NKG2d BI-specific t cell engager and chimeric antigen receptor / t cell receptor cell delivery systems and methods of making and using same

Abstract

The present invention provides NKG2D fusion proteins comprising a domain of NKG2D and a scFv specific for CDS and methods of making and using the same. The fusion proteins provided herein may be administered in combination with CAR T cells. In addition, CAR T cells or other immune cells engineered to express the NKG2D fusion proteins described herein are also provided and may be used in the methods described herein.

Description

[0001] NKG2D BI-SPECIFIC T CELL ENGAGER AND CHIMERIC ANTIGEN RECEPTOR / T CELL RECEPTOR CELL DELIVERY SYSTEMS AND METHODS OF MAKING

[0002] AND USING SAME

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 557,644 filed on February 26, 2024, the contents of which is incorporated by reference in its entireties.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The contents of the electronic sequence listing (15555400762.xml; Size: 53,968bytes; and Date of Creation: February 25, 2025) is herein incorporated by reference in its entirety.

[0007] BACKGROUND

[0008] Advances in immunotherapy have revolutionized the cancer treatment landscape. Immune checkpoint inhibition (ICI) and chimeric antigen receptor (CAR) T cells, in particular, have achieved remarkable success in some solid cancers and in hematological malignancies, respectively. However, many solid tumors, including glioblastoma (GBM), have thus far responded poorly. Hence, GBM still remains nearly uniformly fatal, with a median survival of just 15-18 months. Immunotherapeutic failures have been attributed to significant intratumoral antigenic heterogeneity, as well as to severe tumor-induced local and systemic immunosuppression.

[0009] GBM, like many solid tumors, possesses few tumor-specific antigens (TSA) and exhibits neoantigen expression profiles that vary even cell to cell. EGFRvIII, a mutated form of the epidermal growth factor receptor (EGFR), is the most prominent example of a GBM-specific antigen. It is present, however, in only 30-50% of newly diagnosed tumors, and then in just 30- 50% of cells. As a result, antigen-negative cells frequently persist following, for example, EGFRvIII-directed CAR T cell treatment. Indeed, the inventors own pre-clinical and clinical experiences demonstrate that tumors possessing as few as 5-10% EGFRvIII-negative cells will readily escape EGFRvIII-targeted CARs. Accordingly, antigenic heterogeneity has been identified as a key reason for the failure of EGFRvIII-specific CAR T cells in clinical trials (NCT02664363, NCT03283631). Newer approaches are needed that do not rely on ubiquitous expression of a tumor antigen.

[0010] SUMMARY

[0011] As described herein, the inventors have developed a novel bispecific fusion protein comprising anti-CD3 bound to the NKG2D receptor as a tumor-targeting moiety. Further, the inventors show NKG2D bispecific fusion protein can be combined with a CAR T cell, or a CAR T cell which lacks costimulatory signaling and can secrete the bispecific protein.

[0012] One aspect of the present invention provides an NKG2D fusion protein comprising a NKG2D domain linked to a single chain variable fragment (scFv) specific for CD3. In some embodiments, the NKG2D domain comprises SEQ ID NO: 5 or a sequence having 95% identity to SEQ ID NO: 5, and the scFv specific for CD3 is capable of binding to and activating a T cell receptor. In some embodiments, the scFv specific for CD3 comprises complementarity determining regions of SEQ ID NOs: 36-41 or sequences with at least 95% identity to SEQ ID NO: 36-41, in some embodiments the scFV specific for CD3 comprises SEQ ID NO: 3 or a sequence with at least 95% identity to SEQ ID NO: 3. In some embodiments, the fusion protein comprises a signal sequence. In some embodiments, the fusion protein comprises a tag. In some embodiments, the fusion protein comprises SEQ ID NO: 1 or a sequence with at least 95% identity to SEQ ID NO: 1. In some embodiments, a pharmaceutical composition comprising theNKG2D fusion protein described herein is provided.

[0013] In some embodiments, a construct comprising a polynucleotide sequence encoding the NKG2D fusion protein is provided. In some embodiments, the construct additional comprises a sequence encoding a extracellular domain and a transmembrane domain, wherein the extracellular domain comprises an antigen binding region. In some embodiments, the antigen binding region is capable of binding both a wildtype and or an EGFR VIII variant. In some embodiments, the antigen binding region is capable of binding TIM-4. In some embodiments, the antigen binding region comprises SEQ ID NO: 8, 15 or 25 or a sequence with at least 95% identity to SEQ ID NO: 8, 15 or 25. In some embodiments, the construct further comprises at least one co-stimulatory domain. In some embodiments, the construct comprises a chimeric antigen receptor (CAR), the CAR comprising an extracellular domain, a transmembrane domain, and at least one intracellular signaling domain. In some embodiments, the CAR comprises SEQ ID NO: 6 or SEQ ID NO: 16 or SEQ ID NO: 41, a sequence with at least 95% identity to SEQ ID NO: 6, 16 or 41. In some embodiments, the CAR and the NKG2D fusion protein are connected via a self-cleavage site or an inter ribosome entry site. In some embodiments, the construct comprises a polynucleotide encoding a sequence selected from SEQ ID NO: 19, 20, 22, 23, 26 or 42 or a sequence with at least 95% sequence identity to SEQ ID NO: 19, 20, 22, 23, 26 or 42.

[0014] In some embodiments an immune cell comprising a construct described herein is provided. In some embodiments, the immune cell is a T lymphocyte, a macrophage cell or a natural killer (NK) cell.

[0015] Another embodiment of the present disclosure provides a method for treating cancer. In some embodiments, the method comprises administering a therapeutically effective amount of a NKG2D fusion protein described herein or a pharmaceutical composition comprising an NKG2D fusion protein with a pharmaceutically acceptable excipient, carrier and / or diluent to a subject, wherein the subject has cancer. In some embodiments, the method additionally comprises administered T lymphocytes. In some embodiments, the T lymphocytes are CAR-T cells. In some embodiments, a method of treating cancer is provided, the method comprises administering a therapeutically effective amount of an immune cell described herein and a pharmaceutically acceptable excipient, carrier and / or diluent to a subject, wherein the subject has cancer. In some embodiments a method for treating caner is provided, the method comprising administer a cell comprising a construct provided herein. In some embodiments, the cancer is a EGFR-associated cancer or a phosphatidylserine (PS) associated cancer. In some embodiments, the treatment of the cancer results in the induction of an anti-tumor immune response to the EGFR or PS-associated cancer.

[0016] Anther aspect of the present disclosure proves a method of detecting cancer. In some embodiments, the method comprises contacting a sample comprising cells or tissue from a subject with a fusion protein described herein or the NKG2D domain of SEQ ID NO:5 linked to a tag, detecting the presence or accumulation of the tag on the sample, and detecting cancer in the sample, wherein the accumulation or presence of the tag allows for ex-vivo detection of cancer. In some embodiments, the cancer detected is a glioma, glioblastoma, medulloblastoma, ependymoma, diffuse intrinsic pontine glioma (DIPG), a brain metastases, head and neck, ovarian, cervical, bladder or esophageal cancer.

[0017] Another aspect of the present disclosure provides a method of inducing an immune response in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a fusion protein, pharmaceutical composition or immune cell described herein, wherein an antitumor immune response is induced in the subject. In some embodiments, the immune response is an antitumor immune response.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying Figures and Examples are provided by way of illustration and not by way of limitation. The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying example figures (also “FIG ”) relating to one or more embodiments.

[0020] Figure 1. NKG2D BEAR redirects T cells to kill U87 glioma cells. A. Schematic showing the predicted structure of NKG2D BEAR consisting of the extracellular domain of NKG2D, a (Glycine4Serine)3 linker, and anti-CD3scFv. The predicted structure of NKG2D alone is overlaid to show that the NKG2D structure is unaltered by the fusion to anti-CD3scFv. B. Human CD3+T cells were co-cultured at a 10: 1 ratio with CellTrace Far Red-labeled U87 human glioma cells. Increasing volumes of NKG2D BEAR HEK293 expression supernatant or control media was added to the co-culture. After 24 hours, remaining U87 cells were quantified by flow cytometry and normalized to untreated samples (Tumor + T cells only) to give a “Percent Survival”.

[0021] Figure 2. CARE-BEAR Design. Schematics showing the design of human (A) and mouse (B) VIII CARE-BEAR. The VIII CAR is encoded upstream of the NKG2D BEAR in a bicistronic message, with the CAR and BEAR being separated by a 2A self-cleavage site to produce two separate proteins. The VIII CAR was used as a proof-of-concept but this domain can be interchanged with any other CAR or TCR or antigen-targeting moiety.

[0022] Figure 3. Generation of VIII CARE-BEAR. Activated human T cells were retrovirally transduced with VIII CAR or VIII CARE-BEAR, or left non-transduced (NT). 7 days later, T cells were stained with PEPvIII-AF647 to detect VIII CAR (A) or anti-human NKG2D to detect surface bound NKG2D BEAR (B).

[0023] Figure 4. VIII CARE-BEAR kills EGFRvIII-negative U87 glioma and M202 melanoma in vitro. 10,000 U87-GFP cells (A) or 10,000 CellTrace Far Red-stained M202 cells (B) were plated in 96-well plates. The appropriate number of NT T cells, VIII CAR, or VIII CAREBEAR was added to each well to give the indicated E:T Ratio. After 24 hours, remaining tumor cells were quantified via flow cytometry and normalized to Tumor Only wells to give a “Percent Survival”.

[0024] Figure 5. VIII CARE-BEAR is superior to VIII CAR in killing EGFRvIII heterogeneous tumors in vitro. 7,000 U87-GFP cells and 3,000 CellTrace Far Red-stained U87vIII cells were plated in 96-well plates. The appropriate number of NT T cells, VIII CAR, or VIII CARE-BEAR was added to each well to give the indicated E:T Ratio. After 24 hours, remaining tumor cells were quantified via flow cytometry and normalized to Tumor Only wells to give a “Percent Survival”. Survival is shown for the entire tumor (A), and the U87 (B) and U87vIII (C) subsets from the heterogeneous tumor.

[0025] Figure 6: Generation and functional characterization of murine VIII CARE-BEAR. A. Activated mouse T cells were retrovirally transduced with VIII CAR or VIII CARE-BEAR or nontransduced (NT T cells). 72 hours post-transduction, T cells were stained with PepVIII-AF647 to detect surface VIII CAR expression. B. 10,000 CT2A murine glioma cells (EGFRvIII-negative) were co-cultured with murine NT T cells, murine VIII CAR, or murine VIII CARE-BEAR at the designated E:T ratios. After 24 hours, remaining CT2A cells were quantified via flow and normalized to Tumor Only wells to give a “Percent Survival”.

[0026] Figure 7: Murine VIII CARE-BEAR prolongs survival compared to VIII CAR in an immunocompetent glioma model in vivo. A mixture of 37,500 CT2A and 12,500 CT2AvIII was implanted intracranially in C57BL / 6 mice. 7 days later, 2 x 106VIII CAR or VIII CARE-BEAR were administered at the tumor site (or mice were left untreated). Mice were followed for survival. The median survival for untreated, VIII CAR, and VIII CARE-BEAR were 17.5, 17, and 20.5 days, respectively.

[0027] Figure 8: Human VIII CARE-BEAR has superior efficacy compared to VIII CAR when treating U87vIII heterogenous glioma in vivo. A mixture of 17,500 U87 and 7,500 U87vIII human glioma cells was implanted intracranially in NSG mice. 7 days later, 2 x 106VIII CAR or VIII CARE -BEAR were administered at the tumor site (or mice were left untreated). Mice were followed for survival. The median survival for untreated, VIII CAR, and VIII CARE -BEAR were 21, 25, and 38 days, respectively.

[0028] Figure 9: VIII CARE-BEAR has superior efficacy compared to VIII CAR against a patient-derived glioblastoma. A-B. Ex vivo expanded patient-derived glioblastoma cells (GBM PDX) were stained for expression of EGFRvIII (A) or the NKG2DLs MICAZB (B). C. 10,000 CellTrace Far Red-stained GBM PDX cells were plated in 96-well plates. The appropriate number of NT T cells, VIII CAR T cells, or VIII CARE-BEAR T cells was added to each well to give the indicated E:T Ratio. After 24 hours, remaining tumor cells were quantified via flow cytometry and normalized to Tumor Only wells to give a “Percent Survival”.

[0029] Figure 10: CARE-BEAR is effective against human lung and breast cancer in vitro. A,C. A549 human non-small cell lung cancer (NSCLC) cells (A) or MDA-MB-468 human triple negative breast cancer (TNBC) cells (C) were stained for expression of MICA / B. B,D. 10,000 CellTrace Far Red-stained A549 lung cancer cells (B) or MDA-MB-468 TNBC cells (D) were plated in 96-well plates. The appropriate number of NT T cells or VIII CARE-BEAR was added to each well to give the indicated E:T Ratio. After 24 hours, remaining tumor cells were quantified via flow cytometry and normalized to Tumor Only wells to give a “Percent Survival”.

[0030] Figure 11. D2C7 CARE-BEAR kills EGFR-negative U87 glioma and in vitro. A. Schematic of D2C7 CARE-BEAR B. 10,000 CellTrace Far Red-stained U87EGFRko cells were plated in 96-well plates. The appropriate number of NT T cells, D2C7 CAR, or D2C7 CAREBEAR was added to each well to give the indicated E:T Ratio. After 24 hours, remaining tumor cells were quantified via flow cytometry and normalized to Tumor Only wells to give a “Percent Survival”.

[0031] Figure 12: D2C7 CARE-BEAR has superior efficacy compared to D2C7 CAR when treating EGFR heterogenous glioma in vivo. A mixture of 17,500 U87EGFRko and 7,500 U87 human glioma cells was implanted intracranially in NSG mice. 7 days later, 2 x 106VIII CAR or VIII CARE-BEAR were administered at the tumor site (or mice were left untreated). Mice were followed for survival. The median survival for untreated, VIII CAR, and VIII CARE-BEAR were 35, 87, and 126 days, respectively.

[0032] Figure 13: VIII CARE-BEAR modified to include VIII CAR targeting but not VIII CAR signaling. A. Schematic showing the design of VIII CARE-BEAR with the CAR signaling domain deleted (VIII CARE-BEAR CARsigdel). B,C. NT T cells, VIII CARE-BEAR T cells, or VIII CARE-BEAR CARsigdel T cells were co-cultured with a GBM PDX (B) or A549 lung cancer (C) cells at the indicated effectortarget ratio. After 24 hours, remaining tumor cells were quantified via flow cytometry and normalized to Tumor Only wells to give a “Percent Survival”.

[0033] Figure 14: Engineering a non-tumor-targeting T cell to secrete NKG2D BEAR. A. Schematic showing the design of a T cell (No CAR) designed to secrete NKG2D BEAR (T BEAR). B. Validation that T BEAR lacks expression of CAR on the surface (top) but still secretes NKG2D BEAR (bottom). C. NT T cells, VIII CAR T cells, VIII CARE-BEAR T cells, or T BEAR T cells were co-cultured with U87 GBM cells at the indicated effectortarget ratio. After 24 hours, remaining tumor cells were quantified via flow cytometry and normalized to Tumor Only wells to give a “Percent Survival”.

[0034] Figure 15: NKG2D BEAR is secreted by T cells and mediates killing of CAR-antigen- negative tumor cells. A. VIII CAR, VIII CAREBEAR, and T BEAR T cells were expanded for 3 days. The supernatant from each T cell culture was concentrated and then incubated with normal CD3+T cells for 30 minutes at 4C. T cells were washed extensively to remove excess NKG2D BEAR and then stained with anti-NKG2D. B. Normal CD3+T cells were co-cultured at a 5: 1 ratio with U87 tumor cells in the presence of the indicated volume of concentrated T cell supernatant from VIII CAR, VIII CAREBEAR, or T BEAR cultures. After 24 hours, remaining tumor cells were quantified via flow cytometry and normalized to “Oul” wells to give a “Percent Survival”.

[0035] Figure 16: CAR signaling and targeting is not required for CAREBEAR efficacy when administered directly to the tumor site. A. A 70:30 mixture of U87:U87vIII (25,000 cells total) was implanted IC in NSG mice. 7 days later, 2xl06VIII CAR T cells, VIII CAREBEAR T cells, VIII CAREBEAR CARsigdel T cells, or T BEAR T cells were administered at the tumor site. B. 25,000 U87 cells were implanted IC in NSG mice. 7 days later, 2xl06VIII CAR T cells, VIII CAREBEAR T cells, or T BEAR T cells were administered at the tumor site. Mice were monitored for survival.

[0036] DETAILED DESCRIPTION

[0037] Finding a targetable antigen that is expressed on the surface of all tumor cells remains a goal of antigen-specific T cell-based immunotherapies. Non-classical MHC-I consists of multiple proteins that are upregulated on stressed cells, including tumor cells and have low expression on healthy tissue. As described herein, the inventors have developed a novel bispecific fusion protein comprising anti-CD3 bound to the NKG2D receptor as a tumor-targeting moiety. The inventors term this therapeutic fusion protein “Bi-specific Engager without Antigen Requirements,” or BEAR. Further, the inventors show BEAR can be combined with a CAR T cell, which can deliver or secrete the BEAR, which will 1) provide an additional effector T cell population to be bound and activated by BEAR and 2) reduce the potential for systemic toxicity by restricting BEAR to the tumor microenvironment (TME). This platform the inventors term, “CARs Engineered with BEAR,” or CARE-BEAR™. The cytotoxic function of NKG2D on CD8+ T cells normally relies on the concurrent activation of the T cell receptor (TCR). However, by linking the extracellular domain of the NKG2D receptor to a CD3 activating domain, the inventors are obviating the need for concurrent TCR activation. Further, targeting non-classical MHC-I maintains target specificity as non-classical MHC-I is highly and ubiquitously expressed by tumor cells. Lastly, non-classical MHC-I is made an even more attractive target clinically because its expression on tumors is enhanced by cellular stress from radiation and chemotherapy, both of which are standard of care for GBM and many other solid tumors.

[0038] Fusion proteins and constructs

[0039] One aspect of the present disclosure provides a NKG2D fusion protein comprising a NKG2D domain linked to a single chain variable fragment (scFv) specific for CD3. As used herein, a “fusion protein” may also be called a chimeric protein and are proteins created through the joining of two or more genes or portions thereof that originally encoded for separate proteins. Translation of the fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. Fusion proteins may occur in the body by transfer of DNA between chromosomes or be made recombinantly by combining genes or parts of genes via genetic engineering methods such that a single transcript is formed encoding both polypeptides in frame from the same or different organisms.

[0040] NKG2D (natural killer group 2, member D) is a receptor belonging to the NKG2 family of C-type lectin-like receptors. NKG2D is encoded by KLRK1 gene which is located in the NK-gene complex. The NKG2D domain of the present disclosure may comprise SEQ ID NO: 5, or a sequence with at least 95% identity to SEQ ID NO: 5. One mechanism for tumor escape tumor MHC1 downregulation. The inventors previously demonstrated that CD8+ T cells maintain the capacity to kill tumor cells that are entirely devoid of MHC-I expression. This capacity proves to be dependent instead on interactions between T cell natural killer group 2D (NKG2D) and tumor NKG2D ligands (NKG2DLs), the latter of which are highly expressed on MHC-loss variants.

[0041] In some embodiments, the NKG2D domain is linked to a single chain variable fragment (scFv) specific for CD3. CD3 (cluster of differentiation 3) is a protein complex and T cell coreceptor that is involved in activating both the cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). It is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD36 chain, and two CD3e chains. These chains associate with the T- cell receptor (TCR) and the CD3-zeta ((^-chain) to generate an activation signal in T lymphocytes. The recombinant NKG2D fusion protein of the present disclosure may be linked via a linker to a single-chain variable fragment (scFv) specific for CD3. A scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker. The scFv specific for CD3 may comprise complementarity determining regions (CDR) of SEQ ID NO: 36 (VH CDR1), SEQ ID NO: 37 (VH CDR2), SEQ ID NO: 38 (VH CDR3), SEQ ID NO: 39 (VL CDR1), SEQ ID NO: 40 (VL CDR2), and SEQ ID NO: 41 (VL CDR3) or a sequence with at least 95% identity to any one of SEQ ID NOs: 36-41. The anti-CD3 scFv of the present disclosure may comprise SEQ ID NO: 3 or a sequence with at least 95% identity to SEQ ID NO: 3. The scFv specific for CD3 is capable of binding to and activating T cells via CD3. The NKG2D fusion protein may comprise the scFv specific for CD3 or another antigen binding fragment specific for CD3. For example, an antigen-binding fragment selected from the group consisting of a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), and a singlechain antibody molecule (scFv).

[0042] In some embodiments, the NKG2D domain is linked to a single chain variable fragment (scFv) specific for CD3 via a linker peptide. A peptide linker is a short peptide sequence that occurs between protein domains. Linkers are often composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Linkers can be varied in length and content as necessary. In some embodiments, the linker is a glycine serine linker. In some embodiments, the linker comprises at least one copy of SEQ ID NO: 4. In some embodiments, the linker comprises three copies of SEQ ID NO: 4, such that SEQ ID NO: 4 is repeated three times.

[0043] The NKG2D fusion protein may further comprise an N-terminal signal sequence. The N- terminal signal sequence may comprise a secretion signal sequence. A secretion signal sequence or peptide comprises a short peptide present at the N-terminus of proteins that are destined toward the secretory pathway. These proteins include those that reside either inside certain organelles, secreted from the cell, or inserted into cellular membranes. Signal sequences may be removed or cleaved off from the fusion protein. Thus, when administered as a protein, the NKG2D fusion protein will generally lack the signal sequence, but the signal sequence may be needed to allow for adequate expression and secretion of the protein. The nucleotide sequence encoding the protein will generally encode for the signal sequence. In some embodiments, the secretion signal sequence includes a signal sequence of SEQ ID NO: 7 or a sequence with at least 90% identity to SEQ ID NO: 7. Signal sequences are generally 15-22 amino acids in length and those of skill in the art will appreciate that one signal sequence can be removed and another signal sequence used in its place.

[0044] The fusion proteins provided herein may additionally comprise a tag. The tag may by attached to the protein for various purposes. The tag may comprise any known tag in the art and may be used for the purposes of purification, tracking or imaging the fusion protein. Types of tags may comprise an affinity tag, a solubilization tag, chromatography tag, epitope tag, fluorescent tag or enzymatic modification tag. In some embodiments, the tag may comprise a His tag, a FLAG tag, a fluorescent tag or a M c tag.

[0045] The fusion protein may comprise a NKG2D domain, a linker and a CD3 scFv. The NKG2D domain and the CD3scFv may be in any order relative to each other. For example, the NKG2D domain may be on the N-terminus of the fusion protein and the CD3 scFv may be on the C- terminus. Alternatively, the CD3 scFv may be on the N-terminus and the NKG2D domain may be on the C-terminus. For example, the fusion protein may comprise SEQ ID NO: 3, 4 and 5 or a sequence with at least 95% identity to any one of SEQ ID NO: 3, 4 or 5, wherein SEQ ID NO: 3 and SEQ ID NO: 5 are linked in any order relative to each other via SEQ ID NO: 4. The fusion protein may comprise SEQ ID NO: 2, 3, 4 and 5 or a sequence with at least 95% identity to any one of SEQ ID NO: 2, 3, 4 or 5. The fusion protein may comprise SEQ ID NO: 1. The fusion protein may comprise a sequence with at least 95% identity to any one of SEQ ID NO: 3, 4 or 5 and a tag, or a sequence with at least 95% identity to any one of SEQ ID NO: 2, 3, 4 or 5 and a tag, or a sequence with at least 95% identity to SEQ ID NO: 1 and a tag. A NKG2D fusion protein may be called a Bi-specific Engager without Antigen Requirements,” or BEAR.

[0046] As used herein, the term “bispecific T-cell engaging molecule” refers to a molecule designed to harness a subject's T cells to kill cancer cells by targeting T cells to the tumor cells expressing a desired molecule. In certain embodiments, the desired molecule is the NKG2D ligand. In other embodiments, the bispecific T cell engaging molecules comprises an NKG2D domain capable of binding the NKG2D ligand linked to an scFv capable of binding to T cells such as the CD3 specific scFv. As used herein, “polypeptide,” “peptide,” and “protein” are used interchangeably and include reference to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms also apply to polymers containing conservative amino acid substitutions such that the protein remains functional.

[0047] As used herein, “nucleic acid” or “nucleic acid sequence” includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof as well as conservative variants, i.e., nucleic acids present in wobble positions of codons and variants that, when translated into a protein, result in a conservative substitution of an amino acid.

[0048] As used herein, “encoding” with respect to a specified nucleic acid, includes reference to nucleic acids which comprise the information for translation into the specified protein. The information is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. However, variants of the universal code, such as is present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum (Proc. Nat'l Acad. Sci. USA 82:2306-2309 (1985), or the ciliate Macronucleus, may be used when the nucleic acid is expressed in using the translational machinery of these organisms. Any codon for an amino acid may be substituted for another codon for the same amino acid without changing the encoded protein. Translation efficiency may, however, be modified.

[0049] As used herein, “expressed” includes reference to translation of a nucleic acid into a protein. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane or be secreted into the extracellular matrix or medium.

[0050] The present disclosure provides pharmaceutical compositions comprising one or more of the compositions or fusion proteins as described herein and an appropriate carrier, excipient or diluent. The exact nature of the carrier, excipient or diluent will depend upon the desired use for the composition and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use. The composition may optionally include one or more additional compounds. When used to treat or prevent a disease or symptoms of a disease, such as cancer, the compositions described herein may be administered singly, as mixtures of one or more compounds or in mixture or combination with other agents (e.g., therapeutic agents) useful for treating such diseases and / or the symptoms associated with such diseases. The compounds may be administered in the form of compounds (i.e. proteins) per se, or as pharmaceutical compositions comprising a compound.

[0051] Pharmaceutical compositions comprising the compositions may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the compositions into preparations which can be used pharmaceutically. Pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.

[0052] The compositions described herein will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent cancer. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and / or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also generally includes halting or slowing the progression of the disease, regardless of whether improvement is realized. Treating cancer includes, but is not limited to, reducing the number of cancer cells or the size of a tumor in the subject, reducing progression of a cancer to a more aggressive form, reducing proliferation of cancer cells or reducing the speed of tumor growth, killing of cancer cells, reducing metastasis of cancer cells or reducing the likelihood of recurrence of a cancer in a subject. Treating a subject as used herein refers to any type of treatment that imparts a benefit to a subject afflicted with a disease or at risk of developing the disease, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay the onset of symptoms or slow the progression of symptoms, etc.

[0053] The amount of composition administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular composition, the conversion rate and efficiency of delivery under the selected route of administration, etc.

[0054] Determination of an effective dosage for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage for use in animals may be formulated to achieve a circulating blood or serum concentration of the composition that is at or above an IC50 of the particular composition as measured in an in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular composition via the desired route of administration is well within the capabilities of skilled artisans. Initial dosages can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and / or metabolism of compositions are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine dosages suitable for human administration.

[0055] Dosage amounts will typically be in the range of from about 0.0001 mg / kg / day, 0.001 mg / kg / day or 0.01 mg / kg / day to about 100 mg / kg / day, but may be higher or lower, depending upon, among other factors, the activity of the active composition, the bioavailability of the composition, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels which are sufficient to maintain therapeutic or prophylactic effect. For example, the compositions may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of compositions may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.

[0056] The dose of the NKG2D fusion protein administered to an individual (such as a human) may vary with the particular composition, the mode of administration, and the type of disease being treated. In some embodiments, the amount of the composition is effective to result in an objective response (such as a partial response or a complete response). In some embodiments, the amount of a NKG2D fusion protein in the composition is included in a range of, e.g., about 0.001 pg to about 1000 pg. In some embodiments of any of the above aspects, the effective amount of a NKG2D fusion protein in the composition is in the range of about 0.1 pg / kg to about 100 mg / kg of total body weight.

[0057] The NKG2D fusion protein can be administered to an individual (such as human) via various routes, including, for example, intravenous, intra-arterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intra-tracheal, subcutaneous, intradermal, intraocular, intrathecal, transmucosal, intratumoral, and transdermal. In some embodiments, sustained continuous release formulation of the composition may be used.

[0058] In some embodiments a construct comprising a polynucleotide sequence encoding any NKG2D fusion protein described herein is provided. The term "construct" or "polynucleotide construct" is a polynucleotide, either DNA or RNA, which allows the encoded sequence to be replicated and / or expressed in the target cell. A construct may contain an exogenous promoter, operably linked to any one of the polynucleotides described herein. As used herein, a polynucleotide is “operably connected” or “operably linked” when it is placed into a functional relationship with a second polynucleotide sequence. The term "operably linked" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0059] As used herein, the terms “heterologous promoter,” “promoter,” “promoter region,” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5’ or 3’ side of polynucleotides described herein, or within the coding region of said polynucleotides. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3’ direction) coding sequence. The typical 5’ promoter sequence is bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0060] In some embodiments, the construct is in an expression construct, a vector or a viral vector. A vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence, typically DNA into another cell, where it can be replicated and / or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Expression constructs comprise a heterologous promoter and the nucleic acid sequence encoding protein of interest (e.g., NKG2D, CD3 scFv) which is capable of expression in the cell in which it is introduced. The expression constructs include vectors which are capable of directing the expression of exogenous genes to which they are operatively linked. Such vectors are referred to herein as "recombinant constructs," "expression constructs," "recombinant expression vectors" (or simply, "expression vectors" or "vectors") and may be used interchangeably. Suitable vectors are known in the art and contain the necessary elements in order for the gene encoded within the vector to be expressed as a protein in the host cell. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, specifically exogenous DNA segments encoding the fusion proteins provided herein. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Viral vectors are incorporated into viral particles that are then used to transport the viral polynucleotide encoding the protein of interest into the target cells. Certain vectors are capable of autonomous replication in a host cell into which they are introduced. Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., lentiviral vectors). Moreover, certain vectors are capable of directing the expression of exogenous genes to which they are operatively linked. In general, vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification "vector" include expression vectors, such as viral vectors (e.g., replication defective retroviruses (including lentiviruses), adenoviruses and adeno-associated viruses (AAV)), which serve equivalent functions.

[0061] The vectors are heterogeneous exogenous constructs containing sequences from two or more different sources. Suitable vectors include, but are not limited to, plasmids, expression vectors, lentiviruses (lentiviral vectors), adeno-associated viral vectors (rAAV), among others and includes constructs that are able to express the protein of interest in cells. A preferred vector is a lentiviral vector or adeno-associated vector. Suitable methods of making viral particles are known in the art to be able to transform cells in order to express the protein of interest in cells. The cells may be immune cells, including, but not limited to a T lymphocyte, natural killer (NK) cell, macrophage, and B lymphocytes. The cells may be CAR T cells. The CAR T cells may be capable of targeting the cancer. Those of skill in the art can determine other CAR T cells that may be used in combination with the NKG2D fusion proteins and constructs encoding the NKG2D fusion proteins provided herein.

[0062] In some embodiments the present disclosure provides a construct described herein operably linked to the polynucleotide sequence encoding the NKG2D fusion protein. Heterologous promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally regulated, chemically regulated, tissuepreferred, tissue-specific promoters and cell- type specific. The heterologous promoter may be a plant, animal, bacterial, fungal, or synthetic promoter. Suitable promoters are known and described in the art. In mammalian cells, typical promoters include, without limitation, promoters for Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), SV40 virus, as well as the translational elongation factor EF-la promoter, actin, or ubiquitin promoter.

[0063] In some embodiments a construct comprising a polynucleotide sequence encoding any NKG2D fusion protein described herein may further comprise a targeting sequence encoding an extracellular domain and a transmembrane domain. The extracellular domain of the targeting sequence comprises an antigen binding region capable of targeting a cell engineered to express it to a cancer cell. In some embodiments, the extracellular domain is a tumor-associated antigen or a tumor specific antigen. In some embodiments, the construct further comprises a self-cleavage site, or internal ribosome entry site between the transmembrane domain / targeting sequence and the NKG2D domain. In some embodiments, a construct may comprise: a promoter, a signal peptide, an extracellular domain, a transmembrane domain, a self-cleavage site, and an NKG2D fusion protein described herein comprising a NKG2D domain, a linker and an CD3 scFv. In some embodiments, the antigen binding region is specific for a cancer antigen or target. In some embodiments the antigen binding region is capable of binding to a wildtype EGFR and or an EGFRVIII variant. In some embodiments, the antigen binding region is capable of binding TIM- 4.

[0064] A construct or fusion protein comprising an extracellular domain and a transmembrane domain may also be called a CARsigdel. In some embodiments, a CARsigdel comprises a promoter, an extracellular domain, a hinge and transmembrane domain, a self-cleavage site and a NKG2D fusion protein described herein, and may be referred to as a CAREBEAR CARsigdel. A CAREBEAR CARsigdel is a variation of the CAREBEAR described herein, which retains antigen specificity but lacks costimulatory and CD3 signaling of a traditional chimeric antigen receptor.

[0065] A construct or fusion protein of the present disclosure may comprise an extracellular domain comprising an antigen binding region which binds to a wild type and or a variant of the epidermal growth factor receptor (EGFR). EGFR is also known as ErbB-1, FIERI and is a receptor that becomes activated by binding to specific ligands, including members of the EGF family of extracellular ligands. EGFR vIII is a variant of EGFR with an extracellular deletion mutation. EGFR vIII is expressed in some tumors, and considered a tumor specific antigen in glioblastoma tumors, despite being present in only 30% of tumors, and only approximately 30-50% of cells in the tumors. EGFRvIII means a mutant form of the epidermal growth factor receptor recognized by MR1 scFv and characterized by an 801 base pair in frame deletion of exons 2 to 7 near the amino terminal. This form of the receptor is known in the art, as exemplified by the Wickstrand et al., Moscatello et al., and Lorimer et al. references cited in the Background. Due to a change in terminology, EGFRvIII was originally termed a Type II mutation in some earlier work in the field, as exemplified by U.S. Pat. No. 5,212,290.

[0066] An antigen binding region that binds to both a wild-type and an EGFRVIII variant may be referred to as D2C7. A CAR or CARsigdel of the present disclosure may include an antigen binding agent capable of binding the EGFR VIII variant or D2C7. The extracellular domain specific for EGFRVIII may comprise SEQ ID NO: 8, or a sequence with at least 95% identity to SEQ ID NO: 8. The extracellular domain for D2C7 may comprise SEQ ID NO: 15, or a sequence with at least 95% identity to SEQ ID NO: 15. The CAR or CARsigdel of the present disclosure comprises an extracellular domain comprising an antigen binding region which binds to T cell membrane protein 4, or TIM-4 (also may be known as T cell membrane protein 4 or T-cell immunoglobulin and mucin domain containing 4). TIM-4 is a protein in humans encoded by the TIMD4 gene. TIM-4 is a phosphatidylserine (PS) receptor that is expressed on various immune cell and macrophage subsets. PS is a phospholipid typically present on the cytoplasm-facing side of the plasma membrane, where it remains out of view to the immune system. Cellular stresses can lead to dysregulation of processes that keep PS facing internally and instead promote PS exposure on the cell surface. Tumor cells frequently lose the capacity to regulate their plasma membrane and exhibit detectable levels of PS on their surface, as may normal cells undergoing apoptosis. PS can have a tolerizing effect on the immune system when exposed on the cell surface. While PS exposure thus can be tumor-adaptive, it also leaves the tumor with an “Achilles Heel” that can be targeted immunologically or otherwise. For instance, TIM family proteins bind PS with varying affinity, the strongest binder being TIM-4. The extracellular domain for TIM-4 may comprise SEQ ID NO: 25, or a sequence with at least 95% identity to SEQ ID NO: 25.

[0067] The extracellular domain antigen binding region of the present disclosure may comprise a single chain variable fragment (scFv) which is comprised of six complementarity determining regions. Complementary determining regions, or CDR, or CDRs, are hypervariable domains that determine specific antigen binding. scFv are polypeptides that contain the variable light chain and variable heavy chain of an antibody connected by a flexible linker peptide. The scFv of the present disclosure may comprise the scFv of any one of SEQ ID NOs: 8 or 15 or sequences with at least 95% identity to any one of SEQ ID NOs: 8 or 15.

[0068] The linker may be 10-25 amino acids long and made up of glycine and serine amino acids, as well as optionally with dispersed hydrophilic residues to increase solubility. The linker keeps each of the variable regions at a distance that favors proper folding and formation of the antigenbinding site while also minimizing oligomerization of the scFv. A simple form of a linker is the hinge region of IgGl . The linker of the present disclosure may be a linker of SEQ ID NO: 9 or at least one copy of SEQ ID NO: 4, or a sequence with at least 95% identity to either SEQ ID NO: 9 or 4.

[0069] The CAR or CARsigdel of the present disclosure may comprise a transmembrane and hinge sequence. A hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence may be positioned between the antigen recognition moiety or extracellular domain (e.g. EGFR / EGFRvIII scFv or TIM-4 extracellular domain; SEQ ID NO: 25) and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

[0070] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon , CD45, CD4, CD5, CD8 (e.g. , CD8 alpha, CD8 beta), CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD11 a, CD18) , ICOS (CD278) , 4-1 BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1 ) , CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 11 d, ITGAE, CD 103, IT GAL, CD 11 a, LFA- 1 , ITGAM , CD11 b, ITGAX, CD11 c, ITGB1 , CD29, ITGB2, CD 18, LFA- 1 , ITGB7, TNFR2, DNAM 1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM 1 , CRTAM , Ly9 (CD229) , CD160 (BY55) , PSGL1 , CD 100 (SEMA4D) , SLAMF6 (NTB-A, Lyl08) , SLAM (SLAMF1 , CD 150, IPO-3) , BLAME (SLAMF8) , SELPLG (CD 162) , LTBR, and PAG / Cbp. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. A short oligo- or polypeptide linker, such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the endoplasmic domain of the CAR. In some embodiments, the CAR has more than one transmembrane domain, which can be a repeat of the same transmembrane domain or can be different transmembrane domains. The CAR of the present disclosure may comprise a hinge and transmembrane domain of SEQ ID NO: 10 or sequences with at least 95% identity to SEQ ID NO: 10.

[0071] In some embodiments, the construct may comprise a polynucleotide sequence encoding an NKG2D fusion protein and a signal peptide of SEQ ID NO: 7, an antigen binding domain of SEQ ID NO: 8, SEQ ID NO: 15 or SEQ ID NO: 25, a linker of SEQ ID NO: 9, and a hinge of SEQ ID NO: 10. In some embodiments, the construct may comprise a polynucleotide sequence encoding an NKG2D fusion protein and SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 42 or a sequence with at least 95% identity to SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 42. In some embodiments, the construct may comprise SEQ ID NO: 21, SEQ ID NO:24, SEQ ID NO: 27 or a sequence with at least 95% identity to SEQ ID NO: 21, SEQ ID NO:24, SEQ ID NO: 27.

[0072] In some embodiments a construct comprising a polynucleotide sequence encoding any NKG2D fusion protein described herein may further comprise a sequence encoding a chimeric antigen receptor (CAR) is provided. The CAR and the NKG2D fusion protein may be expressed using distinct promoters or may be expressed using a single promoter and may be on a single construct or expression vector or may be on separate constructs or expression vectors to transfect or be introduced into a cell. The term "chimeric antigen receptor" or “chimeric receptor” or "CAR" or "CARs" as used herein refers to a polypeptide having a pre-defined binding specificity to a desired target and operably connected to (e.g., as a fusion or as separate chains linked by one or more disulfide bonds, etc.) the intracellular part of a T-cell activation domain. More particularly, CAR are engineered receptors, which, when expressed graft an antigen specificity onto a cytotoxic cell, for example T cells, NK cells or macrophages. For example, CAR proteins are engineered to give T cells the new ability to target a specific protein. The CARs of the present invention may comprise an extracellular domain with at least one antigen specific targeting region, a transmembrane domain (TM), an intracellular domain including one or more co-stimulatory domains (CSD) and a signaling domain in a combination that is not naturally found together on a single protein. This particularly includes receptors wherein the extracellular domain and the cytoplasmic domain are not naturally found together on a single receptor protein. Further, the chimeric receptor is different from the TCR expressed in the native T cell lymphocyte.

[0073] The term “CAR- immune cell” as used herein refers to an immune cell (for example a T lymphocyte, NK cell, or macrophage) or population thereof, which has been modified through molecular biological methods to express a chimeric antigen receptor (CAR) on the cell surface. In some embodiments the CAR-immune cell is a CAR-T cell. The CAR is a polypeptide having a pre-defined binding specificity to a desired target expressed and operably connected to (e.g., as a fusion, separate chains linked by one or more disulfide bonds, etc.) to the intracellular part of a T- cell activation domain. By bypassing MHC class I and class II restriction, CAR engineered T cells of both CD8 and CD4+subsets can be recruited for redirected target cell recognition. The most common CARs are fusions of immunoglobulin binding functionality e.g., as a single-chain variable fragment (scFv) derived from a monoclonal antibody) to CD3-zeta (CD3Q transmembrane and endodomain. Such molecules result in the transmission of a zeta signal in response to recognition by the immunoglobulin binding functionality of its target. There are, however, many alternatives. By way of example, an antigen recognition domain from native T- cell receptor (TCR) alpha and beta single chains may be used as the binding functionality. Alternatively, receptor ectodomains (e.g. CD4 ectodomain) or cytokines (which leads to recognition of cells bearing the cognate cytokine receptor) may be employed. All that is required of the binding functionality is that it binds a given target with high affinity in a specific manner.

[0074] An extracellular domain is external to the cell or organelle and functions to recognize and respond to a ligand. A transmembrane domain spans the membrane of a cell, and an intracellular domain is situated inside a cell. Intracellular co-stimulatory domains provide secondary signals to the cell. They can recruit signaling molecules, cytoskeletal mobilization or induce cell proliferation, differentiation or survival. In the present disclosure a CAR may include an antigen specific extracellular domain, a transmembrane domain and one or more intracellular domains with one or more co-stimulatory domains. The antigen binding domain of a CAR may bind to a single target, or multiple targets.

[0075] A NKG2D fusion protein of the present disclosure may be combined with any CAR known in the art. The CAR of the present disclosure may comprise an extracellular domain comprising an antigen binding region which binds to a wild type and or a variant of EGFR. EGFR vIII is a variant of EGFR with an extracellular deletion mutation. An antigen binding region that binds to both a wild-type and an EGFRVIII variant may be referred to as D2C7. A CAR of the present disclosure may include an EGFR VIII variant or D2C7. The extracellular domain for EGFRVIII may comprise SEQ ID NO: 8, or a sequence with at least 95% identity to SEQ ID NO: 8. The extracellular domain for D2C7 may comprise SEQ ID NO: 15, or a sequence with at least 95% identity to SEQ ID NO: 15.

[0076] The CAR of the present disclosure comprises an extracellular domain comprising an antigen binding region which binds to TIM-4. The extracellular domain for TIM-4 may comprise SEQ ID NO: 25, or a sequence with at least 95% identity to SEQ ID NO: 25. The CAR of the present disclosure may comprise a transmembrane and hinge sequence. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule. The CAR of the present disclosure may comprise a hinge and transmembrane domain of SEQ ID NO: 10 or a sequence with at least 95% identity to SEQ ID NO: 10.

[0077] The intracellular signaling domain of the CAR-immune cell provided herein is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR- has been placed. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term "intracellular signaling sequence" is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0078] The CAR of the present disclosure may comprise at least one intracellular signaling domain, region or co-stimulatory molecule. The intracellular signaling domain may be a costimulatory domain. A costimulatory domain is required for an efficient antigen response in immune cells. In particular embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3(^ (TCR zeta, GenBank aceno. BAG36664.1). T-cell glycoprotein CD3 zeta (CD3(^ chain, also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is a protein that in humans is encoded by the CD247 gene. CD3(^ of the present disclosure may comprise SEQ ID NO: 13, or a sequence with at least 95% identity to SEQ ID NO: 13.

[0079] The CAR of the present invention may also optionally comprise additional co-stimulatory domains, including CD28, 4- IBB, OX-40, ICOS or other members of the TNF receptor superfamily or immunoglobulin (Ig) superfamily. Members of the TNF superfamily form trimeric structures, and their monomers are composed of beta-strands that orient themselves into a two- sheet structure. The TNF superfamily ligands include lymphotoxin alpha, tumor necrosis factor, lymphotoxin beta, 0X40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, CD 137 ligand, TNF -related apoptosis-inducing ligand, receptor activator of nuclear factor kappa-B ligand, TNF-related weak inducer of apoptosis, a proliferation-inducing ligand, B-cell activating factor, LIGHT, vascular endothelial growth factor, TNF superfamily member 18 and ectodysplasin A. These ligands then bind to receptors in the TNF superfamily. Ig superfamily members are characterized based on shared structural features with immunoglobulins (aka antibodies), including an immunoglobulin domain with a characteristic Ig-fold. The Ig domain is reported to be one of the most populous family of proteins in the human genome with over 700 members identified and known in the art. Co- stimulatory domains of the present disclosure may comprise SEQ ID NO: 11 or SEQ ID NO: 12, or a sequence with at least 95% identity to SEQ ID NO: 11 or SEQ ID NO: 12. These co-stimulatory domains may be used in isolation or in any combination.

[0080] While the CAR of the present disclosure is exemplified with the above mentioned costimulatory molecules, other co-stimulatory domains, including CD27, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, and BTNL3 can be used alone or in combination with other co-stimulatory molecules.

[0081] The CAR of the present disclosure may comprise a signal sequence or signaling domain. A signal sequence plays a determinant role in protein distribution and can allow the CAR to be glycosylated and anchored in the cell membrane. In some embodiments, the signal sequence comprises SEQ ID NO: 7 or a sequence having at least 95% identity to SEQ ID NO: 7.

[0082] By way of example, but not by way of limitation, in some embodiments, the disclosed CAR may comprise a signal peptide, the scFv of EGFRVIII, a CD8 hinge & transmembrane domain, CD28 and 4- IBB co-stimulatory domains and a CD3(^ signaling domain. The CAR of the present disclosure may comprise SEQ ID NO.: 7, 8, 10, 11, 12 and 13 or a sequence having at least 95% identity to SEQ ID NO: 7, 8, 10, 11, 12 and 13. The CAR of the present disclosure may comprise SEQ ID NO.: 6 or a sequence having at least 95% identity to SEQ ID NO: 6.

[0083] By way of example, but not by way of limitation, in some embodiments, the disclosed CAR may comprise a signal peptide, the scFv of D2C7, a CD8 hinge & transmembrane domain, CD28 and 4- IBB co-stimulatory domains and a CD3(^ signaling domain. The CAR of the present disclosure may comprise SEQ ID NO.: 7, 15, 10, 11, 12 and 13 or a sequence having at least 95% identity to SEQ ID NO: 7, 15, 10, 11, 12 and 13. The CAR of the present disclosure may comprise SEQ ID NO.: 16 or a sequence having at least 95% identity to SEQ ID NO: 16. A description of a D2C7 CDRs can be found in US Patent No. 11,311628 and its use in a CAR can be found in PCT Publication No. WO2023 / 279095, which are incorporated by reference herein in their entirety. By way of example, but not by way of limitation, in some embodiments, the disclosed CAR may comprise a signal peptide, the extracellular domain of TIM-4, a CD8 hinge & transmembrane domain, CD28 and 4-1BB co-stimulatory domains and a CD3^ signaling domain. The CAR of the present disclosure may comprise SEQ ID NO.: 7, 25, 10, 11, 12 and 13 or a sequence having at least 95% identity to SEQ ID NO: 7, 25, 10, 11, 12 and 13. The CAR of the present disclosure may comprise SEQ ID NO.: 41 or a sequence having at least 95% identity to SEQ ID NO: 41 . A description of a TIM-4 CAR can be found in PCT Publication No. WO2023 / 201221 and US Patent Publication No. 2024 / 0182540, which are incorporated by reference herein in their entirety.

[0084] In some embodiments a construct provided herein may comprise an NKG2D fusion protein and a CAR, wherein the CAR and NKG2D fusion protein may be in any orientation to each other. For example, the CAR may be N-terminal to the NKG2D fusion protein, or the NKG2D fusion protein may be N-terminal to the CAR. In some embodiments, a CAR and an NKG2D fusion protein described herein are connected via a self-cleavage site. A self-cleavage site is any sequence that will result in the generation of multiple proteins from a bicistronic polynucleotide. Selfcleavage sites are known in the art and include but are not limited to 2A peptides, and internal ribosome entry sites. In some embodiments the self-cleavage site is SEQ ID NO: 18. In some embodiments, a CAR and aNKG2D fusion protein may be referred to as a CAREBEAR.

[0085] In some embodiments, the construct may comprise a EGFRVIII CAR and an NKG2D fusion protein separated by a self-cleavage site. In some embodiments, the construct may comprise SEQ ID NO: 19, where the EGFRVIII is at the N-terminal end, or SEQ ID NO: 20, wherein the NKG2D fusion protein is at the N-terminal end, or a sequence with at least 95% identity to SEQ ID NOs: 19 or 20.

[0086] In some embodiments, the construct may comprise a D2C7 CAR and an NKG2D fusion protein separated by a self-cleavage site. In some embodiments, the construct may comprise SEQ ID NO: 22, where the D2C7 is at the N-terminal end, or SEQ ID NO: 23, wherein the NKG2D fusion protein is at the N-terminal end, or a sequence with at least 95% identity to SEQ ID NOs: 22 or 23.

[0087] In some embodiments, the construct may comprise a TIM-4 CAR and an NKG2D fusion protein separated by a self-cleavage site. In some embodiments, the construct may comprise SEQ ID NO: 26 or 42, or a sequence with at least 95% identity to SEQ ID NO: 26 or 42. The CAR of the present disclosure may also comprise additional features to augment the activation, survival, persistence and or functionality of the CAR. In some embodiments, a CAR of the present disclosure may comprise a constitutive or inducible expression cassette containing a transgenic protein such as a cytokine, costimulatory molecule or other immune modifier. In some embodiments, the cassette comprises a nuclear factor of the activated T cell (NF AT)- responsive cassette. By way of example, and not limitation, the cytokine may comprise IL-2, IL-5, IL- 12, IL- 15, IL-18, IL-21, IL-23, IL-36, IFNy, Flt3L, or any combination thereof. In some embodiments, the cytokine is IL-12. In some embodiments, the costimulatory molecule may comprise CD28, OX-40, or 4-1BB. A CAR of the present disclosure may also comprise an intracellular domain which allows for the activation of an intracellular signaling pathway, such as the JAK-STAT pathway.

[0088] In some embodiments, an immune cell comprises any construct described herein. The immune cell may include, but are not limited to a T lymphocyte, natural killer (NK) cell, macrophage, and B lymphocytes. In some embodiments, an immune cell may comprise one or more constructs described herein. For example, an immune cell may comprise a construct encoding a NKG2D fusion protein or an immune cell may comprise a construct encoding a NKG2D fusion protein and a different construct encoding a CAR or CARsigdel; or an immune cell may comprise a construct encoding a NKG2D fusion protein and a CAR or CARsigdel as part of the same construct. In some embodiments, the immune cell is a T-lymphocyte.

[0089] To assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0090] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. These reporter genes may also be used as tags and be encoded as part of the NKG2D fusion protein and be used as a means to ensure the NKG2D protein is being expressed. Suitable reporter genes may include genes encoding luciferase, P-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tel et al., FEBS Letters, 479, 2000). Several of these reporters have split reporter function where a portion of the reporter can be provided in trans to significantly reduce the size of the tag to effect labeling of the fusion protein. Such split reporters are available commercially and are well known to those of skill in the art. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0091] Methods of introducing and expressing genes in a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means. The CRISPR / Cas system may also be used to insert the heterologous polynucleotides into the genome of the cell to allow for stable expression of the genes in the construct in the cell. Methods of CRISPR / Cas based genetic engineering are well known in the art.

[0092] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well- known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, the introduction of a polynucleotide into a host cell is carried out by calcium phosphate transfection.

[0093] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus 1, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362. Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0094] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0095] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays.

[0096] In some embodiments, sequence variants provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody moiety. Amino acid sequence variants of a fusion protein or construct may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the fusion protein, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody moiety. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e g., antigen-binding. Compositions and sequences provided herein, including NKG2D fusion proteins may also comprise species specific variations. For example, SEQ ID NOs: 1, 5, 19, and 22, are human sequences and SEQ ID NOs: 30, 31, 32 and 34 are murine sequences. Species specific sequences and variations may be used depending on the desired subject species and outcomes.

[0097] Methods

[0098] Another aspect of the present disclosure provides a method for treating cancer. In some embodiments, the method comprises administering a therapeutically effective amount of an NKG2D fusion protein or a pharmaceutical composition described herein and a pharmaceutically acceptable excipient, carrier and / or diluent to a subject who has cancer. In some embodiments, the method further comprises administering T lymphocytes, wherein the T-lymphocytes are activated upon binding to the NKG2D fusion protein. In some embodiments, the method comprises administering any NKG2D fusion protein described herein, any CARsigdel or CAREBEAR CARsigdel or CAREBEAR described herein, alone or in combination. In some embodiments, the NKG2D fusion protein used in a method described herein comprises an NKG2D domain of SEQ ID NO: 5 or a sequence with at least 95% identity to SEQ ID NO: 5. In some embodiments, the CARsigdel, CAREBEAR CARsigdel, or CAREBEAR comprise an antigen binding region selected from SEQ ID NO: 8, SEQ ID NO: 15 or SEQ ID NO: 25 or a sequence having at least 95% identity to SEQ ID NO: 8, 15 or 25.

[0099] As used herein, "treatment,” “therapy” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition. As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disease, disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder or condition. Treating cancer in a subject includes the reducing, repressing, delaying or preventing cancer growth, reduction of tumor volume, and / or preventing, repressing, delaying or reducing metastasis of the tumor. Treating cancer in a subject also includes the reduction of the number of tumor cells within the subject. The term "treatment" can be characterized by at least one of the following: (a) reducing, slowing or inhibiting growth of cancer and cancer cells, including slowing or inhibiting the growth of metastatic cancer cells; (b) preventing further growth of tumors; (c) reducing or preventing metastasis of cancer cells within a subject; and (d) reducing or ameliorating at least one symptom of cancer. In some embodiments, the optimum effective amount can be readily determined by one skilled in the art using routine experimentation. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition. The term "effective amount" or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0100] As used herein, the term "administering" an agent, such as a therapeutic entity to composition described herein an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent composition, the term "administering" is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, intratumoral administration and administration by the intranasal or respiratory tract route.

[0101] As is known in the art, cancer is generally considered as uncontrolled cell growth. The compositions and methods of the present invention can be used to treat any cancer, and any metastases thereof, that are associated with EGFRvIII and / or NKG2D expression. Examples include, but are not limited to, lung cancer, head and neck cancer, cervical cancer, urothelial cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia, lymphoma, squamous cell cancer, smallcell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, multiple myeloma, leukemia, salivary gland carcinoma, mesothelioma, kidney cancer, vulval cancer, pancreatic cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma. In certain embodiments, the cancer is brain cancer. In certain embodiments, the cancer comprises glioblastoma.

[0102] In some embodiments, the cancer comprises EGFR-associated cancers which are those cancer associated with EGFR expression. Suitable examples include, but are not limited to, of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, mesothelioma, kidney cancer, vulval cancer, pancreatic cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma. In some embodiments, the cancer comprises glioblastoma. In some embodiments, the cancer is characterized by or associated with phosphatidylserine (PS) expression (herein referred to as an “PS-associated cancer”). PS is a phospholipid that is normally present on the inner leaflet of normal cells. However, apoptotic as well as non-apoptotic cancer cells such as malignant melanoma, leukemia, neuroblastoma, and gastric carcinoma have been shown to widely express PS on their surfaces. PS exposed on the surface of tumor cells contributes to suppression of T-cell activity and blocks tumor clearance. In some embodiments, the cancer comprises those with PS on the cell surface.

[0103] As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The methods and compositions disclosed herein can be used on a sample either in vitro (for example, on isolated cells or tissues) or in vivo in a subject (i.e. living organism, such as a patient). A “subject in need thereof’ as utilized herein may refer to a subject in need of treatment for a disease or disorder associated with a cancer or suspected tumor, such as glioblastoma. A subject in need thereof may include a subject having a cancer that is characterized by gross abnormality visible by X-ray, computerized tomography (CT), or magnetic resonance imaging (MRI), histology or immunofluorescence.

[0104] In some embodiments the method further comprises administering effector cells, in particular, T lymphocytes, wherein T lymphocytes are activated upon binding to the NKG2D fusion protein. The T lymphocytes may be autologous or allogeneic or differentiated T cells from a stem or multipotent cell. The T lymphocytes may be nonspecific for a target or antigen. Exemplary effector cells useful for the present disclosure include, but are not limited to, dendritic cells (including immature dendritic cells and mature dendritic cells), T lymphocytes (such as naive T cells, effector T cells, memory T cells, cytotoxic T lymphocytes, T helper cells, Natural Killer T cells, Treg cells, tumor infdtrating lymphocytes (TIL), and lymphokine-activated killer (LAK) cells), B cells, Natural Killer (NK) cells, monocytes, macrophages, neutrophils, granulocytes, and combinations thereof. Subpopulations of effector cells can be defined by the presence or absence of one or more cell surface markers known in the art (e.g., CD3, CD4, CD8, CD 19, CD20, CD11c, CD123, CD56, CD34, CD14, CD33, etc ).

[0105] T cell activation refers to a process in which mature T cells can express antigen-specific T cell receptors on their surface to recognize their cognate antigens and respond by entering the cell cycle, clonally expand and differentiate, secreting cytokines or lytic enzymes, and initiating the cell-based functions of the immune system. Cytokine release is a consequence of T cell activation and efficacy, it is preferred that at least a portion of the activated T cell produce one or more cytokine such as those selected from the group consisting of IL-1, IL-ip, IL-2, IL-4, IFN-y, IL-10, IL-12, TNF-oc and GM-CSF. Additionally, at least a portion of the activated T cells preferably express one or more surface markers selected from the group consisting of CD2, CD28, CTLA4, CD40 ligand (gp39), CD18, CD25, CD69, CD16 / CD56, MHC Class I, MHC Class II, CD8, CD4, CD3 / TcR, CD54, LFA-1 and VLA-4. The T cell may be activated in any way known in the art prior to administration. The T lymphocytes may also become activated or specifically activated upon binding to the NKG2D fusion protein. T lymphocytes may bind to the scFv anti-CD3 domain of the NKG2D fusion protein. The T lymphocytes may be CAR T cells. The CAR T cells may comprise an EGFR VIII CAR of SEQ ID NO: 6 or a D2C7 CAR of SEQ ID NO: 16 or a TIM-4 CAR of SEQ ID NO: 41 or sequences having at least 95% identity to SEQ ID NOs: 6, 16 or 41.

[0106] Natural Killer (NK) cells are cytotoxic lymphocytes that play a crucial role in the innate immune system. NK cells release cytotoxic granules containing perforin and granzymes, which damage the membranes of target cells, leading to apoptosis. NK cells also patrol the body for cells that have lost MHC class I expression, a sign of infection or malignancy.

[0107] Macrophages are initiators of the immune response. They release cytokines and chemokines that attract other immune cells to the site of infection or injury. They also present antigens to T cells, which helps activate the adaptive immune system. Macrophages regulate inflammation by producing pro- and anti-inflammatory cytokines

[0108] In some embodiments theNKG2D fusion protein may be administered separately from the T lymphocyte or CAR-T cell. In some embodiments, the NKG2D fusion protein may be administered together with T lymphocyte and / or including CAR-T cells. A NKG2D fusion protein of the present disclosure may be administered before, together with or following the administration of T lymphocytes, CAR-T cells or any other cancer therapy including other immunotherapies, chemotherapy, radiation, surgery or any other standard of care cancer treatment. The NKG2D fusion protein of the present disclosure may be administered with a CAR-T cell of any specificity.

[0109] Some embodiments of the present disclosure provide a method for treating cancer comprising administering a therapeutically effective amount of any of the CAR-immune cells described herein and a pharmaceutically acceptable excipient, carrier and / or diluent to a subject. In some embodiments the cancer is a PS-associated cancer or an EGFR-associated cancer including those described herein. Some embodiments of the present disclosure provide a method for treating cancer comprising administering a cell comprising any construct described herein to a subject. In some embodiments the cancer is a PS-associated cancer or an EGFR-associated cancer including those described herein.

[0110] Another aspect of the present disclosure provides a method of detecting cancer comprising administering a fusion protein described herein to a subject suspected of having cancer, detecting the presence or accumulation of a tag associated with the fusion protein in the subject suspected of having cancer, and detecting cancer in the subject, wherein the accumulation or presence of the tag allows for in vivo detection of the cancer. In some embodiments, detection of the tag is indicative or correlated with cancer. In some embodiments detection of the tag can be used to diagnose cancer. A medical diagnosis is the process of determining which disease or condition explains a person's symptoms and signs. The cancer may be a PS-associated cancer or an EGFR- associated cancer including those described herein. The tag can be detected by any means know in the art. By way of example, and not limitation a tag can be detected via blood tests, X-ray. CT scan, MRI, nuclear medicine scans including PET scan, and ultrasound.

[0111] In some embodiments, a method of detecting cancer, the method comprising contacting a sample comprising cells or tissue from a subject with the fusion protein described herein, or the NKG2D domain of SEQ ID NO: 5 linked to a tag, detecting the presence or accumulation of the tag on the sample, and detecting cancer in the sample, wherein the accumulation or presence of the tag allows for ex-vivo detection of cancer. In some embodiments, the NKG2D fusion protein is contacted with a labeled affinity reagent, such as an antibody, capable of binding the fusion protein. The bound, labeled affinity reagent can be detected to detect cancer. The term “sample” refers to a tissue (e.g., tissue biopsy), organ, cell (including a cell maintained in culture), cell lysate (or lysate fraction), biomolecule derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), or body fluid from a subject. Non-limiting examples of body fluids include blood, urine, plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, perspiration, semen, transudate, exudate, and synovial fluid. In some embodiments, detection of the tag is indicative or correlated with cancer. In some embodiments detection of the tag can be used to diagnose cancer. A medical diagnosis is the process of determining which disease or condition explains a person's symptoms and signs. The cancer may be a PS-associated cancer or an EGFR-associated cancer including those described herein. A tag can be detected on a sample by any means known in the art, including, but not limited to immunodetection, histochemistry, or immunofluorescence.

[0112] The tag may comprise any known tag in the art and may be used for the purposes of purification, localization, quantification, tracking or imaging purposes. Types of tags may comprise an affinity tag, a solubilization tag, chromatography tag, epitope tag, fluorescent tag or enzymatic modification tag. In some embodiments, the tag may comprise a His tag, a FLAG tag, a fluorescent tag or a Myc tag.

[0113] In some embodiments, a method of inducing an immune response in a subject is provided. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a fusion protein, pharmaceutical composition, or the immune cells described herein, wherein an antitumor immune response is induced in the subject. The immune response may comprise an antimicrobial immune response, an antiviral immune response, or an antibacterial immune response. In some embodiments, the immune response is an antitumoral immune response.

[0114] An anti-tumor immune response may also be referred to as antitumor immunity. The terms “anti-tumor immune response” and “antitumor immunity” refer to innate and or adaptive immune responses which lead to tumor control. An anti-tumor immune response may comprise a reducing, repressing, delaying or preventing cancer growth, reduction of tumor volume, and / or preventing, repressing, delaying or reducing metastasis of the tumor. An anti-tumor immune response may also include a reduction of the number of tumor cells within the subject. An anti-tumor immune response may include among others, T lymphocytes, including cytotoxic T lymphocytes and helper T lymphocytes, antigen presenting cells, natural killer cells, neutrophils, macrophages, B lymphocytes, as well as secreted factors including cytokines, antibodies, Damage-associated molecular patterns (DAMPs), and other small molecules.

[0115] In some embodiments, a fusion protein, pharmaceutical composition, or the immune cells described herein may be administered by any route to induce an antitumor immune response. As used herein, the term "administering" an agent, such as a fusion protein, pharmaceutical composition, or the immune cells described herein to an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent, the term "administering" is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route.

[0116] Additional definitions

[0117] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps.

[0118] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter.

[0119] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0120] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0121] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0122] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0123] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0124] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0125] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0126] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0127] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0128] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0129] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0130] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0131] EXAMPLES

[0132] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0133] Example 1:

[0134] Chimeric Antigen Receptor T Cells Engineered with Bi-Specific Engagers Without Antigen Requirements (CARE-BEAR) Significance: Tumor heterogeneity is a key barrier to effective antigen-targeted immunotherapies in GBM and other solid tumors.

[0135] Despite numerous treatment advances, GBM remains uniformly lethal. Immunotherapies offer significant promise, but newer immune-based approaches must reflect our gained understanding of barriers to therapeutic success and offer novel means of countering these obstacles. One such barrier is tumor heterogeneity, a roadblock particularly relevant among solid cancers. CAR T and other antigen-specific immunotherapeutic platforms fail in many solid cancers, in part because uniformly expressed antigens have not been identified as they have for hematologic malignancies (i.e. CD19 in B cell lymphoma). In the hands of the inventors, as few as 5-10% antigen loss variants in a tumor (i.e. EGFRvIII-negative cells in the context of EGFRvIII CAR T cell treatment) is sufficient to obviate nearly all survival benefit. Unfortunately, this problem has not been eliminated by personalized medicine strategies aimed at sequencing individual tumors and targeting the uncovered neoantigens. Finding better surface targets is critical for the advancement of effective immunotherapy.

[0136] Significance: Tumor cells express non-classical MHC-I on their surface.

[0137] Finding a targetable antigen that is expressed on the surface of all tumor cells represents the “Holy Grail” of antigen-specific T cell-based immunotherapies. Non-classical MHC-I consists of multiple proteins that are upregulated on stressed cells, including tumor cells. Recently, it was identified that many of these non-classical MHC-I molecules are upregulated in tumor cells lacking MHC-I. Importantly, these antigens have low expression on healthy tissue. Thus, non-classical MHC-I molecules represent a potential tumor antigen that can be safely targeted with immunotherapy. Intriguingly, non-classical MHC-I is expressed on the surface of many different tumor histologies, making a targeted therapy potentially useful for multiple cancers.

[0138] The accepted paradigm for both cellular and antitumor immunity relies upon tumor cell kill by CD8+ T cells recognizing cognate antigens presented in the context of target cell MHC-I molecules. Likewise, a classically described mechanism of tumor immune escape is tumor MHC- I downregulation. It was recently demonstrated that CD8+ T cells maintain the capacity to kill tumor cells that are entirely devoid of MHC-I expression antigen-independent innate killing mechanism involving the NKG2D receptor. However, the cytotoxic function of NKG2D by CD8+ T cells normally relies on the recent activation of the TCR. By linking the extracellular domain of the NKG2D receptor to a CD3 activating domain to generate an NKG2D Bispecific T cell engager, the need for concurrent TCR activation is obviated and the ability of CD8+ T cells to kill through an antigen-agnostic mechanism extended. This has been termed this NKG2D Bispecific T cell engager “Bi-specific Engager without Antigen Requirements,” or BEAR. Further, targeting non- classical MHC-I maintains target specificity as non-classical MHC-I is highly expressed by tumor cells, but not highly expressed on normal tissues. Non-classical MHC-I is also an attractive target clinically because its expression is increased following radiation or chemotherapy, both of which are standard of care for GBM and many other solid tumors.

[0139] Engineering of CAR to secrete NKG2D Bispecific T cell engager in a local fashion .

[0140] Concerns for systemic delivery of BEAR (and Bispecific T cell engagers in general) are: 1) Failure to arrive sufficiently across the blood-brain barrier; 2) off-tumor, on-target toxicity; 3) short half-life; and 4) insufficient intratumoral T cells to provide efficacy. The intratumoral delivery of CAR T cells engineered to locally secrete BEAR addresses all 4 of these potential limitations for the Bispecific T cell engager platform, while providing an additional anti-tumor platform with efficacy of its own in combinatorial fashion. This is one of the first uses of such a therapeutic pre-clinically and potentially clinically. As an initial proof-of-concept, a Vlll-targeting CAR T cell that secretes NKG2D Bispecific T cell engager (VIIICARxNKG2DBispecific T cell engager; VIII CARE-BEAR) was engineered. Next. VIII CAR or VIIICARxNKG2DBispecific T cell engager was co-cultured with CT2A murine glioma cells that lack expression of EGFRvIII. It was hypothesized that VIII CAR alone would not kill CT2A cells, but VIIICARxNKG2DBispecific T cell engager would lead to significant cytotoxicity of CT2A cells. Indeed, VIIICARxNKG2DBispecific T cell engager killed CT2A in a dose-dependent manner; whereas, VIII CAR and control non-transduced T cells (NT T cells) had no effect.

[0141] Data'.

[0142] Identifying ideal target antigens is crucial for the success of antigen-targeting immunotherapies, such as CAR T cells. The ideal target antigen should be universally present on all tumor cells while remaining absent on normal tissue, thereby bypassing barriers imposed by tumor heterogeneity and limiting on-target, off-tumor toxicities. The inventors recently observed and reported the upregulation of NKG2D ligands (NKG2DLs) on multiple tumor types including glioma and melanoma9. Importantly, NKG2DL expression is relatively low or absent on healthy tissue. Moreover, the NKG2DL family consists of multiple different proteins, all of which can interact with their receptor, NKG2D, thus decreasing the likelihood of antigen escape due to antigen loss. These factors make NKG2DLs a potentially ideal target for immunotherapy. Therefore, the inventors hypothesized that redirecting T cells to target NKG2DL on tumor cells would result in potent tumor cell death.

[0143] The inventors developed a fusion protein composed of the extracellular domain of NKG2D linked to a T cell -activating anti-CD3 single chain variable fraction (scFv) (Figure 1A). The inventors termed this molecule NKG2D Bi-specific Engager without Antigen Requirements (BEAR). NKG2D BEAR is predicted to crosslink and activate CD3+T cells in the presence of NKG2DL+tumor cells, leading to tumor cell death. To test this hypothesis, the inventors cocultured human CD3+T cells and U87 human glioma cells in the presence or absence of different concentrations of human NKG2D BEAR (Figure IB). The presence of NKG2D BEAR led to potent dose-dependent death of glioma cells, suggesting that NKG2D BEAR is functionally able to redirect T cells to kill tumor cells.

[0144] Although NKG2DLs are upregulated on tumor cells, NKG2DL expression is not tumorspecific. Therefore, systemic NKG2D BEAR administration has the potential to cause on-target, off-tumor toxicities. To minimize toxicity, the inventors developed a tumor-specific delivery platform for NKG2D BEAR. This platform consists of a chimeric antigen receptor (CAR) T cell that secretes NKG2D BEAR. Ideally, the CAR domain will target a tumor-specific antigen (TSA), localizing NKG2D BEAR to the tumor microenvironment (TME). The inventors have termed this platform Chimeric Antigen Receptor T cells Engineered with Bi-specific Engager without Antigen Requirements (CARE-BEAR). As a proof-of-concept, the inventors used an EGFRvIII-targeting CAR secreting NKG2D BEAR for preliminary studies (VIII CARE-BEAR). CARE-BEAR is encoded as a bicistronic message with the CAR domain expressed upstream of the NKG2D BEAR domain (Figure 2). A self-cleaving 2A site is incorporated between the CAR and BEAR domains so that two separate proteins are expressed from the single message. Both human (Fig. 2A) and murine (Fig. 2B) versions of CARE-BEAR have been designed and developed. Of note, while the inventors initially engineered the CAR domain upstream of the BEAR domain, the order of these domains could be reversed. That goes for each component within each domain as well (e.g. the order of NKG2D and anti-CD3scFv in NKG2D BEAR could be reversed). Also, the inventors initially engineered a P2A site in between CAR and BEAR, but any sequence could be used in between the domains that will result in two separate proteins (e.g. IRES, etc.).

[0145] Initial characterization of VIII CARE-BEAR involved retroviral transduction of human T cells with human VIII CARE-BEAR. Any delivery method that introduces the CAREBEAR gene into T cells or other effector cells (e.g. NK cells, macrophages, etc.) is suitable (e.g. lentiviral, AAV, etc.). 7 days post-transduction, the inventors were able to detect a similar percentage of VIII CAR on the surface of VIII CAR T cells and VIII CARE-BEAR T cells (Figure 3A). To detect if NKG2D BEAR is being expressed by CARE-BEAR, the inventors stained each T cell with an anti-NKG2D antibody (Figure 3B). Human T cells constitutively express low levels of NKG2D as indicated by the roughly 30% NKG2D+T cells in the NT T cells and VIII CAR groups. However, all of the T cells in the CARE-BEAR group are NKG2D+, suggesting that NKG2D BEAR is being secreted by these T cells and binding to CD3.

[0146] Next, the inventors functionally characterized VIII CARE-BEAR via in vitro co-culture assays. These assays were designed primarily to focus on NKG2D BEAR activity, hence the utilization of EGFRvIII-negative tumors. First, U87 glioma cells were co-cultured with NT T cells, VIII CAR, or VIII CARE-BEAR at various effector (E) to target (T) ratios (Figure 4A). After 24 hours, the remaining number of viable tumor cells was quantified with flow cytometry and normalized to Tumor Only conditions to give a “Percent Survival”. NT T cells and VIII CAR had no effect on U87 survival. However, VIII CARE-BEAR potently eradicated U87 cells in a dosedependent manner, suggesting that NKG2D BEAR secreted by VIII CARE-BEAR is able to redirect T cells to kill U87 glioma cells. NKG2DL overexpression is not limited to gliomas. To demonstrate the versatility of the CARE-BEAR platform, the inventors performed the same in vitro cytotoxicity assay as above but with M202 human melanoma cells as the targets (Figure 4B). As expected, NT T cells and VIII CAR did not kill M202 cells. In contrast, VIII CARE-BEAR potently killed M202 melanoma cells in a dose-dependent manner. Together, these data suggest that CARE-BEAR is a therapeutic platform that can treat multiple tumor types.

[0147] The main impetus for engineering the CARE-BEAR platform is to successfully treat heterogeneous tumors where traditional antigen-specific therapies fail. To test this hypothesis, the inventors performed an in vitro cytotoxicity assay using an EGFR / EGFRvIII heterogeneous tumor model. A clinically relevant 70:30 mixture of U87:U87vIII glioma cells were co-cultured with NT T cells, VIII CAR, and VIII CARE-BEAR at various E:T ratios (Figure 5). After 24 hours, the remaining total number of tumor cells were quantified and normalized to Tumor Only controls. When total (U87 and U87vIII) tumor cells are assessed, VIII CARE-BEAR is clearly more effective than VIII CAR at eliminating EGFRvIII heterogeneous tumors (Fig. 5A). Since U87 and U87vIII cells were stained with different markers, the inventors can further breakdown the effect of VIII CARE-BEAR and VIII CAR on U87 and U87vIII within the heterogeneous population. While there is noticeable bystander killing of U87 by VIII CAR, VIII CARE-BEAR eliminated virtually all of the U87 subpopulation (Fig. 5B). As expected, VIII CAR and VIII CARE-BEAR are effective against the U87vIII subpopulation (Fig. 5C). These data suggest that VIII CAREBEAR is superior to VIII CAR in circumstances of EGFRvIII heterogeneity.

[0148] The inventors also developed a fully murine version of VIII CARE-BEAR (Fig. 2B). This allows the inventors to study VIII CARE-BEAR in immunocompetent mouse models. As with human VIII CARE-BEAR, the inventors first validated that the transduction efficiency of VIII CARE-BEAR was similar to VIII CAR by detecting surface VIII CAR expression (Figure 6A). Indeed, VIII CAR and VIII CARE-BEAR both were around 70% VIII CAR1, suggesting similar transduction efficiencies. Next, the inventors functionally validated that NKG2D BEAR was being produced by co-culturing NT T cells, VIII CAR, or VIII CARE-BEAR with CT2A murine glioma cells. Importantly, these CT2A cells lack expression of EGFRvIII. VIII CARE-BEAR killed CT2A cells in a dose-dependent manner; whereas, NT T cells and VIII CAR had no impact on tumor cells. These data suggest that murine VIII CARE-BEAR is operating as expected in vitro.

[0149] To test murine VIII CARE-BEAR in vivo, the inventors utilized an EGFRvIII tumor heterogeneity model. A 70:30 mixture of CT2A:CT2AvIII glioma cells were implanted intracranially (IC) in C57BL / 6 mice. This 70:30 ratio represents a clinically relevant ratio of EGFRvIII tumor cells. 7 days after tumor implantation, 2 x 106VIII CAR or VIII CARE-BEAR were administered IC at the tumor site, or mice were left untreated. Notably, mice did not undergo lymphodepletion preconditioning prior to T cell administration. Untreated mice and mice treated with VIII CAR all died by day 18 post-tumor implantation, with median survivals of 17.5 and 17 days, respectively (Figure 7). VIII CARE-BEAR had a modest survival benefit with VIII CARE- BEAR-treated mice having a median survival of 20.5 days.

[0150] Next, the inventors aimed to demonstrate human CARE-BEAR efficacy in vivo in a human model of heterogeneous GBM. To this end, the inventors implanted a 70:30 heterogeneous mixture of U87:U87vIII cells IC in immunodeficient NSG mice. 7 days post-tumor implantation, 2 x 106 VIII CAR T cells or VIII CARE-BEAR T cells were administered IC at the tumor site, or mice were left untreated. Mice treated with VIII CARE-BEAR had significantly extended survival compared to mice treated with VIII CAR or mice left untreated (Figure 8). The median survival of untreated, VIII CAR treated, and VIII CARE-BEAR treated mice was 21, 25, and 38 days, respectively.

[0151] Thus far, the inventors have shown VIII CARE-BEAR to be effective in heterogeneous models of GBM. However, the heterogeneous environment was artificially created and may not represent the true heterogeneity displayed by GBM clinically. Therefore, the inventors isolated tumor cells from a surgically resected glioblastoma and expanded these tumor cells minimally ex vivo so that they more closely retained their original phenotypic landscape. The inventors call these patient-derived glioblastoma cells GBM PDX. EGFRvIII expression was heterogeneous in this GBM PDX, with expression ranging from absent to low (Figure 9A). Next, the inventors stained GBM PDX for expression of the NKG2DLs MICA and MICB. In contrast to EGFRvIII expression, MICA / B was detectable on all of the GBM PDX cells (Fig. 9B). The heterogeneous expression of EGFRvIII but homogeneous expression of NKG2DLs led to the hypothesis that VIII CARE-BEAR would demonstrate superior killing of GBM PDX compared to VIII CAR. Indeed, when co-cultured with GBM PDX, VIII CARE-BEAR T cells were more effective than VIII CAR T cells, displaying more potent killing (Fig. 9C).

[0152] The inventors already provided evidence that the CARE-BEAR platform is effective against human melanoma (Fig. 4B). However, the inventors wanted to expand this evidence to include other solid tumor types, namely lung and breast cancer. The inventors first validated that the A549 non-small cell lung cancer (NSCLC) and the MDA-MB-468 triple negative breast cancer (TNBC) cell lines express NKG2DLs by staining for MICA / B (Figure 10A,C). Next, the inventors co-cultured A549 and MDA-MB-468 with NT T cells or VIII CARE-BEAR T cells. Both A549 and MDA-MB-468 are EGFRvIII-negative, so all killing will be mediated by the NKG2D BEAR secreted by VIII CARE-BEAR. VIII CARE-BEAR killed both cell lines in a dose-dependent manner (Fig. 10B,D). Altogether, these data provide further evidence that the CARE-BEAR platform has utility against multiple solid tumor types. The inventors show effectiveness against glioma, melanoma, lung, and breast cancers.

[0153] Next, the inventors sought to demonstrate that the “CAR” domain of the CARE-BEAR platform is exchangeable with a CAR with a different specificity. This flexibility would allow CARE-BEAR to be tailored to the specific antigenic phenotype of the tumor. As a proof-of- concept, the inventors chose to exchange the EGFRvIII-targeting CAR domain of VIII CAREBEAR with a D2C7 CAR, thus creating D2C7 CARE-BEAR (Figure 11 A). D2C7 CAR is specific for both EGFRwt and EGFRvIII. The inventors generated D2C7 CARE-BEAR T cells and tested their ability to kill U87 tumor cells that lacked EGFR expression (U87EGFRko). D2C7 CAREBEAR T cells were able to kill these U87EGFRko cells; whereas, D2C7 CAR T cells lacked activity against these cells, as expected (Fig. 11B). The inventors then tested D2C7 CARE-BEAR in vivo in a model of EGFR heterogeneity. A 70:30 mixture of U87EGFRko:U87 cells were administered IC in NSG mice. Of note, U87 cells naturally express EGFRwt. 7 days post-tumor implantation, 2 x 106D2C7 CAR T cells or D2C7 CARE-BEAR T cells were administered IC at the tumor site, or mice were left untreated. Mice treated with D2C7 CARE-BEAR had a significantly improved median survival compared to mice treated with D2C7 CAR (126 vs. 87 days, respectively) (Figure 12). These data provide evidence that the CARE-BEAR platform is flexible and can be modified based on the surface antigen profile of the tumor. The antigen receptor can be an scFv, antibody fragment, T cell receptor, native receptor, or any structure that provides antigen specificity.

[0154] The inventors have shown that the CAR domain of CARE-BEAR is interchangeable by developing VIII CARE-BEAR and D2C7 CARE-BEAR and showing efficacy in multiple tumor models. While the inventors hypothesize that the CAR domain is required for tumor localization of the NKG2D BEAR, the inventors sought to prove this by engineering different variations of CARE-BEAR. First, the inventors designed a VIII CARE -BEAR that lacks the intracellular T cell signaling components of the CAR domain (VIII CARE-BEAR CARsigdel; Figure 13A). These T cells should still retain tumor-targeting capabilities via the CAR scFv, but the CAR antigen will not activate these T cells. Therefore, all target cell killing will be mediated by the secreted NKG2D BEAR. In a set of in vitro cytotoxicity assays, the inventors validated that VIII CARE-BEAR CARsigdel T cells could kill an EGFRvIII-heterogeneous human glioblastoma patient-derived xenograft (GBM PDX) (Fig. 13B) as well as an EGFRvIII-negative A549 human lung cancer cell line (Fig. 13C). The inventors also generated a version of CARE-BEAR that completely lacks a CAR domain. Essentially, these are non-specific T cells that constitutively express NKG2D BEAR (T BEAR; Figure 14A). The inventors confirmed that T BEAR lacked CAR expression (Fig. 14B, top) and secreted NKG2D BEAR (Fig. 14B, bottom). The inventors then validated that T BEAR had similar killing efficacy as VTIT CARE-BEAR when co-cultured with EGFRvIII-negative U87 glioma cells. As expected, VIII CAR was unable to kill U87 cells. These data provide evidence that T BEAR is functional in vitro. Importantly, T BEAR could potentially work against any cancer characterized by NKG2DL expression and would eliminate the need for determining the optimal CAR-antigen target. However, this is likely to be dependent on the administration route (intravenous vs. intratumoral) of the T cells.

[0155] Data thus far has suggested that NKG2D BEAR is being secreted by CAREBEAR and T BEAR given the killing of CAR-antigen negative cells by CAREBEAR and T BEAR T cells (Figs. 4A, 4B, 6B, 10B, 10D, 11B, 14C). However, it remained unclear whether NKG2D BEAR is being secreted in high enough quantities to redirect bystander T cells, or if NKG2D BEAR is simply redirecting the T cells that are producing it. To validate that NKG2D BEAR is being secreted into the extracellular space by CAREBEAR and T BEAR T cells, the inventors collected the supernatant from 3-day continuous cultures of VIII CAR T cells, VIII CAREBEAR T cells, and T BEAR T cells. The supernatant was concentrated from 6 ml to 500 pl using a centrifugal fdter. 50 pl of each supernatant was incubated with 106normal CD3+T cells for 30 minutes. Anti-NKG2D was then used to detect cell-bound NKD2D BEAR. T cells incubated with VIII CAREBEAR and T BEAR supernatant showed 100% positivity for NKG2D, indicating that NKG2D BEAR was indeed being secreted (Figure 15A). Furthermore, when this CAREBEAR and T BEAR supernatant was included in a co-culture of normal CD3 human T cells and U87 tumor cells, tumor cells were potently killed (Fig. 15B). Together, these data provide evidence that CAREBEAR and T BEAR T cells secrete NKG2D BEAR in sufficient quantities to redirect bystander T cells to kill tumor cells.

[0156] Next, the inventors tested the requirement for CAR signaling and CAR-antigen-targeting of CAREBEAR. First, mice bearing a heterogeneous mixture of U87 and U87vIII tumor cells were treated at the tumor site with VIII CAR T cells, VIII CAREBEAR T cells, VIII CAREBEAR CARsigdel T cells, or T BEAR T cells (Figure 16A). In this particular experiment, mice treated with VIII CAREBEAR CARsigdel T cells survived longer than mice in any other treatment group, suggesting that CAR signaling may not be required for CAREBEAR efficacy. To test the requirement for CAR-mediated targeting, the inventors implanted U87 tumors in NSG mice (Fig. 16B). U87 cells do not express EGFRvIII. These mice were administered VIII CAR T cells, VIII CAREBEAR T cells, or T BEAR T cells directly to the tumor site. Interestingly, both VIII CAREBEAR and T BEAR T cell treatment prolonged the median survival of mice over VIII CAR T cell treatment. These results indicate that the CAREBEAR platform remains effective even in the absence of CAR-antigen when treatment is administered directly to the tumor. In contrast, the role of CAR antigen and CAR signaling is likely to be critical when the T cell therapy is administered systemically or intracerebroventricularly, a focus of future studies.

[0157] References

[0158] 1 Topalian, S. L. et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443-2454 (2012).

[0159] 2 Reardon, D. A. et al. OS 10.3 Randomized Phase 3 Study Evaluating the Efficacy and Safety of Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma: CheckMate 143. Neuro Oncol 19, iii21 (2017).

[0160] 3 Davis, M. E. Glioblastoma: Overview of Disease and Treatment. Clin J Oncol Nurs 20, S2-8 (2016).

[0161] 4 Tomaszewski, W ., Sanchez-Perez, L., Gajewski, T. F. & Sampson, J. H. Brain Tumor Microenvironment and Host State: Implications for Immunotherapy. Clin Cancer Res 25, 4202- 4210 (2019).

[0162] 5 Sottoriva, A. et al. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proc Natl Acad Sci USA 110, 4009-4014 (2013).

[0163] 6 Chongsathidkiet, P. et al. Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors. Nat Med 24, 1459-1468 (2018).

[0164] 7 Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396-1401 (2014).

[0165] 8 Sampson, J. H. et al. Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma. J Clin Oncol 28, 4722-4729 (2010). [pii] 10.1200 / JC0.2010.28.6963

[0166] 9 Lerner, E. C. et al. CD8(+) T cells maintain killing of MHC -1-negative tumor cells through the NKG2D-NKG2DL axis. Nat Cancer 4, 1258-1272 (2023).

[0167] 10 Prajapati, K., Perez, C., Rojas, L. B. P., Burke, B. & Guevara-Patino, J. A. Functions of NKG2D in CD8+ T cells: an opportunity for immunotherapy. Cellular & Molecular Immunology 15, 470-479 (2018). 11 Markiewicz, M. A. et al. Costimulation through NKG2D Enhances Murine CD8+ CTL Function: Similarities and Differences between NKG2D and CD28 Costimulation. The Journal of Immunology 175, 2825-2833 (2005).

[0168] 12 Dhar, P. & Wu, J. D. NKG2D and its ligands in cancer. Curr Opin Immunol 51, 55-61 (2018).

[0169] 13 Raulet, D. H. Roles of the NKG2D immunoreceptor and its ligands. Nature Reviews Immunology 3, 781-790 (2003).

[0170] 14 Venkataraman, G. M., Suciu, D., Groh, V., Boss, J. M. & Spies, T. Promoter Region Architecture and Transcriptional Regulation of the Genes for the MHC Class I-Related Chain A and B Ligands of NKG2D. The Journal of Immunology 178, 961-969 (2007).

[0171] 15 Gasser, S. & Raulet, D. H. Activation and self-tolerance of natural killer cells. Immunological Reviews 214, 130-142 (2006).

Claims

CLAIMSWhat is claimed:

1. A NKG2D fusion protein comprising a NKG2D domain linked to a single chain variable fragment (scFv) specific for CD3, wherein the NKG2D domain comprises SEQ ID NO: 5 or a sequence having 95% identity to SEQ ID NO: 5, and wherein the scFv specific for CD3 is capable of binding to and activating a T cell receptor.

2. The fusion protein of claim 1, wherein the scFv specific for CD3 comprises complementarity determining regions (CDR) of SEQ ID NOs: 36-41 or sequences with at least 95% identity to SEQ ID NO: 36-41.

3. The fusion protein of claim 1 or 2, wherein the scFV specific for CD3 comprises SEQ ID NO: 3 or a sequence with at least 95% identity to SEQ ID NO: 3.

4. The fusion protein of any one of claims 1-3, wherein the NKG2D domain is linked to the scFv specific for CD3 via a linker peptide and optionally wherein the linker is a glycine serine linker.

5. The fusion protein of claim 4, wherein the linker comprises at least one copy of SEQ ID NO: 4.

6. The fusion protein of any one of the preceding claims, further comprising an N-terminal signal sequence.

7. The fusion protein of claim 6, wherein the signal sequence comprises SEQ ID NO: 2 or a sequence with at least 95% identity to SEQ ID NO: 2.

8. The fusion protein of any one of claims 1-7, additionally comprising a tag.

9. The fusion protein of claim 8, wherein the tag is selected from the group consisting of a His tag, a FLAG tag, and a fluorescent tag.

10. The fusion protein of any one of the preceding claims, wherein the scFv specific for CD3 is N-terminal to the 74X020 domain.

11. The fusion protein of any one of claims 1-10, wherein the fusion protein comprises the signal sequence of SEQ ID NO: 2, the NKG2D domain of SEQ ID NO: 5, the linker of SEQ ID NO: 4, and the scFv specific for CD3 comprising SEQ ID NO: 35-40.

12. The fusion protein of claim 1 1, wherein the scFv specific for CD3 comprises SEQ ID NO: 3 or a sequence having at least 95% identity to SEQ ID NO: 3.

13. The fusion protein of claim 12, wherein the fusion protein comprises SEQ ID NO: 1 or a sequence with at least 90% identity to SEQ ID NO: 1.

14. A pharmaceutical composition comprising theNKG2D fusion protein of any of the previous claims and a pharmaceutically acceptable excipient, carrier and / or diluent.

15. A construct comprising a polynucleotide sequence encoding the NKG2D fusion protein of any one of claims 1-13.

16. The construct of claim 15, further comprising a promoter operably linked to the polynucleotide sequence encoding the NKG2D fusion protein.

17. The construct of claim 15 or 16, additionally comprising a sequence encoding an extracellular domain and a transmembrane domain, and wherein the extracellular domain comprises an antigen binding region.

18. The construct of claim 17, wherein the antigen binding region is capable of binding both a wildtype EGFR and or an EGFR VIII variant.

19. The construct of claim 18, wherein the antigen binding region comprises SEQ ID NO: 8 or a sequence with at least 95% identity to SEQ ID NO: 8.

20. The construct of claim 18, wherein the antigen binding region comprises SEQ ID NO: 15 or a sequence with at least 95% identity to SEQ ID NO: 15.

21. The construct of claim 17, wherein the antigen binding region is capable of binding TIM- 4.

22. The construct of claim 21, wherein the antigen binding region comprises SEQ ID NO: 25 or a sequence with at least 95% identity to SEQ ID NO: 25.

23. The construct of claim 17-22, wherein the transmembrane domain comprises SEQ ID NO: 10 or a sequence with at least 95% identity to SEQ ID NO: 10.

24. The construct of any one of claims 17-23, wherein the extracellular domain comprises an scFv comprising a heavy chain portion and a light chain portion linked via a second linker thesecond linker comprises SEQ ID NO: 9, SEQ ID NO: 4, a sequence with at least 95% identity to SEQ ID NO: 9 or a sequence with at least 95% identity to SEQ ID NO: 4.

25. The construct of any one of claims 17-24, further comprising a sequence encoding a signal peptide.

26. The construct of any one of claims 15-25, wherein the construct comprises a polynucleotide encoding selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 24 or SEQ ID NO: 27 or a sequence with at least 95% sequence identity to SEQ ID NO: 21, 24 or27.

27. The construct of any one of claims 17-26, wherein the construct further comprises at least one co-stimulatory domain.

28. The construct of claim 27, wherein the co-stimulatory domain is selected from CD28, 4- 1BB, 0X40, and combinations thereof.

29. The construct of any of claims 17-28, wherein the construct comprises a chimeric antigen receptor (CAR), the CAR comprising an extracellular domain, a transmembrane domain, and at least one intracellular signaling domain.

30. The construct of claim 29, wherein the extracellular domain comprises an antigen binding region capable of binding both a wildtype EGFR and or an EGFR VIII variant.

31. The construct of claim 29, wherein the CAR comprises SEQ ID NO: 6 or SEQ ID NO: 16 or a sequence with at least 95% identity to SEQ ID NO: 6 or 16.

32. The construct of claim 29, wherein the extracellular domain comprises an antigen binding region capable of binding TIM-4.

33. The construct of claim 32, wherein the CAR comprises SEQ ID NO: 41 or a sequence with at least 95% identity to SEQ ID NO: 41.

34. The construct of any one of claims 29-33, wherein the CAR and the NKG2D fusion protein are connected via a self-cleavage site or an internal ribosome entry site.

35. The construct of claim 24, wherein the construct comprises a polynucleotide encoding a sequence selected from SEQ ID NO: 19, 20, 22, 23, 26 or 42 or a sequence with at least 95% sequence identity to SEQ ID NO: 19, 20, 22, 23, 26 or 42.

36. The construct of any one of claims 15-35, wherein the construct is included in a lentiviral, retroviral or AAV vector.

37. An immune cell comprising the construct of any one of claims 15-36.

38. An immune cell comprising the construct of claim 15, further comprising a second construct, wherein the second construct comprises a polynucleotide encoding a CAR, the CAR comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

39. The immune cell of claim 38, wherein the CAR comprises a sequence selected from SEQ ID NO: 6, 16 or 41 , or a sequence with at least 95% identity to SEQ ID NO: 6, 16 or 41 .

40. The immune cell of any one of claims 37-39, wherein the immune cell is a T lymphocyte, a macrophage cell or a natural killer (NK) cell.

41. The immune cell of any one of claims 37-40, wherein the immune cell is a T lymphocyte.

42. A method for treating cancer, the method comprising administering a therapeutically effective amount of the NKG2D fusion protein of any one of claims 1-13 or the pharmaceutical composition of claim 14 and a pharmaceutically acceptable excipient, carrier and / or diluent to a subject, wherein the subject has cancer.

43. The method of claim 42, additionally comprising administering T lymphocytes, wherein T lymphocytes are activated upon binding to the NKG2D fusion protein.

44. The method of claim 43, wherein the T lymphocytes are CAR T cells.

45. The method of claim 44, wherein the CAR T cells comprise an antigen binding region selected from SEQ ID NO: 8, SEQ ID NO: 15 or SEQ ID NO: 25 or a sequence having at least 95% identity to SEQ ID NO: 8, 15 or 25.

46. A method for treating cancer, the method comprising administering a therapeutically effective amount of the immune cell of any one of claims 37-41 and a pharmaceutically acceptable excipient, carrier and / or diluent to a subject, wherein the subject has cancer.

47. A method for treating cancer, the method comprising administering a cell comprising the construct of any one of claims 15-36 to a subject, wherein the subject has cancer.

48. The method of any one of claims 42-47, wherein the cancer is an EGFR-associated cancer or a phosphatidylserine (PS) associated cancer.

49. The method of claim 48, wherein the EGFR or PS-associated cancer comprises a glioma, glioblastoma, medulloblastoma, ependymoma, diffuse intrinsic pontine glioma (DIPG), a brain metastases, head and neck, ovarian, cervical, bladder or esophageal cancer.

50. The method of any one of claims 42-49, wherein the treatment of the cancer results in induction of an anti-tumor response to the EGFR or PS-associated cancer.

51. A method of detecting cancer, the method comprising administering the fusion protein of any one of claims 8 or 9 to a subject suspected of having cancer, detecting the presence or accumulation of a tag in the subject suspected of having cancer, and detecting cancer in the subject, wherein the accumulation or presence of the tag allows for in vivo detection of the cancer.

52. A method of detecting cancer, the method comprising contacting a sample comprising cells or tissue from a subject with the fusion protein of any one of claims 8 or 9 or the NKG2D domain of SEQ ID NO:5 linked to a tag, detecting the presence or accumulation of the tag on the sample, and detecting cancer in the sample, wherein the accumulation or presence of the tag allows for ex-vivo detection of cancer.

53. The method of claim 51 or 52, wherein the cancer detected is a glioma, glioblastoma, medulloblastoma, ependymoma, diffuse intrinsic pontine glioma (DIPG), a brain metastases, head and neck, ovarian, cervical, bladder or esophageal cancer.

54. A method of inducing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the fusion protein of any one of claims 1-13, the pharmaceutical composition of claim 14, or the immune cells of any one of claims 37-41, wherein an antitumor immune response is induced in the subject.

55. The method of claim 54, wherein the immune response is an antitumor immune response.

Images