonography. Animals are sacrificed when they appear moribund. Tumor and spleen are harvested from all treatment groups and MUC1 expression is evaluated in these organs (in time point studies, from 5 mice of each group). Macrophage depletion is confirmed using flow cytometry for the peripheral blood and tumor. The effect of macrophage depletion on NK and CD8 T cell activity is examined in anti-MUC1.lgE+ polylCLC treated mice. The function of NK and CD8 T cells in the macrophage depleted tumor-bearing mice is also examined. Also, tumor cell death I proliferation is evaluated using cleaved caspase-3 and Ki67 staining, respectively.

Outcomes

[00351] Saline control mice are expected to fail to restrict tumor growth and will die within a month of tumor implantation. Macrophage depletion is expected to significantly impact the anti-tumor efficacy of Anti-MUC1.lgE + polylCLC in tumor bearing mice. A significant increase in tumor growth and attenuated survival in macrophage-depleted and anti-MUC1 .IgE+polylCLC-treated tumor-bearing mice is expected. Macrophage depletion is expected to impair NK cells function in anti- MUC1. IgE+polylCLC-treated tumor-bearing mice. [00352] While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.

SEQUENCE TABLE

Italic underlined = amino acid sequence of variable region from FR1 to FR4

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