Non-natural NKG2d receptors that do not directly signal the cells to which they are attached

Abstract

To provide non-natural NKG2D receptors attached to mammalian cell surfaces.SOLUTION: There is provided a non-natural, modified NKG2D receptor, wherein the receptor binds to non-natural, modified α1-α2 domains of NKG2D ligands but not natural ligands of NKG2D, wherein the modified NKG2D receptor attached to a mammalian cell serves as a surrogate high affinity receptor for the delivery to the cell of a heterologous atom or molecule binding to the modified α1-α2 domains, and wherein the modified NKG2D receptor does not directly activate or signal the mammalian cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] 2. Background of the Invention FIELD OF THEINVENTION The present application relates generally to a non-natural ectodomain of a non-natural NKG2D receptor that has been modified to specifically bind to the non-natural NKG2D receptor and that does not directly activate or directly signal the mammalian cell when a non-natural NKG2D ligand binds to the receptor, wherein a heterologous molecule is attached to the modified α1-α2 domain of the NKG2D ligand. [Background technology]

[0002] Background information NKG2D is an activating receptor expressed as a type II homodimeric integral protein on the surface of natural killer (NK) cells and certain T cells and macrophages. Upon binding to one of its eight natural ligands, which are primarily expressed on the surface of stressed cells, NKG2D activates NK cells to kill the stressed cells, or, in the case of T cells, ligand-occupied NKG2D co-stimulates activated T cells to execute their effector functions. The three-dimensional structures have been solved for the ectodomain of human native NKG2D, some of its soluble natural ligands, and, in some cases, the bound complex of soluble ligand and receptor ectodomain. The monomeric α1-α2 domain of the NKG2D ligand specifically binds to the two ectodomains of the native NKG2D homodimer. Summary of the Invention

[0003] Summary of the Invention The present disclosure relates to a non-natural NKG2D receptor inserted on the surface of a mammalian cell, which does not directly signal or activate the cell when a cognate non-natural α1-α2 domain of an NKG2D ligand that has been modified to specifically bind to the non-natural NKG2D receptor binds to the non-natural receptor. The non-natural α1-α2 domain of the NKG2D ligand may be bound to a heterologous atom or molecule, including a polypeptide, in some embodiments a cytokine or modified cytokine, an antibody or a fragment of an antibody. Direct activation of the cell or direct signaling to the cell is not mediated by the inserted non-natural NKG2D receptor, even if an immunological synapse occurs. BRIEF DESCRIPTION OF THE DRAWINGS [Brief description of the drawings]

[0004] [Figure 1] 1A-1B: (Figure 1A) Alignment of native NKG2D.wt ectodomain (SEQ ID NO: 17) with NKG2D.YA (SEQ ID NO: 18) and NKG2D.AF (SEQ ID NO: 25) non-natural variants. The positions of Y152 and Y199 are indicated, and the mutated residues present in the non-natural variants are highlighted in grey. (Figure 1B) Alignment of native / wild type ULBP2

[0005] [ka]

[0006] and an alignment of non-natural variants of ULBP2, including ULBP2.R80W (SEQ ID NO: 108). Residues important for binding of non-natural ULBP2 variants to the non-natural NKG2D.YA or NKG2D.AF receptors are highlighted in grey. 2.S3, SEQ ID NO: 127) or NKG2D.AF (ULBP2.C, SEQ ID NO: 111; ULBP2.R, SEQ ID NO: 113; ULPB2.AA, SEQ ID NO: 115; and ULBP2.AB, SEQ ID NO: 117). [Diagram 2] Figure 2: Size exclusion chromatography comparison of non-native Fc-NKG2D fusion proteins analyzed on an Akta HiLoad 16 / 600 Superdex 200 column. The migration of properly assembled material is illustrated by a separate symmetrical peak that elutes at a higher volume, while aggregated material elutes earlier at a lower volume. The site and nature of the modifications are indicated by amino acid number Y152, Y199, or both (starting at the top of the figure, SEQ ID NOs: 48, 43, 42, 58, 41, and 40). [Diagram 3] Figure 3: Size-exclusion chromatographic profiles of non-native Fc-eNKG2D variants with one or two amino acid changes were analyzed on an Akta Superdex 200 Increase 10 / 300 GL column. The migration of properly assembled material is illustrated by a separate symmetrical peak that elutes at a higher volume, while aggregated material (characterized by a broad peak or series of peaks of low amplitude) elutes at a lower volume. The letters in brackets represent the amino acids at positions 152 and 199, respectively (SEQ ID NOs: 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, and 40, from top to bottom). [Figure 4] Figure 4: ELISA binding of ULBP2 wild-type, MICwed- and MIC25-Rituximab MicAbodies to Fc-eNKG2D candidates. Legends are shown at the top of the figure, but since many of the curves overlap, individual curves are also labeled in each graph. [Diagram 5] Figure 5: Binding of eNKG2D variants to wild-type ligand. Wild-type ligand (all Fc fusion format) was captured on an Octet AHC biosensor and titrated from 300 nM to 0.41 nM with native NKG2D as an Fc fusion, NKG2D.Y152A, or eNKG2D5 (Y152A / Y199F), respectively. Maximum binding response was quantified by Octet (note different vertical scales for each graph). [Figure 6]Figure 6: Titration ELISA of individual phage variants to confirm selective binding to Fc-NKG2D.AF and reduced or abolished binding to Fc-NKG2D.wt. Mutations are detailed in Figure 22. [Figure 7] Figure 7: ELISA data for four non-native α1-α2 ULBP2 variant MicAbodies binding to NKG2D.wt, NKG2D.YA, and NKG2D.AF. Fc-NKG2D variants were used as capture agents. MicAbodies were titrated in and detected with HRP-conjugated anti-human κ. [Figure 8-1] Figure 8: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHCI immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHC4.0 Server and queried against HLA supertype representatives. For input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was included along with 9 residues upstream and downstream (total of 24 residues) and a 9-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides that are strongly predicted to bind to the MHCI pocket (defined as having a % rank <0.5) and therefore have a strong likelihood of being presented. Light grey boxes correspond to predicted weak binders (% rank <2). See Example 5 main text for further details. [Figure 8-2]Figure 8: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHCI immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHC4.0 Server and queried against HLA supertype representatives. For input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was included along with 9 residues upstream and downstream (total of 24 residues) and a 9-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides that are strongly predicted to bind to the MHCI pocket (defined as having a % rank <0.5) and therefore have a strong likelihood of being presented. Light grey boxes correspond to predicted weak binders (% rank <2). See Example 5 main text for further details. [Figure 9-1] Figure 9: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHC class II immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHCII 2.3 Server and queried against HLA-DR, HLA-DQ, and HLA-DP. For the input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was entered along with 15 residues upstream and downstream (total of 36 residues) and a 15-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides strongly predicted to bind to the MHCII pocket and therefore likely to be presented and immunogenic. Light grey boxes correspond to predicted weak binders. [Figure 9-2]Figure 9: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHC class II immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHCII 2.3 Server and queried against HLA-DR, HLA-DQ, and HLA-DP. For the input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was entered along with 15 residues upstream and downstream (total of 36 residues) and a 15-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides strongly predicted to bind to the MHCII pocket and therefore likely to be presented and immunogenic. Light grey boxes correspond to predicted weak binders. [Figure 9-3] Figure 9: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHC class II immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHCII 2.3 Server and queried against HLA-DR, HLA-DQ, and HLA-DP. For the input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was entered along with 15 residues upstream and downstream (total of 36 residues) and a 15-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides strongly predicted to bind to the MHCII pocket and therefore likely to be presented and immunogenic. Light grey boxes correspond to predicted weak binders. [Figure 9-4]Figure 9: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHC class II immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHCII 2.3 Server and queried against HLA-DR, HLA-DQ, and HLA-DP. For the input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was entered along with 15 residues upstream and downstream (total of 36 residues) and a 15-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides strongly predicted to bind to the MHCII pocket and therefore likely to be presented and immunogenic. Light grey boxes correspond to predicted weak binders. [Figure 9-5] Figure 9: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHC class II immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHCII 2.3 Server and queried against HLA-DR, HLA-DQ, and HLA-DP. For the input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was entered along with 15 residues upstream and downstream (total of 36 residues) and a 15-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides strongly predicted to bind to the MHCII pocket and therefore likely to be presented and immunogenic. Light grey boxes correspond to predicted weak binders. [Figure 9-6]Figure 9: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHC class II immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHCII 2.3 Server and queried against HLA-DR, HLA-DQ, and HLA-DP. For the input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was entered along with 15 residues upstream and downstream (total of 36 residues) and a 15-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides strongly predicted to bind to the MHCII pocket and therefore likely to be presented and immunogenic. Light grey boxes correspond to predicted weak binders. [Figure 9-7] Figure 9: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHC class II immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHCII 2.3 Server and queried against HLA-DR, HLA-DQ, and HLA-DP. For the input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was entered along with 15 residues upstream and downstream (total of 36 residues) and a 15-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides strongly predicted to bind to the MHCII pocket and therefore likely to be presented and immunogenic. Light grey boxes correspond to predicted weak binders. [Figure 9-8]Figure 9: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHC class II immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHCII 2.3 Server and queried against HLA-DR, HLA-DQ, and HLA-DP. For the input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was entered along with 15 residues upstream and downstream (total of 36 residues) and a 15-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides strongly predicted to bind to the MHCII pocket and therefore likely to be presented and immunogenic. Light grey boxes correspond to predicted weak binders. [Figure 9-9] Figure 9: ULBP2.C (SEQ ID NO:111), ULBP2.R (SEQ ID NO:113), ULBP2.AA (SEQ ID NO:115), and ULBP2.AB (SEQ ID NO:117) were examined for changes in peptide-MHC class II immunogenicity compared to wild-type ULBP2 (SEQ ID NO:4) using the NetMHCII 2.3 Server and queried against HLA-DR, HLA-DQ, and HLA-DP. For the input sequences, the variable region for each variant (residues 154-159 according to the alignment in Figure 1B) was entered along with 15 residues upstream and downstream (total of 36 residues) and a 15-mer peptide window was examined for predicted immunogenicity. Dark grey boxes correspond to peptides strongly predicted to bind to the MHCII pocket and therefore likely to be presented and immunogenic. Light grey boxes correspond to predicted weak binders. [Figure 10]Figures 10A-10B: ELISA-measured binding of Rituximab-MicAbodies containing ULBP2.wt (wild type), ULBP2.R80W (with enhanced affinity for wild type NKG2D), ULPB2.S3 (NKG2D.YA selected orthogonal variant), or ULBP2.R (NKG2D.AF selected orthogonal variant) to native NKG2D.wt, NKG2D.YA, and NKG2D.AF. (Figure 10A) ELISA curves. The decrease in 458 nm absorbance for some assays at higher concentrations is an artifact often seen for higher concentrations of high affinity engagers due to precipitation of the TMB-Ultra ELISA chromogenic reagent. (Figure 10B) EC50 values ​​(reported in nM) determined in GraphPad Prism based on the curves in (A). nd = not determined due to lack of relationship between increased concentration and binding. [Figure 11] Figures 11A-11C: In vitro cytolysis assays using CD8 effector cells either untransduced or transduced with NKG2D.wt, NKG2D.YA, or NKG2D.AF CAR constructs consisting of the CD8a hinge / transmembrane domain and intracellular 4-1BB and CD3 zeta signaling domains. Target cells were preloaded with calcein and exposed to effector cells at increasing effector to target (E:T) ratios. Released calcein was quantified after 5 hours. (Figure 11A) Cytolysis of HeLa cells. (Figure 11B) Lysis of HeLa cells transfected to overexpress surface ULBP1. (Figure 11C) Cytolysis of HeLa cells expressing non-native ULBP2.R on their surface. Error bars correspond to the standard deviation of technical replicates in an experiment. [Figure 12]Figures 12A-12B: MicAbody-directed cytolysis of tumor lines by NKG2D-CAR CD8 T cells. (Figure 12A) Ramos cells (expressing CD20, the target of Rituximab) were preloaded with calcein and exposed to NKG2D.AF- or NKG2D.YA-CAR cells at an E:T ratio of 20:1 with increasing concentrations of either ULBP2.S2 or ULBP2.R Rituximab-Micabody. Levels of cytolysis were quantified after 2 hours of co-incubation. (Figure 12B) The mouse tumor line CT26, transfected to express human Her2, was used as a cytolysis target in parallel with Ramos cells. NKG2D.AF-CAR CD8 T cells were preloaded with saturating concentrations (5 nM) of Rituximab-ULBP2.R, Trastuzumab-ULBP2.R, or an equimolar mixture of the two MicAbodies. Unbound MicAbody was removed by washing and CD8 cells were added to the target cells at two different E:T ratios. Cytolysis was measured after 2 hours. [Figure 13]Figures 13A-13D: Illustration of candidate MicAbody and MicAdaptor formats. (Figure 13A) Various antibody Fc variants utilized in the development of MicAbody and MicAdaptor reagents include (a) wild-type human IgG1 Fc, (b) two mutations that render the Fc ADCC-deficient, and (c) electrostatic steering mutations in the respective Fc—Fc1 or Fc2—that allow for the generation of heterodimeric Fc molecules that also contain ADCC-deficient mutations. (Figure 13B) Examples of methods to fuse orthogonal ligands to the C-terminus of (a) the heavy chain or (b) the light chain to generate MicAbody reagents. (Figure 13C) Examples of MicAdaptors with direct orthogonal ligand fusions without antibody components. (FIG. 13D) Illustration of various MicAdaptor molecules that can be generated in the context of human IgG1 Fc to enhance serum stability, with or without (a, b, c, referred to as "Fc1 / Fc2" in text and figure legends) heterodimer-Fc mutations depending on the desired valency of the molecule for either heterologous cargo or orthogonal ligand. This can also involve utilizing the complete antibody structure, and can be a fusion of either the heavy or light chain, depending on the desired cargo, valency, and functionality (f, g). [Figure 14] Figure 14: Schematic diagram of CAR constructs, silent CARs, and other CAR variants. SEQ ID NOs for each construct are shown. [Figure 15]Figures 15A-15C: Selective delivery of cytokine fusions to CD8 T cells expressing NKG2D.YA-CAR. (Figure 15A) CD8 cells expressing either NKG2D.wt-CAR (SEQ ID NO: 151) or NKG2D.YA-CAR SEQ ID NO: 153) were exposed to 30 or 300 IUe / mL of recombinant human IL2 (rhIL2) or IL15 (rhIL15), or direct fusions of mutant-IL2 / -IL15 orthogonal ligands or variants of the fusions in the context of heterodimeric Fc (Fc1 / Fc2). After 3 days, proliferation was quantified by WST cell proliferation reagent. Data here are shown normalized to a no cytokine control. (Figure 15B) Human T cells transduced with a vector encoding NKG2D.YA-CAR were exposed to various ligand-cytokine fusion molecules for 7 days and the percentage of GFP+CAR- cells in the cultures was followed over time. (FIG. 15C) Orthogonal ligands enhance delivery of IL21 and mutant IL21 reagents to NKG2D.YA-CAR cells and promote their expansion over 3 days of culture compared to non-transduced cells as determined by WST assay. IUe / mL of cytokine or cytokine-MicAdaptor are shown in parentheses in the horizontal axis legend. See FIG. 24 for MicAdaptor sequence numbers. [Figure 16] Figures 16A-16C: Data investigating the sufficiency of the NKG2D.YA ectodomain alone in promoting delivery of orthogonal ligand-cytokine-fusions to cells. (Figure 16A) The NKG2D.YA ectodomain alone (SEQ ID NO: 157) was unable to direct killing of Ramos cells by Rituximab-ULBP2.S3 MicAbody (SEQ ID NOs: 98 and 129). (Figure 16B) WST proliferation assays after 3 days of exposure to various cytokine reagents. (Figure 16C) WST proliferation assays demonstrated that engagement of the NKG2D.YA-CAR with the orthogonal ligand was insufficient to promote cell proliferation (but in the absence of fusion cytokine or cytokine variants). See Figure 24 for MicAdaptor SEQ ID NOs. The IUE / mL amounts of cytokine and cytokine-MicAdaptor are shown in parentheses in the horizontal axis legend. [Figure 17] Figures 17A-17C: (Figure 17A) WST proliferation assays of various costimulatory domain mutants (SEQ ID NOs: 161, 163, and 165) after 3 days of incubation with the indicated cytokines or cytokine-MicAdaptor. (Figure 17B) The same costimulatory domain CAR mutants examined for their ability to effectively lyse calcein-loaded Ramos target cells in the presence of rituximab-ULBP2.S3 MicAbod (SEQ ID NOs: 98 and 129). (Figure 17C) Co-expression of the NKG2D.YA ectodomain (NKG2D.YA-ecd) with the full CD19scFv-CAR is sufficient to promote proliferation as assessed by WST assays after 3 days of incubation with cytokine reagents. See Figure 24 for MicAdaptor SEQ ID NOs. [Figure 18] FIG. 18: Summary of mutational and protein aggregation properties of candidate non-native Fc-eNKG2D variants as determined by size exclusion chromatography (SEC). [Figure 19] Figure 19: Saturation percentage (Rmax) of eNKG2D variants normalized to wild-type NKG2D binding by either MICwed-MicAbody or MIC25-MicAbody. Wild-type Fc-NKG2D and each Fc-eNKG2D receptor were captured on an AHC biosensor and then exposed to 20 nM of trastuzumab-specific MicAbody. Dissociation rates were monitored and the Rmax values ​​of Fc-eNKG2D fusions were ranked. Samples that were not tested (nt) were due to either severe aggregation or insufficient amounts of material expressed or recovered after SEC fractionation. [Figure 20] Figure 20: EC50 values ​​(nM) for the Fc-eNKG2D ELISA shown in Figure 3. nt = not tested; nb = no binding, or very low binding even at 300 nM, EC50 value not calculated. [Figure 21-1]Figure 21: A subset of combinatorial mutations in ULBP2 that resulted in phage clones with selective binding to NKG2D.AF versus native NKG2D.wt as confirmed by spot ELISA. Mutations were ranked by frequency of occurrence among selected phage. [Figure 21-2] Figure 21: A subset of combinatorial mutations in ULBP2 that resulted in phage clones with selective binding to NKG2D.AF versus native NKG2D.wt as confirmed by spot ELISA. Mutations were ranked by frequency of occurrence among selected phage. [Figure 22] Figure 22: Specificity of NKG2D.AF selected ULBP2 variants in Rituximab-MicAbody format retained their binding to NKG2D.AF by quantitative ELISA. Specific amino acid modifications of each ULBP2 variant are shown as the ratio of its binding to Fc-NKG2D.wt fusions versus Fc-NKG2D.AF fusions. The positions of amino acid residues in ULBP2 are those in Figure 1B. [Diagram 23] Figure 23: Selected mutations at the indicated amino acid positions of ULBP2.R80W (Figure 1B; SEQ ID NO: 108) that resulted in Y152A-specific phage clones. [Figure 24] FIG. 24: MicAdaptor sequence numbers and purification methods from transient transfections. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Detailed Description of the Invention Natural killer (NK) cells and specific (CD8+αβ and γδ) T cells of the immune system play an important role in humans and other mammals as the first line of innate defense against neoplasms and infected cells (Cerwenka, A., and LLLanier. 2001. NK cells, viruses and cancer. Nat. Rev. Immunol. 1:41-49). NK cells and certain T cells display on their surface a prominent homodimeric surface immune receptor, NKG2D, which is responsible for target cell recognition and activation of the innate defense against pathological cells (Lanier, LL, 1998. NK cell receptors. Ann. Rev. Immunol. 16:359-393; Houchins JP et al. 1991. DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human NK cells. J. Exp. Med. 173:1017-1020; Bauer, S et al., 1999. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285:727-730).The human NKG2D molecule has a C-type lectin-like extracellular (ecto) domain that binds to its eight different cognate ligands, the best studied of which are the 84% sequence-identical or homologous monomeric MICA and MICB (polymorphic analogs of major histocompatibility complex (MHC) class I chain-related glycoprotein (MIC)) (Weis et al. 1998. The C-type lectin superfamily of the immune system. Immunol. Rev. 163:19-34; Bahram et al. 1994. A second lineage of mammalian MHC class I genes. PNAS 91:6259-6263; Bahram et al. 1996a. Nucleotide sequence of the human MHC class I MICA gene. Immunogenetics 44: 80-81; Bahram and Spies TA. 1996. Nucleotide sequence of human MHC. class I MICB cDNA. Immunogenetics 43:230-233). Non-pathological expression of MICA and MICB is restricted to some intestinal epithelia, keratinocytes, endothelial cells and monocytes, but aberrant surface expression of these MIC proteins occurs in response to many types of cellular stress, such as proliferation, oxidation and heat shock, and characterizes cells as pathological (Groh et al. 1996. Cell stress-regulated human MHC class I gene expressed in GI epithelium. PNAS 93:12445-12450; Groh et al. 1998. Recognition of stress-induced uced MHC molecules by intestinal γδ T cells. Science 279:1737-1740; Zwirner et al. 1999. Differential expression of MICA by endothelial cells, fibroblasts, keratinocytes and monocytes. Human Immunol. 60:323-330). Pathological expression of MIC proteins also appears to be involved in several autoimmune diseases (Ravetch, JV and Lanier LL. 2000. Immune Inhibitory Receptors. Science 290:84-89; Burgess, SJ. 2008. Immunol. Res. 40:18-34). Differential regulation of NKG2D ligands such as polymorphic MICA and MICB is important for providing the immune system with the means to identify and respond to a wide range of emergency cues while still protecting healthy cells from unwanted attack (Stephens HA, (2001) MICA and MICB genes: can the enigma of their polymorphism be resolved? Trends Immunol. 22:378-85; Spies, T. 2008. Regulation of NKG2D ligands: a purposeful but delicate affair.Nature Immunol.9:1013-1015).

[0008] Viral infection is a common inducer of MIC protein expression, identifying virus-infected cells for NK or T cell attack (Groh et al. 1998; Groh et al. 2001. Co-stimulation of CD8+ αβT cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat. Immunol. 2:255-260; Cerwenka, A., and LLLanier. 2001). In fact, to avoid such attacks on their host cells, cytomegalovirus and other viruses have evolved mechanisms to prevent the expression of MIC proteins on the surface of the cells they infect in order to escape targeting by the innate immune system (Lodoen, M., K. Ogasawara, JA Hamerman, H. Arase, JP Houchins, ES Mocarski, and LLLanier. 2003. NKG2D-mediated NK cell protection against cytomegalovirus is impaired by gp40 modulation of RAE-1 molecules. J. Exp. Med. 197:1245-1253; Stern-Ginossar et al., (2007) Host immune system gene targeting by viral miRNA.Science 317:376-381;Stern-Ginossar et al.,(2008)Human microRNAs regulate stress-induced immune responses mediated by the receptor NKG2D.Nature Immunology 9:1065-73;Slavuljica,IA Busche,M Babic,M Mitrovic,I Gasparovic,D Cekinovic,E Markova Car,EP Pugel,A Cikovic,VJ Lisnic,WJ Britt,U Koszinowski,M Messerle,A Krmpotic and S Jonjic.2010.Recombinant mouse cytomegalovirus expressing a ligand for the NKG2D receptor is attenuated and has improved vaccine properties.J.Clin.Invest.120:4532-4545).

[0009] Despite these stresses, many malignant cells, such as those in lung cancer and glioblastoma brain cancer, also evade the expression of MIC proteins and, as a result, evade the innate immune system in an excessive manner. Furthermore, NK cell mediated rejection of experimental human lung cancer by genetic over expression of MHC class I chain-related gene A.Human Gene Therapy 17:135-146;Doubrovina,ES,MM Doubrovin,E Vider,RB Sisson,RJ O'Reilly,B Dupont,and YM Vyas,2003.Evasion from NK Cell Immunity by MHC Class I Chain-Related Molecules Expressing Colon Adenocarcinoma (2003)J.Immunology 6891-99;Friese,M.et al.2003.MICA / NKG2D-mediated immunogene therapy of experimental gliomas.Cancer Research 63:8996-9006;Fuertes,MB,MV Girart,LL Molinero,CI Domaica,LE Rossi, MM Barrio, J Mordoh, GA Rabinovich and NW Zwirner.(2008)Intracellular Retention of the NKG2D Ligand MHC Class I Chain-Related Gene A in Human Melanomas Confers Immune Privilege and Prevents NK Cell-Mediated Cytotoxicity.J.Immunology,180:4606-4614).

[0010] A high-resolution structure of human MICA bound to NKG2D has been solved, demonstrating that the α3 domain of MICA has no direct interaction with NKG2D (Li et al. 2001. Complex structure of the activating immunoreceptor NKG2D andits MHC class I-like ligand MICA. Nature Immunol. 2: 443-451; Protein Data Bank accession code 1HYR). The α3 domain of MICA, like that of MICB, is linked to the α1-α2 platform domain by a short, flexible linker peptide, and as such is naturally positioned as a "spacer" between the platform and the surface of MIC-expressing cells. The three-dimensional structures of human MICA and MICB α3 domains are nearly identical (94C-αα' root mean square distance <1 Å) and functionally interchangeable (Holmes et al. 2001. Structural Studies of Allelic Diversity of the MHC Class I Homolog MICB, a Stress-Inducible Ligand for the Activating Immunoreceptor NKG2D. J Immunol. 169:1395-1400).

[0011] Using gene transfer techniques, T cells, NK cells, and macrophages can be engineered to directly and stably express on their surface the binding domain of an antibody that confers new antigen specificity (Saar Gill & Carl H. June. Going viral: Chimeric Antigen Receptor (CAR) T cell therapy for hematological malignancies.Immunological Reviews 2015.Vol.263:68-89;Wolfgan g Glienke,Ruth Esser,Christoph Priesner,Julia D.Suerth,Axel Schambach,Winfried S.Wels,Manuel Grez,Stephan Kloess,Lubomir Arseniev and Ulrike Koehl.2015.Advantages and applications of CAR-expressing na tural killer cells. Front.Pharmacol.doi:10.3389 / fphar.2015.00021). CAR-T cells are an application of this approach that combines the antigen recognition domain of a specific antibody with the fused intracellular domain of the CD3-zeta chain. The CD3-zeta chain is the main transduction of signals from the ectodomain of the endogenous T cell receptor (TCR) to the intracellular space. CARs constructed with the CD3-zeta chain and costimulatory molecules such as CD27, CD28, ICOS, 4-1BB, or OX40, upon binding to a target antigen, trigger the activation of CAR-T cells in a manner similar to that of the endogenous T cell receptor, but independent of the major histocompatibility complex (MHC).

[0012] Certain non-natural α1-α2 domains of NKG2D ligands have been described that have been engineered to bind to the native human NKG2D receptor with higher affinity than does the native α1-α2 domain (Candice S Engyel, Lindsey J. Willis, Patrick Mann, David Baker, Tanja Kortemme, Roland K. Strong and Benjamin J. McFarland. Mutations Designed to Destabilize the Receptor-Bound Conformation Increase MICA-NKG2D Association Rate and Affinity. Journal of Biological Chemistry Vol. 282, no. 42, pp. 30658-30666, 2007; Samuel H. Henager, Melissa A. Hale, Nicholas J. Maurice, Erin C. Dunnington, Carter J. Swanson, Megan J. Peterson, Joseph J. Ban, David J. Culpepper, Luke D. Davies, Lisa K. Sanders, and Benjamin J. McFarland.Combining different design strategies for rational affinity maturation of the MICA-NKG2D interface.Protein Science 2012 VOL 21:1396-1402. We describe herein the insertion of a non-natural NKG2D receptor into a mammalian cell surface in a format that retains specific binding to a modified non-natural NKG2D ligand-bound heterologous molecule, but the non-natural receptor avoids direct or cis activation of the mammalian cell or intracellular signaling to the mammalian cell, even when the cell forms an immunological synapse with a cell or other surface targeted by the heterologous molecule.The non-native NKG2D receptor itself is mutated at one or two specific sites, each of which results in abolishment or loss of binding to all natural α1-α2 domains of NKG2D ligands (David J. Culpepper, Michael K. Maddox, Andrew B. Caldwell, and Benjamin J. McFarland. Systematic mutation and thermodynamic analysis of central tyrosine pairs in polyspecific NKG2D receptor interactions. Mol Immunol. 2011 January;48(4):516-523; USPTO Application No. 14 / 562,534; USPTO Provisional Application No. 62 / . No. 088,456). The present invention creates CARs that, when inserted into the cell surface of a mammal, provide a silent receptor that can function as a high affinity surrogate receptor for the binding of a heterologous atom or molecule to the cell surface. Thus, via binding non-natural ligands specific for the non-natural modified NKG2D receptor, heterologous molecules, including, for example, defective cytokines, can be specifically delivered to the silent receptor on the surface of a mammalian cell, but not to cells lacking the cognate silent receptor. Upon binding to a cell bearing the silent receptor, the defective heterologous molecule can bind to its respective receptor subunit on the cell surface where binding is retained, thereby directly signaling the cell as if stimulated by a wild-type ligand.

[0013] Of course, CARs composed of inactive non-natural NKG2D, CD3-zeta and costimulatory domains such as CD28, 4-1BB, ICOS, or OX40 on mammalian cells can directly stimulate and activate CAR cells upon immunological synapse formation. The activation of such second or third generation CAR-T cells is dependent on the function of their CD3-zeta domain and at least one costimulatory domain, e.g., 4-1BB or CD28. However, as silent CARs, such CARs can function as high affinity surrogate receptors for the binding of cognate non-natural ligand-bound heterologous molecules that are defective in binding to their respective natural receptor or receptor subunit(s). This high affinity binding allows the heterologous molecules bound to the non-natural ligands to signal the cells through their respective other receptor subunits that retain their binding.

[0014] Importantly, if the CD3-zeta domain of such a directly activatable CAR is selectively inactivated, it still functions as a silent CAR, allowing cognate non-natural ligand-binding heterologous molecules (whose binding to their respective natural receptors or receptor subunits is dysfunctional) to signal the cell through their respective other receptor subunits. If the CAR costimulatory domain, such as 4-1BB, is inactivated and an active CD3-zeta domain is retained, the CAR cannot function as a silent receptor. That is, CD3-zeta is not required, but a functional costimulatory domain is required to allow heterologous molecules, such as dysfunctional cytokines, that are bound to the cognate non-natural ligand bound to the receptor to mediate their respective signals to the CAR cell.

[0015] The present invention reveals the unexpected need for a costimulatory domain, rather than that of CD3-zeta, to allow a dysfunctional heterologous cytokine bound to its cognate non-natural ligand to mediate its respective signal to the CAR cell. Furthermore, the present invention discloses that the costimulatory domain can act in cis or trans on a silent receptor bound by its cognate ligand fused to a dysfunctional heterologous dysfunctional molecule.

[0016] When a heterologous molecule, such as an antibody or antibody fragment targeted to a particular molecule, binds to the cognate non-natural NKG2D ligand, which in turn binds to the silent receptor, the mammalian cell bearing the silent receptor will home to the surface to which the targeting heterologous molecule is directed. Even when a "synapse" is created between the cell bearing the silent receptor and the target cell surface, the former is not activated by the silent receptor.

[0017] Because there are multiple copies of the non-natural NKG2D-based silent receptor of the invention on the cell surface, homing and / or selective activation by heterologous molecules can be multiplexed or sequentially altered during the manufacturing process or processing protocol.

[0018] Cells carrying a silent receptor CAR may also express another receptor(s) or CAR that is orthogonal to the silent CAR and act independently of the silent CAR to specifically and directly activate or otherwise signal to that same cell when appropriately stimulated. The other or "second" orthogonal CAR may be a conventional single-chain Fv (scFv)-CAR, or a second, orthogonal, non-natural, engineered NKG2D-based CAR with its own cognate non-natural α1-α2 ligand(s) (AF Provisional Reference). The ability to create effector cells of the immune system with more than one orthogonal non-natural CAR, silent or active, and multiple cognate non-natural ligands with heterologous molecules or atoms attached, greatly expands the utility, flexibility, and control of adoptive cell therapy (ACT).

[0019] Silent CAR and its cis- or trans-acting costimulatory domains (4-1B In the course of characterizing the dependence of CARs on CD3-binding domains (e.g., CD3+, CD4+, CD5+, CD6+, CD8+, CD9+, and CD10+), human T cells expressing silent CARs with costimulatory domains showed significantly enhanced responses to native IL-2 or to a cognate non-natural ligand fused to either native IL-2 or a mutant IL-2 with reduced affinity for its receptor α subunit, compared to unmodified human T cells. This observation has important utility in the selective ex vivo or in vivo expansion of cells expressing CARs composed of costimulatory domains with or without the CD3-zeta domain.

[0020] As used herein, "soluble MIC protein," "soluble MICA," and "soluble MICB" refer to MIC proteins that contain the α1-α2 domains (with or without the α3 domain) of the MIC protein, but do not contain the transmembrane or intracellular domains. NKG2D ligands ULBP1-6 do not naturally have an α3 domain (Cerwenka A, Lanier LL. 2004. NKG2D ligands: unconventional MHC class I-like molecules exploited by viruses and cancer. Tissue Antigens 61(5):335-43. doi:10.1034 / j.1399-0039.2003.00070.x.PMID 12753652). The "α1-α2 domain" of an NKG2D ligand refers to the protein domain of the ligand that binds to the NKG2D receptor.

[0021] In some embodiments, the α1-α2 domain of the non-natural NKG2D ligand protein of the invention is at least 80% identical or homologous to the native or naturally occurring α1-α2 domain of the NKG2D ligand (SEQ ID NOs: 1-9 for MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and OMCP, respectively). In other embodiments, the modified α1-α2 domain is 85% identical to the native or naturally occurring α1-α2 domain of the NKG2D ligand. In yet other embodiments, the modified α1-α2 domain is 90% identical to the native or naturally occurring α1-α2 domain of the natural NKG2D ligand protein and binds to a non-natural NKG2D.

[0022] Preferably, the modified or non-natural α1-α2 domain of the non-natural MIC proteins of the invention is at least 80% identical or homologous to the native or natural α1-α2 domain of one of the eight human NKG2D ligand proteins (SEQ ID NOs: 1-8) and binds to the non-natural NKG2D ectodomain. In some embodiments, the non-natural α1-α2 domain is at least 85% identical to the native or natural α1-α2 domain of the NKG2D ligand protein and binds to the non-natural NKG2D. In other embodiments, the non-natural α1-α2 platform domain is 90%, 95%, 96%, 97%, 98%, or 99% identical to the native or natural α1-α2 platform of a human natural α1-α2 domain protein and binds to the non-natural NKG2D.

[0023] In some embodiments, heterologous molecular tags may be fused to the N-terminus or C-terminus of the non-native α1-α2 domain of the soluble MIC protein or to the N-terminus or C-terminus of the bound heterologous peptide or protein to aid in the purification of the soluble MIC ligand. Tag sequences include peptides such as polyhistidine, myc peptides, FLAG tags, streptavidin-like tags, or small molecules such as biotin. Such tags may be removed after isolation of the MIC molecule by methods known to those skilled in the art.

[0024] Specific mutations in the α1-α2 domain of an NKG2D ligand can be made to generate a non-natural α1-α2 domain that is engineered to bind to a non-natural NKG2D receptor and that itself has reduced affinity for the natural NKG2D ligand. For example, through genetic engineering. Such modified non-natural NKG2D receptors can be used to generate NKG2D-based CARs capable of binding to molecules comprising the non-natural α1-α2 domains of the invention on the surface of NK cells, T cells, macrophages, or other cells of the immune system. These non-natural NKG2D receptors and their cognate non-natural NKG2D ligands provide important safety, efficacy, and manufacturing advantages for the treatment of cancer and viral infections compared to current CAR-T and CAR-NK cells, as described below. When the intracellular signaling of non-natural NKG2D receptors on the surface of mammalian cells is silenced, as in the present invention, these CARs of the present invention can function as high-affinity surrogate receptors for otherwise non-functional heterologous molecules, such as cytokines, chemokines, lymphokines, cytotoxins, and atoms fused or conjugated to orthogonal NKG2D ligands. This provides for the direct and specific delivery of heterologous molecules to silent receptor-bearing cells without the silent receptor itself directly activating the host cell. Furthermore, heterologous molecules that bind to specific targets and thereby bind to cells or other surfaces bearing such targets can provide specific homing functions to cells bearing silent receptors without unintentionally activating or stimulating them.

[0025] CAR-T or CAR-NK cells that contain an ectodomain of a non-natural NKG2D receptor that does not bind or only weakly binds the natural NKG2D ligand are not subject to activation by any natural ligand and therefore are not as toxigenic as cells expressing a CAR based on the natural NKG2D receptor. Furthermore, the ectodomain of non-native NKG2D receptors on cells is not subject to downregulation by natural NKG2D ligands in soluble format or on myeloid-derived suppressor cells (MDSCs) (Deng W, Gowen BG, Zhang L, Wang L, Lau S, Iannello A, Xu J, Rovis TL, Xiong N, Raulet DH, 2015. Antitumor immunity. A shed NKG2D ligand that promotes natural killer cell activation and tumor rejection. Science. 2015 Apr 3;348(6230):136-9. doi:10.1126 / science.1258867. Epub 2015 Mar 5). However, when such a CAR cell bearing the ectodomain of the non-native NKG2D receptor is engaged by a bispecific molecule with the cognate non-native α1-α2 domain of the invention and its heterologous targeting motif that finds and binds to its intended target, the CAR is activated and the effector functions of the CAR cell are expressed.

[0026] Because CAR-T or CAR-NK cells containing a non-native NKG2D receptor ectodomain are not activated except in the presence of an engaged bispecific molecule containing the cognate non-native α1-α2 domain, their activation can be controlled by administration of the bispecific molecule, which, as a biopharmaceutical, exhibits pharmacokinetics and pharmacodynamics well known in the art. In the event of an adverse event, the physician can simply modify the dosing regimen of the administered bispecific molecule, rather than having to deploy an inducible suicide mechanism to destroy the infused CAR cells, as is currently done (Monica Casucci and Attilio Bondanza. Suicide Gene Therapy to Increase the Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes. J Cancer. 2011;2:378-382). Furthermore, such bispecific molecules with different specific targeting motifs can be administered simultaneously or sequentially to help address tumor resistance and escape as a result of target antigen loss, without having to generate, expand and inject multiple different autologous CAR cells (Gill & June, 2015). All CAR constructs can be identical for all CAR cells, and targeting specificity is simply determined by the targeting motif of the bispecific molecule of the invention administered. Since the manufacturing process is simplified and less expensive,

[0027] Examples of parent or recipient proteins or polypeptides that are candidates for binding to a non-native α1-α2 domain of an NKG2D ligand include, but are not limited to, antibodies, proteins containing an Ig-fold or Ig domain (including modified Fc domains that recruit or do not recruit or bind native molecules), globulins, albumins, fibronectin and fibronectin domains, integrins, fluorescent proteins, enzymes, outer membrane proteins, receptor proteins, T cell receptors, chimeric antigen receptors, viral antigens, viral capsids, viral ligands for cellular receptors, hormones, cytokines and modified cytokines (e.g., interleukins), knottins, cyclic peptides or polypeptides, major histocompatibility (MHC) family proteins, MIC proteins, lectins, and ligands of lectins. Non-protein molecules such as polysaccharides, dendrimers, polyglycols, peptidoglycans, antibiotics, and polyketides can also be attached to modify the α1-α2 domain of an NKG2D ligand.

[0028] Thus, the present invention expands the versatility and utility of this remarkable and highly promising immunological approach to cancer management using CAR-T cells, CAR-NK cells, and CAR-macrophage-like cells, while overcoming many of these currently recognized difficulties.

[0029] As used herein, "peptide", "polypeptide", and "protein" are used interchangeably; and "heterologous molecule", "heterologous peptide", "heterologous sequence" or "heterologous atom" are molecules, peptides, nucleic acid or amino acid sequences, or atoms, respectively, that are not naturally or normally found in physical association with the molecule of interest. As used herein, "non-natural" and "modified" are used interchangeably. As used herein, "natural", "native", and "wild type" are used interchangeably, and "NKG2D" and "NKG2D receptor" are used interchangeably. The term "antibody" is used herein in the broadest sense, and specifically covers monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as it exhibits the desired biological activity. An "antibody fragment" includes a portion of an antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments and insertible Fv; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0030] The term "comprising," which is used interchangeably with "including," "containing," and "characterized by," is an inclusive or open-ended term and does not exclude additional, unrecited elements or method steps. The term "consisting of" excludes any element, step, or ingredient not specified in the claim. The term "consisting essentially of" limits the scope of the claim to those materials or steps specified and that do not materially affect the basic novel characteristics of the claimed invention. This disclosure contemplates embodiments of the compositions and methods of the invention that fall within the scope of each of these terms. Thus, a composition or method that includes recited elements or steps contemplates certain embodiments in which the composition or method consists essentially of or consists of those elements or steps.

[0031] All references cited herein are incorporated by reference in their entirety, whether or not they have been specifically incorporated previously. As used herein, the terms "a," "an," and "any" are each intended to include both the singular and the plural.

[0032] Although the invention has been fully described herein, those skilled in the art will recognize that it can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. Although the invention has been described with reference to specific embodiments thereof, it will be understood that further modifications are possible. This application is generally intended to cover any variation, use, or application of the invention, including departures from the present disclosure that are in accordance with the principles of the invention and that are within known or customary practice within the technical field to which the invention pertains and to which the essential features described above may be applied. EXAMPLES

[0033] Working Example Modified NKG2D receptor ectodomain and modified α1-α2 domain of NKG2D ligand Example 1. Modification of tyrosine 152 to alanine (Y152A) and tyrosine 199 to phenylalanine (Y199F) of the human NKG2D receptor to generate an inactive NKG2D ectodomain

[0034] It has been demonstrated by others that mutations in tyrosine 152 or tyrosine 199 (equivalent to positions 73 and 120 in the NKG2D ectodomain) in human NKG2D (FIG. 1A, SEQ ID NO: 17) can greatly reduce binding to the natural ligand MICA (David J. Culpepper, Michael K. Maddox, Andrew B. Caldwell, and Benjamin J. McFarland. Systematic mutation and thermodynamic analysis of central tyrosine pairs in polyspecific NKG2D receptor interactions. Mol Immunol. 2011 January;48(4):516-523). We reasoned that while mutation of any tyrosine residue would profoundly affect the ability of NKG2D to bind its natural ligands, simultaneous mutations at both tyrosine 152 (Y152) and tyrosine 199 (Y199) would essentially abolish the receptor's ability to engage all native ligands. Therefore, we sought to explore individual and combined Y152 and Y199 substitutions with the goal of identifying both single and double mutation variants that would be unable to engage any natural ligands and to characterize them in terms of their biochemical behavior. We particularly focused on variants that were also well expressed and assembled, as these represented inactive ligands that could be more easily produced for analysis.

[0035] The native NKG2D (wild-type) ectodomain (NKG2D.wt, SEQ ID NO: 17) and candidate non-native NKG2D variant ectodomains (SEQ ID NOs: 18-35) (also referred to as "engineered NKG2D" or "eNKG2D") were cloned as fusions into the C-terminus of human IgG1 Fc (without the Fab domain) via a short factor Xa-recognizable Ile-Glu-Gly-Arg linker (SEQ ID NO: 38). They are interchangeably referred to as Fc-NKG2D.wt or NKG2D.wt and Fc-eNKG2D or eNKG2D (SEQ ID NOs: 40-58). gBlocks® DNA Fragments (Integrated DNA Technologies, San Diego, CA) corresponding to the MHCI signal sequence (SEQ ID NO: 36 and 37), human IgG1 Fc with linker (SEQ ID NO: 39), and NKG2D ectodomain variants (SEQ ID NO: 59-77) were synthesized and inserted into pD2610-V12 (ATUM, Newark, CA). DNA constructs exploring substitutions at Y152, Y199, or combinations of Y152 / Y199 mutations (FIG. 18) were transiently expressed in Expi293™ cells (ThermoFisher Scientific, Waltham, MA), and secreted proteins were purified by protein A affinity chromatography (cat. no. 20334, Pierce Biotechnology, Rockford, IL). The eluted material was characterized by size exclusion chromatography (SEC) on an Akta Pur Superdex column, and properly assembled sized material was fractionated and isolated from the aggregate peak prior to inclusion in the assay.

[0036] SEC characterization of purified NKG2D.Y199A-Fc fusions revealed a composition of predominantly aggregated material (Figure 2). In comparison, both native Fc-NKG2D and Fc-NKG2D.Y152A fusion materials were distinguished by a separate non-aggregated peak that was easily distinguished from the more rapidly migrating aggregates. The effect of the Y199A mutation on aggregation was also evident in the Y152A / Y199A double mutant Fc-NKG2D fusion variant, indicating that it has a major impact on protein misfolding (Figure 2). This aspect of including Y199A with any combination of Y152 mutations in NKG2D variants therefore presented a challenge for the production of material necessary for subsequent engineering efforts and raised concerns about assembly and presentation on the cell surface. As a result, efforts were made to explore other substitutions at Y152 and Y199 that could be combined to yield more robust molecules. eNKG2D combination Y152 and Y199 mutant candidates were examined as Fc fusions and are detailed in Figure 18. Additionally, all purified and expressed Fc-eNKG2D fusion candidates were profiled by SEC and the chromatograms revealed varying levels of aggregate formation (Figures 2 and 3, Figure 18). Among the single amino acid substitutions explored at residue 152, alanine, serine, threonine, and valine all had no effect on the assembly of the Fc-NKG2D molecule, whereas Y152-leucine (Y152L) led to highly aggregated material. Similar to alanine, neither glutamic acid nor aspartic acid was tolerated at position 199, but only phenylalanine moderately increased aggregate formation. Of the mutation combinations explored, Y152A / Y199F, Y152S / Y199F, Y152T / Y199F, and Y152F / Y199F did not negatively affect the desired dimer formation, while other combinations led to increased aggregation (Figure 18, Figures 2 and 3).

[0037] Example 2: Generation of antibody-based bispecific molecules "MicAbody" using non-natural NKG2D ligand variants To generate non-natural MicA variants fused to human IgG1, for example, DNA polynucleotides encoding the α1-α2 domains of MICwed (SEQ ID NO: 79) and MIC25 (SEQ ID NO: 81) were PCR amplified using primers that also introduced polynucleotides encoding either the APTSSSGGGGS linker for fusion to the C-terminal kappa light chain (SEQ ID NO: 84) or the GGGS linker for fusion to the C-terminal heavy chain (SEQ ID NO: 82) of human IgG1. In addition, two mutations were introduced in the CH2 domain of the heavy chain, D265A / N297A (Kabat numbering; Figures 13A and 13B), which reduce binding to all FcγR receptors and thus abolish antibody-dependent cellular cytotoxicity (ADCC) function (Shields et al., 2001 JBC, 276:6591-6604). A polynucleotide encoding the α1-α2 domain of wild-type ULBP2 without GPI linkage (ULBP2.wt) (SEQ ID NO: 12) was similarly cloned and fused to a DNA polynucleotide encoding a linker and an IgG1 heavy or light chain. These bispecific antibodies (referred to in the singular as "MicAbody™" and in the plural as "MicAbodies") are bivalent for the fused α1-α2 domain. Examples of antibodies used to generate MicAbodies for the purposes of exploring eNKG2D engineering include, but are not limited to, trastuzumab (SEQ ID NOs: 94 and 96) and rituximab (SEQ ID NOs: 98 and 100), which were subsequently referred to as "trastuzumab-MicAbody" (e.g., SEQ ID NOs: 102 and 104) and "rituximab-MicAbody" (e.g., SEQ ID NO: 106), respectively. The fusion constructs were individually inserted into pD2610-V12 (ATUM, Newark, Calif.) via Gibson cloning (New England Biolabs Inc., Ipswich, Mass.). For a given antibody to be recognized, a plasmid encoding the heavy chain and a plasmid encoding the light chain fused to either a natural or non-natural NKG2D ligand were co-transfected for transient expression in Expi293™ cells (ThermoFisher Scientific, Waltham, Mass.). Alternatively, a plasmid encoding the heavy chain fused to either a natural or non-natural NKG2D ligand and a plasmid for the light chain were co-transfected. Secreted bispecific antibodies were purified by Protein A affinity chromatography (cat. no. 20334, Pierce Biotechnology, Rockford, Ill.) and the eluted material was purified using Akta Purification. Characterization was performed by size-exclusion chromatography (SEC) on a Superdex column and fractionation was performed as required, and purified samples were subjected to SDS-PAGE analysis to confirm the expected molecular weights of the fusion heavy and light chain species.

[0038] Example 3: Identification of modified NKG2D variants that have enhanced binding to wild-type NKG2D and cannot bind to either natural NKG2D-binding ligands or non-natural ligands The binding affinity of α1-α2 variants to the extracellular domain of native (wild-type) NKG2D and non-native eNKG2D proteins was analyzed using a plate-based ELISA method. Each of the SEC-fractionated native Fc-NKG2D and non-native Fc-eNKG2D fusions was coated onto separate wells of a Nunc Maxisorp 96-well plate (Thermo Fisher Scientific, Waltham, MA) overnight at 4°C using a coating concentration of 1 μg / mL in phosphate-buffered saline (PBS). The plates were washed three times in PBS / 0.05% Tween-20 (PBS-T) at 20-22°C and blocked with 0.5% bovine serum albumin in PBS (PBS-B) for 2 h at 20-22°C. MicAbodies were titrated against plate-bound native or non-native Fc-NKG2D fusions for 60 min at 20-22°C in PBS / 0.5% bovine serum albumin (BSA) / 0.05% Tween-20 (PBS-BT), washed three times with PBS-T at 20-22°C, and bound bispecific proteins were detected using HRP-conjugated anti-human κ (Abcam, Cambridge MA) in PBS-BT and developed with 1-Step™ Ultra TMB ELISA Substrate Solution (Thermo Fisher Scientific, Waltham, MA). Binding of ULBP2.wt rituximab-MicAbody (SEQ ID NOs: 98 and 106) discriminated between wild-type NKG2D and eNKG2D variants with reduced binding to the latter, and the ligand variants (MICwed (SEQ ID NOs: 96 and 102) and MIC25 (SEQ ID NOs: 96 and 104)) were more stringent in identifying eNKG2D variants with abolished ligand binding. Binding behavior for each eNKG2D variant to all three dual specific ligands revealed combinations of NKG2D modifications that led to the greatest reduction in binding of wild-type and variant ligands, and were the lead inert It is now possible to select NKG2D variants.

[0039] Further biophysical analysis of binding of eNKG2D variants to ligands was also performed using Bio-Layer Interferometry (BLI) using a ForteBio Octet system (all ForteBio LLC, Fremont, CA). For these experiments, human NKG2D ligands MICA-Fc, MICB-Fc, ULBP1-Fc, ULBP2-Fc, ULBP3-Fc, and ULBP4-Fc were purchased from R&D Systems, Inc. (Minneapolis, MN). Ligands in MicAbody format were captured on an anti-human IgG Fc capture (AHC) biosensor chip. After establishing a baseline, the chip was exposed to a titration series of Fc-eNKG2D fusion proteins ranging from 300 nM to 0.41 nM, and association / dissociation rates were monitored (all steps performed in PBS-BT). Fc-eNKG2D fusion protein was then captured on the AHC chip and MicAbody titrated to characterize binding kinetics.

[0040] To determine the maximum response defined by the binding of native NKG2D to either MICwed or MIC25, native Fc-NKG2D fusions were captured on the AHC biosensor and incubated with 20 nM trastuzumab-MICwed or 20 nM trastuzumab-MIC25 MicAbody for 2 min, then the dissociation rate was observed for 30 s. Binding analysis under the same conditions was then performed using Fc-eNKG2D fusion receptor as the capture agent, and the binding level for each eNKG2D was ranked as a percentage of the maximum binding response established by Fc-NKG2D.wt (Figure 19). For MICwed, the response of all single mutant Fc-eNKG2D variants except Y199F was reduced to 50%. Y199F maintained a 100% binding response. However, all double mutant Fc-eNKG2D variants completely lost binding to MICwed. For MIC25, all single mutant Fc-eNKG2D variants and Y152V / Y199F maintained 100% binding response compared to wild-type Fc-NKG2D binding, but binding was reduced to 50% for some of the double mutant Fc-eNKG2D variants, including Y152A / Y199F, Y152S / Y199F, and Y152T / Y199F.

[0041] ELISA assays using Fc-eNKG2D fusions as capture agents were performed with ULBP2.wt, MICwed, and MIC25 MicAbodies titrated starting at 300 nM (Figure 4). When possible, EC50 values ​​were calculated using GraphPad Prism (Figure 20). Native NKG2D bound to ULBP2, MICwed, and MIC25-based MicAbodies with affinities calculated as Kd values ​​of 1.4, 0.007, and 0.005 nM, respectively. Affinity for ULBP2 and MICwed MicAbodies was reduced for all single mutant eNKG2D candidates, while MIC25 binding to eNKG2D candidates was retained. However, all double mutant eNKG2D candidates had abolished or significantly reduced binding to all three ligands (ULBP2, MICwed, and MIC25) in Micabody format.

[0042] The eNKG2D variants eNKG2D5 (Y152A / Y199F), eNKG2D7 (Y152S / Y199F), eNKG2D8 (Y152T / Y199F), and eNKG2D9 (Y152V / Y199F) had reduced or abolished binding to ULBP2, MICwed, and MIC25-based MicAbodies by both Octet analysis and ELISA (Figures 19 and 20). Furthermore, eNKG2D5, 7, and 8 had minimal amounts of aggregation, suggesting a tighter protein assembly upon 293T expression (Figure 18). eNKG2D5 (SEQ ID NO: 48) was analyzed by Octet analysis and ELISA. Binding to wild-type ligands as MicAbodies captured on AHC chips was examined more rigorously. The single mutant Fc-NKG2D.Y152A (SEQ ID NO: 41) had reduced binding to all native ligands compared to native (SEQ ID NO: 40) NKG2D (Figure 5). The response curve for binding of eNKG2D5(Y152A / Y199F) was further reduced compared to Y152A eNKG2D. eNKG2D5(Y152A / Y199F, hereafter referred to as "AF" or "NKG2D.AF") was selected as the lead NKG2D variant for engineering cognate selective, orthogonal, non-natural ligands.

[0043] Example 4: Construction of orthogonal non-natural α1-α2 domains with selective binding to non-natural NKG2D.AF ectodomains We used phage display to engineer an orthogonal non-native α1-α2 domain that exhibits selective binding to the NKG2D.AF (SEQ ID NO: 48) receptor. As a starting point, the non-native ULBP2.R80W α1-α2 domain (FIG. 1B; SEQ ID NO: 108), which has high affinity for the native wild-type NKG2D (NKG2D.wt) ectodomain, was selected as the parent domain for further mutagenesis and screening by phage display. A synthetic DNA library was generated for the α1-α2 domain of ULBP2.R80W (SEQ ID NO: 108) with an additional C8S mutation to eliminate disulfide bond potential. Codons for amino acid residues of the ligand that are located proximal to positions Y152 and Y199 on the native NKG2D receptor in the bound state were replaced with NNK codons; the library consisted of NNK codons at positions 154-159 (FIG. 1B, SEQ ID NO: 110). The library was cloned as a fusion to the pIII minor coat protein of M13 phage, and phage particles displaying the mutagenized α1-α2 domain variants were produced in SS320 E. coli cells according to standard methods (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, CF, 3rd. (2011)). These α1-α2 phage display libraries were sorted for high binding affinity to the non-native NKG2D.AF receptor by selectively capturing phage clones that bound to biotinylated Fc-NKG2D.AF protein in the presence of non-biotinylated native Fc-NKG2D.wt competitor protein. Selective clones were enriched by multiple rounds of competitive selection with increasing concentrations of non-biotinylated native Fc-NKG2D.

[0044] After four rounds of selection, phage clones were individually arrayed in a 96-well format and spot ELISA was performed to confirm preferential differential binding to plate-bound non-native NKG2D.AF versus NKG2D.wt. Bound phages were detected using biotinylated M13 phage coat protein monoclonal antibody E1 (ThermoFisher Scientific, Waltham, MA), streptavidin-HRP detection (R&D Systems, Minneapolis, MN), and 1-Step Ultra TMB ELISA color development (ThermoFisher Scientific, Waltham, MA). The spot ELISA signal for each clone was expressed as the ratio of NKG2D.AF-binding phages to NKG2D.wt-binding phages. Phages with a ratio of 14 or greater were sequenced to identify specific mutations within the NNK mutagenized region. Figure 21 shows selected amino acid residues for each α1-α2 phage variant that selectively bound to NKG2D.AF. When multiple clones representing the same sequence were identified, the ratios of ELISA signals were plotted and the consistency of the phage clones was confirmed by clustering of data points (data not shown).

[0045] Thirty variants identified in the ELISA were expanded in separate monocultures to generate high titer microbatches of phage. Purified phage concentrations were normalized to OD268=0.5 and then subjected to a 1:3 dilution series against plate-bound Fc-NKG2D.AF or Fc-NKG2D.wt, and phage detection and ELISA development were performed as described above. All 30 variants assayed in this manner consistently demonstrated selective binding to NKG2D.AF, with little to no binding to NKG2D.wt, even at the highest concentrations of phage assayed (Figure 6). Selected phage also showed a 2 or more log shift in the phage concentration at which half-maximal binding was achieved between NKG2D.AF and NKG2D.wt.

[0046] To confirm that the NKG2D.AF selective α1-α2 domain variants retained specific binding properties in the context of antibody fusions, 21 variants (Figure 22, e.g., SEQ ID NOs: 111-118) were cloned as C-terminal fusions with an APTSSSGGGGS linker to the light chain of the rituximab antibody (SEQ ID NOs: 119-126). The resulting fusions were cloned via Gibson cloning (New England Biolabs Inc., Ipswich, MA) into the mammalian expression vector pD2610-V12 (ATUM, Newark, CA) and co-expressed with the parent antibody heavy chain (SEQ ID NO: 99) as paired complete IgG antibodies. Transient expression was performed in Expi293™ cells (ThermoFisher Scientific) according to the manufacturer's protocol. Fc, Waltham, MA) and purified using standard protein A affinity chromatography (cat. no. 20334, Pierce Biotechnology, Rockford, IL). ELISA measuring binding of each variant ULBP2 α1-α2 antibody fusion to non-native Fc-NKG2D.AF and native Fc-NKG2D.wt demonstrated their significantly higher binding affinity towards NKG2D.AF compared to native NKG2D.wt (FIG. 22). Taken together, these data demonstrate the invention of non-native orthogonal α1-α2 domains with high affinity binding to the non-native NKG2D.AF receptor and significantly reduced binding affinity to the native NKG2D receptor. Furthermore, fusions of these orthogonal α1-α2 domains to antibody polypeptides retained their selective binding properties and were used, for example, in the context of chimeric antigen receptor (CAR) T cells to redirect the non-native NKG2D.AF receptor towards a specific antigen.

[0047] Example 5: Identification of non-natural NKG2D ligands that can discriminate between non-natural NKG2D receptor variants by selectively binding to one or the other Phage display to engineer an orthogonal non-native α1-α2 domain with selective binding to the NKG2D.Y152A (hereafter referred to as NKG2D.YA) receptor was performed using the non-native ULBP2.R80W α1-α2 domain (SEQ ID NO: 108) as a starting point as described above. The α1-α2 phage display library was panned for high binding affinity to the non-native Fc-NKG2D.YA receptor by selectively capturing phage clones that bound to biotinylated Fc-NKG2D.YA (SEQ ID NO: 41) protein in the presence of non-biotinylated native Fc-NKG2D.wt (SEQ ID NO: 40) competitor protein. Further phage clone validation studies identified variants with preferential binding to Fc-NKG2D.YA relative to Fc-NKG2D.wt (FIG. 23). For example, ULBP2.S3 (SEQ ID NO: 127) consistently demonstrated selective binding to non-native NKG2D.YA compared to native NKG2D.wt by ELISA and Octet analysis (both in monomeric His-tagged and bispecific antibody fusion formats). This represents a distinct embodiment of the non-native orthogonal α1-α2 domain invention with high affinity binding to a non-native NKG2D receptor (NKG2D.YA in this case versus NKG2D.AF in Example 4). Furthermore, fusion of the orthogonal α1-α2 domain to an antibody polypeptide retained its selective binding properties and was used to selectively redirect the non-native NKG2D receptor toward a specific molecule determined by the fused heterologous peptide, such as an antibody.

[0048] To determine whether a non-natural α1-α2 domain with selective binding to NKG2D.YA (ULBP2.S3, SEQ ID NO: 127) and a non-natural α1-α2 domain with selective binding to NKG2D.AF could discriminate between these two non-natural receptor variants, a titration ELISA was performed. All 21 selected α1-α2 variants that bound to NKGD2.AF were directly compared for binding to NKG2D.AF versus NKG2D.YA. Four of these showed the following characteristics: inability to bind to NKG2D.wt, strong affinity for NKG2D.AF, and greatly reduced (15-20 fold) or abolished binding to NKG2D.YA compared to NKG2D.AF (FIG. 7). These four non-natural ULBP2 α1-α2 variants (ULBP2.C, ULBP2.R, ULBP2.AA, and ULBP2.AB (SEQ ID NOs: 111, 113, 115, and 117)) were also analyzed for changes in predicted immunogenicity profiles compared to the wild-type ULBP2 peptide sequence (SEQ ID NO: 4) using the NetMHC4.0 Server (for peptide-MHC class I binding querying against all HLA supertype representatives using 9-mer peptide analysis; http: / / www.cbs.dtu.dk / services / NetMHC / ) and the NetMHCII 2.3 Server (for peptide-MHC class I binding querying against HLA-DR, HLA-DQ, and HLA-DP haplotypes using 15-mer peptide analysis; http: / / www.cbs.dtu.dk / services / NetMHC / ). for Gapped sequence alignment using artificial neural networks: application to The MHC class I system, 2016 Bioinformatics, 32:511, PMID:26515819; Jensen KK, Andreatta M, Marcatili P, Buus S, Greenbaum JA, Yan Z, Sette A, Peters B, and Nielsen M, Improved methods for predicting peptide binding affinity to MHC class I molecules, 2018 Immunology, PMID:29315598). Mutations engineered into ULBP2.C, ULBP2.R, and ULBP2.AB did not increase predicted immunogenicity, but the predicted immunogenicity of ULPB2.AA was slightly increased for a few haplotypes (Figures 8 and 9). As a result of the specificity of ULBP2.R for NKG2D.AF and its predicted lack of immunogenicity, ULBP2.R was selected for further ELISA analysis to directly compare its binding behavior to ULBP2.S3 (NKG2D.YA selected non-natural orthogonal ligand), ULBP2.R80W (non-natural ligand with enhanced affinity for wild-type NKG2D), and wild-type ULBP2 (ULBP2.wt).Four rituximab-MicAbody reagents (SEQ ID NOs: 98 and 121, 98 and 129, 131 and 100, and 98 and 106 as heavy and light chains for ULBP2.R, ULBP2.S3, ULBP2.R80W, and ULBP2.wt, respectively) were assayed against wild-type NKG2D (NKG2D.wt) and the two inactive non-natural variants NKG2D.YA and NKG2D.AF (FIG. 10). The data demonstrated that the NKG2D.YA selected variant ULBP2.S3 as a MicAbody bound with high affinity to NKG2D.YA but did not engage NKG2D.AF or native NKG2D. Additionally, the NKG2D.AF selected variant ULBP2.R in the MicAbody format bound with high affinity to NKG2D.AF but did not engage NKG2D.YA or native NKG2D.These results demonstrate the enormous potential for exploring the NKG2D-MIC ligand axis and for developing novel, selective and unique pairs of non-natural NKG2D receptors and their respective cognate non-natural MIC ligand binding partners.

[0049] Example 6: Targeting and killing activity of CAR-T cells expressing a non-native NKG2D.AF ectodomain is controlled by an orthogonal α1-α2 domain fused to a heterologous targeting polypeptide A means of selectively controlling CAR-T cell therapy is highly sought after to reduce toxicity and improve efficacy against tumors (Gill and June, supra). Previous attempts have been made to develop CARs using the ectodomain of CD16, which is then engaged via the Fc domain of a therapeutic monoclonal antibody to allow antibody-based control of CAR-T targeting (Chang et al., supra). However, CD16-based CAR-T cells can recognize nearly all endogenous antibody molecules in blood and tissues, and therapeutic antibodies used to control these cells encounter competition from endogenous CD16 receptors on NK cells, PMNs, monocytes, and macrophages. Both of these characteristics contribute to the problems of off-tumor toxicity and poor pharmacokinetics, respectively.

[0050] Natural NKG2D ligands are present on certain healthy and many stressed tissues, creating a significant risk of toxicity using current NKG2D CAR approaches (VanSeggelen et al. 2015). The Y152A non-natural NKG2D receptor specifically binds non-natural α1-α2 domain NKG2D ligands, thereby Thus, they constitute an example of the means by which the activity of a non-natural NKG2D CAR can be selectively controlled using bispecific proteins comprising a non-natural α1-α2 domain of an NKG2D ligand of the invention.

[0051] We engineered CAR-T cells with receptors comprising the modified Y152A / Y199F ("AF") ectodomain of NKG2D, which lacks binding to all native NKG2D ligands or the previously described non-native α1-α2 domain that is orthogonal and cognate to the Y152A modified NKG2D (NKG2D.YA). The cognate non-native α1-α2 domain of the invention bound with high affinity to the non-native NKG2D.AF ectodomain and avoided binding to native NKG2D ectodomain and NKG2D.YA ectodomain. Thus, the engineered α1-α2 domain, which showed strong selectivity for the non-native NKG2D.AF ectodomain compared to native NKG2D and non-native NKG2D.YA, represents an ideal system for selective control of non-native NKG2D CAR receptors, or any receptor or protein fused to a non-native NKG2D ectodomain that can be selectively engaged by the non-native α1-α2 domain of the invention. The invention further enables a single cell to express two different CARs, one containing NKG2D.YA and the other containing NKG2D.AF, each signaling using a distinct intracellular domain, with independent dual control of the activity of the cell by extracellular exposure to their respective cognate orthogonal MicAbodies or separate non-antibody fusion polypeptides.

[0052] To demonstrate selective control of CAR-T cells constructed with chimeric receptors in which a non-native NKG2D.AF ectodomain was placed, we based our previous work using the 4-1BB / CD3-zeta CAR construct (Campana Patent No. 8,399,645) on the fusion of each NKG2D ectodomain to the CD8 hinge region of the CAR and constructed CARs with either the native NKG2D.wt (SEQ ID NO: 135), the non-native NKG2D.YA (SEQ ID NO: 137), or the non-native NKG2D.AF (SEQ ID NO: 139) ectodomain (SEQ ID NO: 151, 153, 155). These constructs (SEQ ID NO: 152, 154, 156) were cloned into lentiviral vectors and expressed in primary human CD8 positive T cells using lentiviral transduction. HeLa cells have constitutively upregulated levels of MIC ligands on their surface, including MICA, MICB, ULBP3, and ULBP2 / 5 / 6 (the antibody used to confirm this cannot distinguish between these three ULBPs; Human ULBP-2 / 5 / 6 Antibody, R&D Systems, Minneapolis, MN). HeLa cells were transfected to overexpress either native ULBP1 or the NKG2D.AF selected variant ULBP2.R on their surface, and these cells were used as targets for in vitro killing assays. HeLa target cells were preloaded with calcein and exposed to NKG2D.wt-CAR, NKG2D.YA-CAR, or NKG2D.AF-CAR CD8 cells at increasing effector-to-target (E:T) ratios for 5 hours, after which the amount of calcein released in the supernatant was quantified and normalized to the total calcein released upon detergent treatment (Figure 11). Due to elevated levels of MIC ligands naturally expressed on the surface of HeLa cells, CD8 cells expressing native NKG2D (NKG2D.wt) as a CAR engaged HeLa cells via this overexpressed native ligand and led to cell lysis.However, both NKGD.YA- and NKG2D.AF-CAR transduced CD8 cells showed very little lysis of native HeLa cells even at high E:T ratios (activity levels comparable to non-transduced CD8 T cells). When ULBP1 was overexpressed on the surface of HeLa cells, only NKG2D.wt-CAR CD8 T cells significantly lysed it. There is some additional killing at high E:T ratios for NKG2D.YA-CAR cells, but not for NKG2D.AF-CAR cells, indicating that the double mutation Y152A / Y199F renders NKG2D more inactive than the single Y152A mutation. NKG2D. In AF-selected HeLa cells overexpressing non-native ULBP2.R, NKG2D.wt-CAR cells directed lysis (due to recognition of endogenous MIC ligands), whereas NKG2D.AF-CAR cells directed significant levels of lysis, consistent with engagement of the receptor and its selective ligand.

[0053] To demonstrate that lysis of either NKG2D.YA- or NKG2D.AF-CAR cells could only be directed by the appropriate cognate targeting MicAbody, Ramos cells were used as targets for cell lysis in combination with a Rituximab-based MicAbody bound to either the non-natural ULBP2.S3 or ULBP2.R orthogonal ligand. As demonstrated in FIG. 12A, the Rituximab-ULBP2.S3 MicAbody was able to direct the cell killing activity of NKG2D.YA-CAR CD8 cells, but not NKG2D.AF-CAR cells, whereas the Rituximab-ULBP2.R MicAbody was able to direct the activity of NKG2D.AF-CAR cells, but not NKG2D.YA-CAR cells. This further demonstrates the selectivity of the two non-natural ULBP2 variants for their cognate non-natural NKG2D variants for which they were engineered as preferred partners. To demonstrate the specificity of the antibody portion of the MicAbody, an in vitro killing assay was performed with NKG2D.AF-CAR CD8 T cells preloaded by incubation with saturating total concentrations of MicAbody, either Rituximab-ULBP2.R, Trastuzumab-ULBP2.R (SEQ ID NOs: 95 and 133, heavy and light chains, respectively), or an equimolar combination of the two. After removing unbound MicAbody by washing, the CD8 cells were subjected to either Ramos cells (expressing CD20, the target of Rituximab) or CT26-Her2 (a murine cell line transfected to express human Her2) preloaded with calcein. After 2 hours of incubation at two different E:T ratios, the amount of calcein released was quantified. As shown in Figure 12B, when cells were preloaded with Rituximab-MicAbody, only Ramos cells were lysed, whereas Trastuzumab-MicAbody directed cytolytic activity only against CT26-Her2 cells. When CD8 cells were preloaded with both rituximab- and trastuzumab-ULBP2.R MicAbodies simultaneously, both target cell lines were lysed, demonstrating that these CAR cells (by virtue of the selective and privileged partnering engineered between receptor and ligand) can be easily multiplexed and thereby directed to engage different tumor targets simultaneously.

[0054] Example 7: Orthogonal α1-α2 domains as a means to selectively deliver cytokines to engineered non-native NKG2D-expressing T cells Bispecific MicAbodies (FIG. 13B) exploiting the privileged interactions between the antigen targeting Fv domain of an antibody and the orthogonal α1-α2 domain with engineered non-natural NKG2D.YA or NKG2D.AF (SEQ ID NOs: 137 and 139) can effectively direct the cytolytic capacity of T cells bearing NKG2D.YA or NKG2D.AF-CARs (SEQ ID NOs: 153 and 155, respectively) to eliminate antigen-expressing target cells. Furthermore, the highly selective interaction between the cognate orthogonal ligand and non-natural modified (also referred to as "engineered") NKG2D (eNKG2D) can be exploited to selectively deliver molecules to cells bearing the eNKG2D-ectodomain (eNKG2D-ecd). If the heterologous atom or molecule (payload / cargo) to which the orthogonal ligand is fused has inherent biological activity with potentially undesirable function when delivered to non-eNKG2D expressing cells, mutations in the biologically active molecule that reduce interaction with the native receptor or target can be examined. Thus, when fused to the orthogonal ligand, the orthogonal ligand can be expressed in the presence of cells bearing the cognate eNKG2D receptor. Except Thus, the final molecule is effectively inactive for all biological functions. Only if an affinity interaction occurs, which promotes interaction between the affinity-reduced variant of the bioactive molecule and its natural target or receptor, will the remaining function of the bioactive molecule be enabled. All orthogonal ligand fusions to entities for directing them to eNKG2D-expressing cells are collectively referred to as MicAdaptors, and in principle can take the form of a direct protein fusion of the payload and the orthogonal ligand at either the N- or C-terminus as a single polypeptide, with or without a linker or tag (e.g., His-tag) for assay or purification (Figure 13C). Furthermore, if improved serum stability is desired, an antibody Fc domain can be included, either as just the CH2-CH3 domain or in the form of a complete antibody, depending on the number of unique payloads desired, the valency of either the orthogonal ligand or the heterologous cargo, and a general, experimentally determined architecture that promotes maximum function of all components (Figure 13D).

[0055] To determine whether cytokines could be selectively delivered to NKG2D.YA-CAR expressing cells (SEQ ID NO: 153), the cognate orthogonal ligand ULBP2.S3 (U2S3, SEQ ID NO: 127) was expressed fused to the N-terminus of Fc1, which had been altered by two residues to express a negatively charged aspartic acid residue at the Fc homodimerization interface (SEQ ID NO: 189). A mutant form of human IL2 (mutIL2) containing two mutations R38A / F42K that significantly reduces the affinity of cytokines to the IL2R-alpha complex (KM Eaton, G. Ju, and EAGrimm, Human Interleukin 2 Analogues That Preferentially Bind the Intermediate-Affinity Interleukin 2 Receptor Lead to Reduced Secondary Cytokine Secretion:Implications for the Use of These Interleukin 2 Analogues in Cancer Immunotherapy,1993 Cancer Res 53:2597,PMID:8495422;K.Sauve et al.,Localization in Human Interleukin 2 of the Binding Site to the Alpha Chain (P55) of the Interleukin 2 Receptor, 1991 PNAS 88:4636, PMID: 2052547) was fused to the C-terminus of Fc2, which had been modified with two residues to express a positively charged lysine residue at the interface of Fc homodimerization (SEQ ID NO: 183). Both were independently cloned into the mammalian expression vector pD2610-V12 (ATUM, Newark, CA), co-transfected into Expi293™ cells (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's protocol, and purified using standard Protein A affinity chromatography (cat.no.20334, Pierce Biotechnology, Rockford, IL)). The purified material was fractionated by size exclusion chromatography (SEC) on an Akta Pur Superdex column. The negatively charged residues of Fc1 and the positive charges of Fc2 provided an electrostatic steering effect that promoted heterodimer assembly of the molecule that was monovalent for the orthogonal U2S3 ligand and mutIL2 (Kannan Gunasekaran et al., Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects: Applications to Bispecific Molecules and Monovalent IgG, 2010 J Biol. Chem 285:19637, PMID:20400508) (Figure 13Da). Additionally, MicAdaptors composed of other combinations of fusions of ULBP2 orthogonal ligands and cytokines were explored, either as heterodimeric Fc1 / Fc2 fusions or as single polypeptides with both components directly fused, and their DNA was similarly cloned, co-expressed, and purified as detailed in Figure 24 and as described above. Recombinant human IL2 (rhIL2, Peprotech) and recombinant human IL15 ( rhIL15, Peprotech) was included as an assay control where appropriate.

[0056] CD8 human T cells were transduced to express either the NKG2D.wt-CAR construct (SEQ ID NO: 151) or the NKG2D.YA-CAR construct (SEQ ID NO: 153) and exposed to 30 IUe / mL of control cytokine or various MicAdaptors for 3 days, and the level of cell proliferation was quantified with WST-1 Cell Proliferation Reagent (Millipore Sigma). Control rhIL2 promoted proliferation of both CAR-expressing cells, but mutIL2 alone did not, as expected from its reduced ability to engage IL2R-alpha and therefore signal through IL2R-beta / gamma-C (Figure 15A). When the MicAdaptor was configured with the nonselective U2R80W ligand (U2R80W-mutIL2, SEQ ID NO: 177), which engages both wild-type and modified NKG2D.YA receptors with high affinity, cells expressing either CAR responded by proliferating. However, when mutIL2 was fused to an orthogonal ligand (MicAdaptor, U2S2-mutIL2, which engages only NKG2D.YA), such as ULBP2.S2 (SEQ ID NO: 179), which selectively engages only NKG2D.YA, only NKG2DYA-CAR cells responded by proliferation. Similar results were obtained when the cytokine fused to U2S2 was a mutant form of IL15 with a V49D mutation that reduces engagement with IL15R-alpha (Bernard, J. et al., Identification of an Interleukin-15α Receptor-binding Site on Human Interleukin-15, 2004 Journal of Biological IL15R-αChemistry, 279:24313, PMID:15039446) (Figure 15A).When NKG2D.YA-CAR-T cells were co-cultured with these reagents for 7 days, the %GFP+ (indicating the percentage of CAR-expressing cells present compared to untransduced GFP-negative cells) increased, but only in the presence of the orthogonal ULBP2.S2 ligands (U2S2-mutIL2 or U2S2-Fc1 / Fc2-mutIL2, Figure 24 for SEQ ID NOs), but not the non-selective U2R80W variant (which bound to the native wild-type NKG2D receptor constitutively present on human CD8 cells) (Figure 15B). IL21 was explored both as a wild-type fusion (IL21.wt) with a single mutation (D18A or E109R) affecting IL21R-alpha binding, as well as a form containing both mutations (D18A / E109R) (Kang, L. et al., Rational Design of Interleukin-21 Antagonist through Selective Elimination of the γC Binding Epitope,). 2010 Journal of Biological Chemistry,285:12223,PMID:20167599). In a comparison between non-transduced CD8 cells or NKG2D.YA-CAR expressing cells, only NKG2D.YA bearing cells responded to all types of IL21 fusions including IL21.wt fusions (see FIG. 24 for SEQ ID NOs) (FIG. 15C). Interestingly, both non-transduced and NKG2D.YA-CAR cells expanded in response to rhIL2 compared to the cytokine-free control, but the amount of proliferation was higher for NKG2D.YA-CAR cells. The proliferative response of NKG2D.YA-CAR expressing cells was independent of generalized binding or engagement of the NKG2D.YA domain, as U2S3-Fc1 / Fc2 (a heterodimeric Fc molecule with a single U2S3 domain and no attached cytokine or cytokine variant) and rituximab-MicAbody (bivalent for the U2S3 domain but lacking any cytokine component, FIG. 13Bb) did not induce proliferation over the course of a 3-day culture, even at IUe / mL concentrations much higher than rhIL2 controls (FIG. 16C).

[0057] Example 8: The presence of intracellular costimulatory domains, either in cis or trans, enhances the expression of non-native modified NKG2D-bearing cells to cytokines and cytokine MicAdaptors Promotes responsiveness Having demonstrated that the engineered NKG2D.YA domain, in the context of a chimeric antigen receptor construct, does indeed function as a highly selective docking site for the delivery of heterologous cargo conjugated to an orthogonal ligand, we sought to determine whether the NKG2D.YA receptor is not only necessary but also sufficient for targeted delivery of cytokines that may then act on the recipient cell. The NKG2D.YA extracellular domain (NKG2D.YA-ecd) was expressed as a transmembrane domain with all intracellular components removed, except for the retention of an intracellular eGFP tag (Figure 14, SEQ ID NO: 157). CD8 cells were transduced to express this "silent CAR" and demonstrated that, as expected, without the costimulatory domain, it was unable to direct the killing of Ramos target cells in the presence of the rituximab-ULBP2.S3 MicAbody (SEQ ID NOs: 98 and 129) (Figure 16A). Importantly, cells expressing this completely inactive or silent CAR did not proliferate when exposed to U2S3-Fc1 / Fc2-mutIL2 (SEQ ID NOs: 189 and 193), but responded at a level comparable to untransduced cells (Figure 16B). This observation - (a) the consistent observation of higher levels of NKG2D.YA-CAR proliferation with rhIL2 compared to untransduced cells (Figures 15C, 16B, 16C), plus (b) the observation that only NKG2D.YA-CAR cells responded to all forms of IL21-MicAdaptor, including IL21.wt, led us to speculate that the intracellular domain present in the NKG2D.YA-CAR construct enhanced responsiveness to these cytokines and cytokine MicAdaptor.

[0058] To test this, we generated a series of CAR constructs in which signaling motifs in the intracellular domain of the CAR were mutated - the two TRAF2 consensus binding sites in 4-1BB (SEQ ID NO: 161), the three pairs of ITAM motifs in CD3-zeta (SEQ ID NO: 163), or a combination of 4-1BB / CD3-zeta mutants (SEQ ID NO: 165). These constructs (Figure 14) were transduced into CD8 cells, co-incubated with the indicated cytokine reagents, and proliferation was quantified after 3 days (Figure 17A). NKG2D.YA-BB-CD3 ΔITAM -GFP (SEQ ID NO: 163) maintained a comparable proliferative response to the NKG2D.YA-CAR under all conditions tested, demonstrating that the CD3-zeta domain (SEQ ID NO: 145) is not required for responsiveness to both cytokines and cytokine-MicAdaptor in the context of the CAR. However, NKG2D.YA-BB ΔTRAF2 - CD3 zeta-GFP receptor expressing cells (SEQ ID NO: 161) carry mutations in both intracellular domains of NKG2D.YA-BB ΔTRAF2 -CD3 ΔITAM As with the -GFP (SEQ ID NO: 165) receptor, responsiveness to all cytokines and cytokine-MicAdaptor was greatly reduced. This indicated that the costimulatory 4-1BB (SEQ ID NO: 143) plays a role in the responsiveness of NKG2D.YA-CAR cells to both cytokines and cytokine-MicAdaptor, and that silent CARs expressed on the cell surface must also have an intracellular costimulatory domain present to promote cytokine-MicAdaptor responsiveness. The inability of the NKG2D.YA-BB silent CAR (SEQ ID NO: 169, FIG. 14) to direct MicAbody-mediated cell killing (FIG. 17B) demonstrated that this CAR architecture lacking CD3-zeta is suitable as a silent docking site for MicAdaptor, allowing cytokine-MicAdaptor signaling, but does not function to mediate cytolysis of MicAbody target cells.

[0059] To further examine how the 4-1BB domain contributes to cytokine and cytokine-MicAdaptor responsiveness, a construct (SEQ ID NO: 173) was generated in which the CD19scFv-CAR (based on FMC63 Fv) contains a functional 4-1BB domain and the full complement of the CD3-zeta domain. The CD19scFv-CAR was co-expressed with NKG2D.YA-ecd (Figure 14). Both components were expressed using independent GMCSFR antigens. The CD19scFv-CAR construct was expressed as a single polypeptide with a T2A self-cleaving peptide motif separating the upstream CD19scFv-CAR construct from the downstream NKG2D.YA-ecd with its lupha chain signal sequence, CD8a hinge, and CD8a transmembrane domain. The construct was transduced into CD8 T cells and co-expression of CD19scFv-CAR and NKG2D.YA-ecd on the surface was confirmed by flow cytometry verifying GFP signal and phycoerythrin-conjugated MicAbody staining, respectively. The cells were incubated with cytokines and cytokine-MicAdaptor for 3 days and proliferation was quantified by WST assay. NKG2D.YA-CAR responded to both rhIL2 and U2S3-Fc1 / Fc2-mutIL2 as expected, whereas control cells containing only CD19scFv-CAR (SEQ ID NO: 171) proliferated more with rhIL2 and to a lesser extent to the highest concentration (300 IUe / mL) of U2S3-Fc1 / Fc2-mutIL2 (Figure 17C). Co-expression of NKG2D.YA-ecd with CD19scFv-CAR expressing cells (SEQ ID NO: 173) showed a greater proliferative response to U2S3-Fc1 / Fc2-mutIL2 cytokine-MicAdaptor than cells expressing only CD19scFv-CAR (SEQ ID NO: 171). In this context, the 4-1BB domain was constitutively provided in trans to NKG2D.YA-ecd. These data demonstrated that the responsiveness of NKG2D.YA-ecd expressing cells to cytokines and cytokine-MicAdaptor was promoted by the 4-1BB domain either in cis or trans and that the NKG2D.YA-ecd domain can be co-expressed with costimulatory 4-1BB-containing CAR cells to provide additional diverse functions to engineered cells in adoptive cell therapy strategies.This function is not only limited to surface receptor engagement upon ligand / silent CAR engagement, but can be extended to intracellular delivery by incorporating cytoplasmic sequence motifs that facilitate turnover of non-native NKG2D variants such that any bound MicAdaptor is co-internalized and heterologous cargo is delivered intracellularly (KN Andey, Functional roles of short sequence motifs in the endocytosis of membrane receptors, 2009 Front Biosci,14:5339,PMID:19482617).

Claims

1. A non-naturally occurring modified NKG2D receptor, which binds to a non-naturally occurring modified α1-α2 domain of an NKG2D ligand but does not bind to a natural NKG2D ligand, wherein the modified NKG2D receptor inserted into a mammalian cell functions as a high affinity surrogate receptor for the delivery of a heterologous atom or molecule bound to the modified α1-α2 domain to the cell, and wherein the modified NKG2D receptor does not directly activate or signal mammalian cells.

2. The heterologous molecule of claim 1, wherein the heterologous molecule, when delivered to a mammalian cell by the modified α1-α2 domain, is capable of activating or signaling the mammalian cell via the respective receptor subunit on or within the mammalian cell.

3. 2. The heterologous atom or molecule of claim 1, wherein said heterologous atom or molecule, when delivered to a mammalian cell by the modified α1-α2 domain, can directly affect the mammalian cell independent of any receptor on or within the cell other than the non-native modified NKG2D receptor.

4. The heterologous atom or molecule of claim 1, wherein the heterologous atom or molecule, when delivered to a mammalian cell by the modified α1-α2 domain, can be internalized by the mammalian cell, and the internalized heterologous atom or molecule, either bound to the modified α1-α2 domain or unbound, has an effect on the mammalian cell.

5. 2. The heterologous atom or molecule of claim 1, which, when delivered to a mammalian cell by the modified α1-α2 domain, can provide the mammalian cell with a marker detectable by an in vivo, ex vivo, or in vitro detection or imaging system.

6. 2. The non-naturally occurring modified NKG2D receptor of claim 1, wherein the costimulatory domain enhances the function of an effector molecule introduced into a mammalian cell, and wherein the effector molecule is not bound to the modified α1-α2 domain, and the costimulatory domain is bound intracellularly.

7. 4. The heterologous atom or molecule of claim 3, which, when delivered to a mammalian cell by the modified α1-α2 domain, can damage the cell by releasing energy or disrupting the cell membrane, such as by promoting the formation of the complement-dependent macromolecular attack complex.

8. A non-naturally occurring modified NKG2D receptor inserted into a mammalian cell, wherein a costimulatory domain is bound to the modified NKG2D receptor in the absence of an active CD3-zeta domain within the cell, and the receptor binds to the non-naturally occurring modified α1-α2 domain of an NKG2D ligand, but does not bind to a natural NKG2D ligand.

9. 9. The non-naturally occurring modified α1-α2 domain of claim 8 to which a heterologous atom or molecule is attached, said heterologous atom or molecule being capable of being internalized by the mammalian cell when delivered to the mammalian cell by the modified α1-α2 domain, and the internalized heterologous atom or molecule, either bound to the modified α1-α2 domain or unbound, has an effect on the mammalian cell.

10. 9. The non-naturally occurring modified α1-α2 domain of claim 8 having attached thereto a heterologous atom or molecule, said heterologous atom or molecule providing a targeting or homing function to the mammalian cell that facilitates delivery of the mammalian cell to a target carrier cell or target carrier surface when the mammalian cell reaches the target surface or environment without directly leading to activation of the mammalian cell.

11. The NKG2D-binding costimulatory domain of claim 8, wherein the costimulatory domain enhances the function of an effector molecule introduced into a mammalian cell, and the effector molecule is not linked to a modified α1-α2 domain.

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