An unnatural NKG2D receptor that does not directly signal to the inserted cells

Abstract

The present disclosure relates to a non-native NKG2D receptor inserted on the surface of a mammalian cell, wherein the receptor does not directly signal or activate the cell when bound to a cognate non-native α1-α2 domain of an NKG2D ligand that has been modified to specifically bind to the non-native NKG2D receptor. The non-native α1-α2 domain of the NKG2D ligand may have attached to it a heterologous atom or molecule, including a polypeptide, in some embodiments a cytokine or modified cytokine, an antibody, or a fragment of an antibody.

Description

Technical Field

[0001] Background of the Invention Field of the Invention This application generally relates to an unnatural extracellular domain of an unnatural NKG2D receptor inserted into a mammalian cell, which is modified to specifically bind to the unnatural NKG2D receptor, and when a heterologous molecule binds to the modified α1-α2 domain of an NKG2D ligand and the unnatural NKG2D ligand binds to the receptor, does not directly activate or directly signal to the mammalian cell, and relates to the extracellular domain.

Background Art

[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. Primarily, when bound to one of its eight natural ligands expressed on the surface of stressed cells, NKG2D activates NK cells to lyse the stressed cells, or, in the case of T cells, NKG2D occupied by the ligand costimulates activated T cells to execute their effector functions. The three-dimensional structures have been elucidated for the extracellular domain of human natural NKG2D, some of its soluble natural ligands, and, in some cases, the binding complexes of the soluble ligand and the receptor extracellular domain. The monomeric α1-α2 domain of the NKG2D ligand specifically binds to the two extracellular domains of the natural NKG2D homodimer.

Summary of the Invention

[0003] Summary of the Invention The present disclosure relates to a non-natural NKG2D receptor inserted into the cell surface of a mammal, wherein when a cognate non-natural α1-α2 domain of an NKG2D ligand modified to specifically bind to the non-natural NKG2D receptor binds to the non-natural receptor, the receptor does not directly signal to the cell or directly activate the cell. 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 a 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 and does not occur even when an immunological synapse occurs. Brief Description of the Drawings

Brief Description of the Drawings

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Figure 1

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Chemical

[0006] and non-natural variants of ULBP2 (including ULBP2.R80W (SEQ ID NO: 108)). Residues important for the binding of non-natural ULBP2 variants to the non-natural NKG2D.YA or NKG2D.AF receptors are highlighted in gray. The positions of the M154-F159 region and residue R80 are shown for orthogonality variants that bind to NKG2D.YA (ULBP2.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).

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[0007] Detailed Description of the Invention Natural killer (NK) cells of the immune system and certain (CD8+ αβ and γδ) T cells play important roles as the first line of innate defense against neoplastic and infected cells in humans and other mammals (Cerwenka, A., and L. L. Lanier. 2001. NK cells, viruses and cancer. Nat. Rev. Immunol. 1:41-49). NK cells and certain T cells display on their surface NKG2D, a prominent homodimeric surface immunoreceptor that is responsible for the recognition of target cells and the activation of innate defense against diseased cells (Lanier, L. L., 1998. NK cell receptors. Ann. Rev. Immunol. 16:359-393; Houchins J. P. 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 (outer) domain that binds to its eight different cognate ligands, and the most studied ligands are the monomers MICA and MICB, which are 84% sequence identical or homologous (polymorphic analogs of major histocompatibility complex (MHC) class I chain-related glycoproteins (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). The constitutive expression of MICA and MICB is restricted to some intestinal epithelium, keratinocytes, endothelial cells, and monocytes, but the 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 the 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 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).The 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 a means to identify and respond to a wide range of distress signals 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 and identifies virus-infected cells for attack by NK or T cells (Groh et al. 1998; Groh et al. 2001. Co-stimulation of CD8+ αβ T cells by NKG2D via engagement by MIC induced on virus-infected cells. Nat. Immunol. 2:255-260; Cerwenka, A., and L.L. Lanier. 2001).Indeed, 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, J. A. Hamerman, H. Arase, J. P. Houchins, E. S. Mocarski, and L. L. Lanier. 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, I A 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 of lung cancer and glioblastoma brain cancer, also avoid the expression of MIC proteins and, as a result, may be particularly aggressive because they evade the innate immune system excessively (Busche, A et al. 2006, 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] The high-resolution structure of human MICA bound to NKG2D has been elucidated, demonstrating that the α3 domain of MICA does not have a direct interaction with NKG2D (Li et al. 2001. Complex structure of the activating immunoreceptor NKG2D and its 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 is itself naturally positioned as a "spacer" between the platform and the surface of MIC-expressing cells. The three-dimensional structures of the human MICA and MICB α3 domains are nearly identical (root mean square distance of C-αα' of 94% < 1 Å) and are 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] T cells, NK cells, and macrophages can be modified to directly and stably express on the surface the binding domain of an antibody conferring new antigen specificity using gene transfer technology (Saar Gill & Carl H. June. Going viral: Chimeric Antigen Receptor (CAR) T cell therapy for hematological malignancies. Immunological Reviews 2015. Vol. 263:68-89; Wolfgang 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 natural 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 major transducer of signals from the extracellular domain of the endogenous T cell receptor (TCR) into the intracellular space. CARs constructed with the CD3-zeta chain and costimulatory molecules such as CD27, CD28, ICOS, 4-1BB, or OX40, when binding to the target antigen, are similar to the endogenous T cell receptor but cause activation of CAR-T cells in a manner independent of the major histocompatibility complex (MHC).

[0012] Specific non-natural α1-α2 domains of NKG2D ligands are described that are modified to bind to the native human NKG2D receptor with higher affinity than the native α1-α2 domain (Candice S. E. Lengyel, 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 non-natural NKG2D receptors into the mammalian cell surface in a form that retains specific binding to binding heterologous molecules of the modified non-natural NKG2D ligand, but the non-natural receptor avoids direct or cis-activation of mammalian cells or intracellular signaling to mammalian cells even when the cell forms an immunological synapse with a cell or other surface targeted by the heterologous molecule.The non-natural NKG2D receptor homologs are mutated at one or two specific sites, each of which results in the inactivation 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 / 088,456)). The present invention creates a CAR that provides a silent receptor that, when inserted into the cell surface of a mammal, can function as a high-affinity alternative receptor for the binding of heterologous atoms or molecules to the cell surface. Thus, via a binding non-natural ligand specific for the non-natural modified NKG2D receptor, a heterologous molecule, such as a defective cytokine, can be specifically delivered to the silent receptor on the surface of mammalian cells, but not to cells lacking the cognate silent receptor. When binding to cells carrying the silent receptor, the defective heterologous molecule binds to its respective receptor subunit on the cell surface where the binding is retained, thereby allowing it to directly signal to the cell as if stimulated by the wild-type ligand.

[0013] Of course, chimeric antigen receptors (CARs) composed of inactive non-natural NKG2D, CD3-zeta, and co-stimulatory domains such as CD28, 4-1BB, ICOS, or OX40 on mammalian cells can directly stimulate and activate CAR cells during immunological synapse formation. Activation of such second-generation or third-generation CAR-T cells depends on the function of their CD3-zeta domain and at least one co-stimulatory domain, such as 4-1BB or CD28. However, such CARs can function as silent CARs as high-affinity alternative receptors for the binding of cognate non-natural ligand-binding heteromolecules that are defective in binding to their respective natural receptor or receptor subunit(s). This high-affinity binding enables heteromolecules bound to non-natural ligands to signal to the cell via their respective other receptor subunits to which the binding is retained.

[0014] Importantly, when the CD3-zeta domain of such directly activating CARs is selectively inactivated, it still functions as a silent CAR, allowing cognate non-natural ligand-binding heteromolecules (whose binding to their respective natural receptor or receptor subunit does not function properly) to signal to the cell via their respective other receptor subunits. When a CAR co-stimulatory 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 necessary, but a functional co-stimulatory domain is required for heteromolecules such as cytokines that do not function properly and are bound to cognate non-natural ligands bound to the receptor to be able to mediate their respective signals to the CAR cells.

[0015] The present invention has revealed an unexpected requirement for co-stimulatory domains, rather than CD3-zeta, in order for dysfunctional heterologous cytokines bound to cognate non-natural ligands to be able to mediate their respective signals to CAR cells. Furthermore, the present invention discloses that the co-stimulatory domain can act in cis or in trans with respect to a silent receptor to which a cognate ligand fused to a dysfunctional, heterologous dysfunctional molecule is bound.

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

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

[0018] Cells carrying a silent receptor CAR also express another receptor(s) or CAR orthogonal to the silent CAR and act independently of the silent CAR to specifically and directly activate or otherwise signal the same cells when appropriately stimulated. The other or "second" orthogonal CAR can be a conventional single-chain Fv (scFv)-CAR or a second, orthogonal, non-natural, modified NKG2D-based CAR (having its own cognate non-natural α1-α2 ligand(s)) (AF provisional reference). The ability to generate effector cells of the immune system using more than one orthogonal non-natural CAR, either silent or active, and multiple cognate non-natural ligands to which heterologous molecules or atoms are bound greatly expands the utility, flexibility, and control of adoptive cell therapy (ACT).

[0019] In the process of characterizing the dependence of silent CAR on cells and its cis- or trans-acting co-stimulatory domains (such as 4-1BB), compared with unmodified human T cells, human T cells expressing silent CAR together with co-stimulatory domains showed significantly enhanced responses to either native IL-2 or a cognate non-native ligand fused to a mutant IL-2 with low affinity for native IL-2 or its receptor α subunit. This observation has important utility in the ex vivo or in vivo selective amplification of cells expressing CAR composed of co-stimulatory 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 domain of the MIC protein (with or without the α3 domain) but do not contain a transmembrane or intracellular domain. The 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 present invention is at least 80% identical or homologous to the native or natural 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 natural α1-α2 domain of the NKG2D ligand. In still other embodiments, the modified α1-α2 domain is 90% identical to the native or natural α1-α2 domain of the native NKG2D ligand protein and binds to non-natural NKG2D.

[0022] Preferably, the modified or non-natural α1-α2 domain of the non-natural MIC protein of the present invention is at least 80% identical or homologous to the native or natural α1-α2 domain of one of the 8 human NKG2D ligand proteins (SEQ ID NOs: 1-8) and binds to the non-natural NKG2D extracellular domain. 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 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 the human native α1-α2 domain protein and binds to non-natural NKG2D.

[0023] In some embodiments, to assist in the purification of the soluble MIC ligand, a heterologous molecular tag may be fused to the N-terminus or C-terminus of the non-natural α1-α2 domain of the soluble MIC protein or to the N-terminus or C-terminus of a linked heterologous peptide or protein. Examples of tag sequences include peptides, such as polyhistidine, myc peptide, FLAG tag, streptavidin-like tag, 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 can be made in the α1-α2 domain of the NKG2D ligand to create a non-natural α1-α2 domain that binds to the non-natural NKG2D receptor and is engineered to have a reduced affinity for the natural NKG2D ligand itself. This can be done, for example, through genetic engineering. Using such a modified non-natural NKG2D receptor, an NKG2D-based CAR that can bind to a molecule containing the non-natural α1-α2 domain of the present invention can be created on the surface of NK cells, T cells, macrophages, or other immune system cells. 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, as compared to current CAR-T cells and CAR-NK cells, as follows. When the intracellular signaling of the non-natural NKG2D receptor on the surface of mammalian cells is silenced, as in the present invention, these CARs of the present invention can function as high-affinity alternative receptors for heterologous molecules such as cytokines, chemokines, lymphokines, cytotoxins, etc. and atoms fused or bound to an orthogonal NKG2D ligand that would not otherwise function properly. This provides for the direct and specific delivery of heterologous molecules to silent receptor-bearing cells without the silent receptor itself directly activating its host cell. Furthermore, heterologous molecules that bind to a specific target and thereby bind to a cell or other surface bearing such a target can provide a specific homing function to cells bearing the silent receptor without inadvertently activating or stimulating it.

[0025] CAR-T or CAR-NK cells that contain the extracellular domain of a non-natural NKG2D receptor that does not bind or only weakly binds to natural NKG2D ligands are not subject to activation by any natural ligand and thus are not toxicogenic like cells expressing a CAR based on the natural NKG2D receptor. Furthermore, the extracellular domain of the non-natural NKG2D receptor on the cell is not subject to downregulation by soluble forms or natural NKG2D ligands on myeloid-derived suppressor cells (MDSC) (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 CAR cells having the extracellular domain of the non-natural NKG2D receptor are engaged by the dual-specificity molecule with the cognate non-natural α1-α2 domain of the present invention and its heterologous targeting motif that finds and binds to its intended target, the CAR is activated and the effector function of the CAR cells is expressed.

[0026] CAR-T or CAR-NK cells containing non-natural NKG2D receptor extracellular domains are not activated except in the presence of an engaged bispecific molecule containing a cognate non-natural α1-α2 domain, so their activation can be controlled by administration of a bispecific molecule (which exhibits pharmacokinetics and pharmacodynamics well known in the art as a biopharmaceutical). In the event of an adverse event, rather than having to equip the injected CAR cells with a suicide-inducing mechanism to destroy them as is currently done, the physician can simply modify the dosing schedule of the administered bispecific molecule (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 assist in combating 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). Since all CAR constructs can be identical for all CAR cells and the targeting specificity is simply determined by the targeting motif of the bispecific molecule of the present invention that is administered, the manufacturing process is simplified and less expensive.

[0027] Examples of parent or recipient proteins or polypeptides that are candidates for binding to the non-natural α1-α2 domain of NKG2D ligands include, but are not limited to, antibodies, proteins containing Ig fold structures or Ig domains (including those that recruit or do not recruit natural molecules or modified Fc domains that do not bind to natural molecules), globulins, albumin, 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 cell receptors, hormones, cytokines and modified cytokines (e.g., interleukins), notchtins, cyclic peptides or polypeptides, major histocompatibility (MHC) family proteins, MIC proteins, lectins, and ligands for lectins. It is also possible to modify the α1-α2 domain of NKG2D ligands by binding non-protein molecules such as polysaccharides, dendrimers, polyglycols, peptidoglycans, antibiotics, and polyketides.

[0028] Thus, the present invention expands the diversity and practicality 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, the terms "peptide," "polypeptide," and "protein" are used interchangeably; and "heterologous molecule," "heterologous peptide," "heterologous sequence," or "heterologous atom" are, respectively, a molecule, peptide, nucleic acid or amino acid sequence, or atom that physically binds to a target molecule and is not found in nature or normally. As used herein, the terms "non-natural" and "modified" are used interchangeably. As used herein, the terms "natural," "native," and "wild-type" are used interchangeably, and the terms "NKG2D" and "NKG2D receptor" are used interchangeably. As used herein, the term "antibody" is used in the broadest sense so long as it exhibits the desired biological activity, and specifically covers monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. 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 open-ended or non-limiting 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 the specified materials or steps and those that do not materially affect the basic novel characteristics of the claimed invention. The present disclosure contemplates embodiments of the compositions and methods of the invention corresponding to the scope of each of these terms. Thus, a composition or method that includes the recited elements or steps is contemplated to include specific embodiments in which the composition or method consists essentially of or consists of those elements or steps.

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

[0032] Although the present invention has been fully described herein, one of ordinary skill 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 in connection with specific embodiments thereof, it will be understood that further modifications are possible. This application is generally intended to cover any variations, uses, or applications of the present invention that include departures from the present disclosure within the scope of known or customary practice in the technical field to which the invention pertains and in which the essential characteristics described above can be applied in accordance with the principles of the invention.

Examples

[0033] Examples Modification of the extracellular domain of the NKG2D receptor and the modified α1-α2 domain of the 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 extracellular domain

[0034] It has been demonstrated by others that mutations at tyrosine 152 or tyrosine 199 (equivalent to positions 73 and 120 in the extracellular domain of NKG2D) in human NKG2D (Figure 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 mutations in either tyrosine residue would greatly affect the ability of NKG2D to bind its natural ligand, but that simultaneous mutations in both tyrosine 152 (Y152) and tyrosine 199 (Y199) would likely abolish the receptor's ability to engage all native ligands. Therefore, we sought to identify single and double mutant variants that could not engage any natural ligand and characterized their biochemical behavior by searching for individual and combinatorial Y152 and Y199 substitutions. Particular attention was paid to variants that were well - expressed and assembled. This was because they would represent inactive ligands that could be more readily produced for analysis.

[0035] The native NKG2D (wild-type) extracellular domain (NKG2D.wt, SEQ ID NO: 17) and candidate non-native NKG2D variant extracellular domains (SEQ ID NOs: 18-35) (also referred to as "engineered NKG2D" or "eNKG2D") were cloned as fusions to the C-terminus of human IgG1 Fc (without Fab domain) via a short Factor Xa - recognizable Ile-Glu-Gly-Arg linker (SEQ ID NO: 38). They were interchangeably designated 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 MHC I signal sequence (SEQ ID NOs: 36 and 37), human IgG1 Fc with linker (SEQ ID NO: 39), and NKG2D extracellular domain variants (SEQ ID NOs: 59-77) were synthesized and inserted into pD2610-V12 (ATUM, Newark, CA). DNA constructs (Figure 18) exploring substitutions in combinations of Y152, Y199, or Y152 / Y199 mutations were transiently expressed in Expi293™ cells (ThermoFisher Scientific, Waltham, MA), and the 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, appropriately sized material was fractionated and isolated from the aggregate peak prior to inclusion in the assay.

[0036] Characterization of the purified NKG2D.Y199A-Fc fusion by SEC revealed the composition of the predominantly aggregated material (Figure 2). As a comparison, both the native Fc-NKG2D fusion and the Fc-NKG2D.Y152A fusion materials were distinguished by well-separated non-aggregated peaks that migrated more rapidly from the more slowly migrating aggregates. The effect of the Y199A mutation on aggregation was also evident in the Y152A / Y199A double mutant Fc-NKG2D fusion variant. This indicates that it has a major impact on protein misfolding (Figure 2). This aspect of including Y199A along with any combination of Y152 mutations in the NKG2D variants therefore presented challenges for the production of the materials required 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 a more robust molecule. The eNKG2D combination Y152 and Y199 mutant candidates were considered as Fc fusions and are detailed in Figure 18. Furthermore, all purified and expressed Fc-eNKG2D fusion candidates were profiled by SEC, and their chromatograms revealed varying levels of aggregate formation (Figures 2 and 3, Figure 18). Among the single amino acid substitutions explored at residue 152, all of alanine, serine, threonine, and valine had no effect on the assembly of the Fc-NKG2D molecule, but Y152-leucine (Y152L) resulted in highly aggregated material. Similar to alanine, neither glutamic acid nor aspartic acid was tolerated at position 199, but only phenylalanine moderately increased aggregate formation. Among the combinations of mutations explored, Y152A / Y199F, Y152S / Y199F, Y152T / Y199F, and Y152F / Y199F did not negatively affect the desired dimer formation, but other combinations resulted in increased aggregation (Figures 18, 2, and 3).

[0037] Example 2: Generation of an antibody-based bispecific molecule “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 a polynucleotide encoding either an APTSSSGGGGS linker for fusion to the C-terminal κ light chain (SEQ ID NO: 84) of human IgG1, or a GGGS linker for fusion to the C-terminal heavy chain (SEQ ID NO: 82). Further, two mutations, D265A / N297A (Kabat numbering; FIGS. 13A and 13B), which reduce binding to all FcγR receptors and thus abolish the antibody-dependent cell cytotoxicity (ADCC) function, were introduced into the CH2 domain of the heavy chain (Shields et al., 2001 JBC, 276:6591-6604). A polynucleotide (SEQ ID NO: 12) encoding the α1-α2 domain of wild-type ULBP2 without GPI linkage (ULBP2.wt) was similarly cloned and fused with a DNA polynucleotide encoding the linker and the IgG1 heavy or light chain. These bispecific antibodies (referred to singly as "MicAbody™" and plurally as "MicAbodies") are bivalent for the fused α1-α2 domain. Examples of antibodies used to generate MicAbodies for the purpose of exploring eNKG2D engineering include, but are not limited to, trastuzumab (SEQ ID NO: 94 and 96) and rituximab (SEQ ID NO: 98 and 100), which were subsequently referred to as "trastuzumab-MicAbody" (e.g., SEQ ID NO: 102 and 104) and "rituximab-MicAbody" (e.g., SEQ ID NO: 106), respectively. The fusion constructs were individually inserted into pD2610-V12 (ATUM, Newark, CA) via Gibson cloning (New England Biolabs Inc., Ipswich, MA). For a given antibody that recognizes a specific antigen, 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, MA).Alternatively, plasmids encoding the heavy chain fused to either a natural or non-natural NKG2D ligand and a plasmid for the light chain were co-transfected. The secreted bispecific antibody was purified by Protein A affinity chromatography (cat.no.20334, Pierce Biotechnology, Rockford, IL), and the eluted material was characterized by size exclusion chromatography (SEC) on an Akta Pur Superdex column and fractionated if necessary. Further, SDS-PAGE analysis was performed on the purified samples to confirm the expected molecular weights of the fusion heavy chain and fusion light chain species.

[0038] Example 3: Identification of a modified NKG2D variant that cannot bind to either a natural NKG2D-binding ligand or a non-natural ligand and has enhanced binding to wild-type NKG2D The binding affinities of the α1-α2 variants to the extracellular domains of native (wild-type) NKG2D and non-native eNKG2D proteins were analyzed using a plate-based ELISA method. Each of the SEC-fractionated native Fc-NKG2D and non-native Fc-eNKG2D fusions was coated overnight at 4 °C on separate wells of a Nunc Maxisorp 96-well plate (Thermo Fisher Scientific, Waltham, MA) 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 then blocked for 2 h at 20–22 °C with 0.5% bovine serum albumin in PBS (PBS-B). MicAbody was titrated for 60 min at 20–22 °C in PBS / 0.5% bovine serum albumin (BSA) / 0.05% Tween-20 (PBS-BT) against the native or non-native Fc-NKG2D fusions bound to the plate, washed three times in PBS-T at 20–22 °C, and the bound bispecific protein was detected using HRP-conjugated anti-human κ (Abcam, Cambridge MA) in PBS-BT and developed using 1-Step™ Ultra TMB ELISA Substrate Solution (Thermo Fisher Scientific, Waltham, MA). The binding of ULBP2.wt rituximab-MicAbody (SEQ ID NOs: 98 and 106) discriminated between wild-type NKG2D and the eNKG2D variant by a decrease in 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 the identification of eNKG2D variants in which ligand binding was abolished. The binding behavior of each eNKG2D variant to all three bispecific ligands revealed the combination of NKG2D modifications that led to the greatest decrease in the binding of wild-type and variant ligands, and the lead inactive enabled the selection of NKG2D variants.

[0039] Further biophysical analysis of the binding of eNKG2D variants to their ligands was also performed using Bio-Layer Interferometry (BLI) with the ForteBio Octet system (all from ForteBio LLC, Fremont, CA). For these experiments, the 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 the MicAbody format were captured onto anti-human IgG Fc capture (AHC) biosensor chips. After establishing a baseline, the chips were exposed to a titration series of Fc-eNKG2D fusion proteins in the range of 300 nM to 0.41 nM, and the association / dissociation rates were monitored (all steps were performed in PBS-BT). Subsequently, the Fc-eNKG2D fusion protein was captured onto the AHC chip, and the MicAbody was titrated to characterize the binding rate.

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

[0041] ELISA assays using the Fc-eNKG2D fusion as the capture agent were performed starting at 300 nM and titrating ULBP2.wt, MICwed, MIC25 MicAbody (Figure 4). EC50 values were calculated using GraphPad Prism when possible (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. The affinities for ULBP2 and MICwed MicAbodies decreased for all single mutant eNKG2D candidates, but the binding of MIC25 to the eNKG2D candidates was retained. However, all double mutant eNKG2D candidates had lost or significantly decreased binding to all three ligands (ULBP2, MICwed, and MIC25) in the 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 aggregation, suggesting stronger protein assembly upon 293T expression (Figure 18). eNKG2D5 (SEQ ID NO: 48) was more rigorously examined for binding to the wild-type ligand as a MicAbody captured on an Octet AHC chip. The single mutant Fc-NKG2D.Y152A (SEQ ID NO: 41) had reduced binding to all natural 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 a lead NKG2D variant for engineering cognate-selective, orthogonal, non-natural ligands for it.

[0043] Example 4: Construction of an orthogonal non-natural α1-α2 domain with selective binding to the non-natural NKG2D.AF extracellular domain We used phage display to engineer orthogonal non-natural α1-α2 domains that exhibit selective binding to the NKG2D.AF (SEQ ID NO: 48) receptor. As a starting point, a non-natural ULBP2.R80W α1-α2 domain (Figure 1B; SEQ ID NO: 108) with high affinity for the native wild-type NKG2D (NKG2D.wt) extracellular domain was selected as the parental 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) that further has a C8S mutation to remove the potential for disulfide bonding. The codons for the amino acid residues of the ligand that are positioned proximal to positions Y152 and Y199 on the native NKG2D receptor in the bound state were substituted with NNK codons; the library consisted of NNK codons at positions 154-159 (Figure 1B, SEQ ID NO: 110). The library was cloned as a fusion to the pIII minor coat protein of M13 phage, and phage particles presenting 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, C.F., 3rd. (2011)). These α1-α2 phage display libraries were sorted for high binding affinity to the non-natural NKG2D.AF receptor by selectively capturing phage clones that bound to the biotinylated Fc-NKG2D.AF protein in the presence of non-biotinylated native Fc-NKG2D.wt competitor protein. Selective clones were enriched by repeating the competitive selection multiple times while gradually increasing the concentration of non-biotinylated native Fc-NKG2D.

[0044] After four rounds of selection, the phage clones were individually arrayed in 96-well format and subjected to spot ELISA to confirm preferential differential binding to plate-bound non-natural NKG2D.AF compared to NKG2D.wt. Bound phage 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 development (ThermoFisher Scientific, Waltham, MA). The spot ELISA signal for each clone was expressed as the ratio of NKG2D.AF-binding phage to NKG2D.wt-binding phage. Phage with ratios of 14 or greater were sequenced to identify specific mutations within the NNK mutagenesis region. Figure 21 shows the selected amino acid residues for each α1-α2 phage variant that selectively binds to NKG2D.AF. When multiple clones representing the same sequence were identified, the ratio of ELISA signals was plotted and the consistency of the phage clones was confirmed by clustering of the data points (data not shown).

[0045] The 30 variants identified in ELISA were expanded in individual single cultures to generate high-titer microbatches of phage. The purified phage concentration was 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 way showed consistent selective binding to NKG2D.AF with little to no binding to NKG2D.wt, even at the highest concentration of phage assayed (Figure 6). The selected phage also showed a shift of more than two logs in the phage concentration required to achieve half-maximal binding between NKG2D.AF and NKG2D.wt.

[0046] To confirm that the NKG2D.AF selective α1-α2 domain variants retain 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 the APTSSSGGGGS linker to the light chain of the rituximab antibody (SEQ ID NOs: 119-126). The resulting fusions were cloned into the mammalian expression vector pD2610-V12 (ATUM, Newark, CA) via Gibson cloning (New England Biolabs Inc., Ipswich, MA) and co-expressed with the heavy chain of the parental antibody (SEQ ID NO: 99) as paired full IgG antibodies. Transient expression was performed in 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). ELISA measuring the binding of each variant ULBP2 α1-α2 antibody fusion to non-native Fc-NKG2D.AF and native Fc-NKG2D.wt demonstrated its binding affinity towards NKG2D.AF that was significantly higher compared to native NKG2D.wt (Figure 22). Collectively, 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 retain their selective binding properties and were used, for example, in the context of chimeric antigen receptor (CAR) T cells to re-direct the non-native NKG2D.AF receptor towards specific antigens.

[0047] Example 5: Identification of non-native NKG2D ligands capable of discriminating non-native NKG2D receptor variants by selectively binding to one or the other Phage display for engineering orthogonal non-natural α1-α2 domains with selective binding to the NKG2D.Y152A (hereinafter referred to as NKG2D.YA) receptor was performed starting with the non-natural ULBP2.R80W α1-α2 domain (SEQ ID NO: 108) as described above. The α1-α2 phage display library was panned for high binding affinity to the non-natural Fc-NKG2D.YA receptor by selectively capturing phage clones that bound to the biotinylated Fc-NKG2D.YA (SEQ ID NO: 41) protein in the presence of the 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 compared to Fc-NKG2D.wt (Figure 23). For example, ULBP2.S3 (SEQ ID NO: 127) consistently showed selective binding to non-natural NKG2D.YA compared to native NKG2D.wt by ELISA and Octet analysis (both in monomeric His-tagged and bispecific antibody fusion formats). This represents different forms of the invention of non-natural orthogonal α1-α2 domains with high affinity binding to non-natural NKG2D receptors (NKG2D.AF in Example 4, in this case NKG2D.YA). Furthermore, fusions of the orthogonal α1-α2 domains to antibody polypeptides retained their selective binding properties and were used to selectively re-direct non-natural NKG2D receptors towards specific molecules determined by fusion heterologous peptides such as antibodies.

[0048] To determine whether non-natural α1-α2 domains having selective binding to NKG2D.YA (ULBP2.S3, SEQ ID NO: 127) and non-natural α1-α2 domains having selective binding to NKG2D.AF can discriminate between these two non-natural receptor variants, a titration ELISA was performed. All of the 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 characteristics of inability to bind to NKG2D.wt, strong affinity for NKG2D.AF, and greatly reduced (15- to 20-fold) or abolished binding to NKG2D.YA compared to NKG2D.AF (Figure 7).Also, 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 examined for changes in the predicted immunogenicity profile compared to the wild-type ULBP2 peptide sequence (SEQ ID NO: 4) using the NetMHC4.0 Server (peptide-MHC class I binding for querying all HLA supertype representatives using 9-mer peptide analysis; http: / / www.cbs.dtu.dk / services / NetMHC / ) and the NetMHCII 2.3 Server (peptide-MHC class II binding for querying HLA-DR, HLA-DQ, HLA-DP haplotypes using 15-mer peptide analysis; http: / / www.cbs.dtu.dk / services / NetMHCII / ) (both algorithms were developed by the Technical University of Denmark (http: / / www.bioinformatics.dtu.dk / ; Andreatta M and Nielsen M, 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)). The mutations incorporated into ULBP2.C, ULBP2.R, and ULBP2.AB did not increase predicted immunogenicity, although the predicted immunogenicity of ULPB2.AA increased slightly for a few haplotypes (Figs. 8 and 9).As a result of the lack of specificity of ULBP2.R for NKG2D.AF and its predicted immunogenicity, ULBP2.R was selected for further ELISA analysis to directly compare its binding behavior with ULBP2.S3 (the NKG2D.YA-selected unnatural orthogonal ligand), ULBP2.R80W (an unnatural 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 the heavy and light chains for ULBP2.R, ULBP2.S3, ULBP2.R80W, and ULBP2.wt, respectively) were assayed against wild-type NKG2D (NKG2D.wt) as well as two inert unnatural variants NKG2D.YA and NKG2D.AF (Figure 10). The data demonstrated that the NKG2D.YA-selected variant ULBP2.S3 as a MicAbody binds with high affinity to NKG2D.YA but does not engage NKG2D.AF or native NKG2D. Furthermore, 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 great potential for the exploration of the NKG2D-MIC ligand axis and the development of unique pairs of novel and selective unnatural NKG2D receptors and their respective cognate unnatural MIC ligand binding partners.

[0049] Example 6: Targeting and killing activities of CAR-T cells expressing an unnatural NKG2D.AF extracellular domain are controlled by an orthogonal α1-α2 domain fused to a heterologous targeting polypeptide Means for selectively controlling CAR-T cell therapy are highly sought for reducing toxicity and improving efficacy against tumors (Gill and June, supra). Attempts have been made previously to develop CARs using the extracellular domain of CD16, which is then engaged via the Fc domain of a therapeutic monoclonal antibody to enable 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 the therapeutic antibodies used to control these cells encounter competition from endogenous CD16 receptors on NK cells, PMNs, monocytes, and macrophages. Both of these features contribute to the problems of off-tumor toxicity and poor pharmacokinetics, respectively.

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

[0051] We engineered CAR-T cells using a receptor that contains a modified Y152A / Y199F (“AF”) extracellular domain of NKG2D that lacks binding to all natural NKG2D ligands or the Y152A-modified NKG2D (NKG2D.YA) and cross-reactivity to previously described non-natural α1-α2 domains of the same family. The non-natural α1-α2 domain of the invention binds with high affinity to the non-natural NKG2D.AF extracellular domain and avoids binding to the natural NKG2D extracellular domain and the NKG2D.YA extracellular domain. Thus, the engineered α1-α2 domain that shows strong selectivity for the non-natural NKG2D.AF extracellular domain compared to natural NKG2D and non-natural NKG2D.YA represents an ideal system for the selective control of non-natural NKG2D CAR receptors, or any receptor or protein fused to a non-natural NKG2D extracellular domain that can be selectively engaged by the non-natural 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) that each signal using distinct intracellular domains. These different CARs have independent dual control of cell activity by extracellular exposure to their respective cognate orthogonal MicAbody or another non-antibody fusion polypeptide.

[0052] To demonstrate the selective control of CAR-T cells engineered with chimeric receptors bearing non-native NKG2D.AF extracellular domains, we based our work on a previous study using a 4-1BB / CD3-zeta CAR construct (Campana Patent No. 8,399,645), fusing each NKG2D extracellular domain to the CD8 hinge region of the CAR to generate CARs using either the native NKG2D.wt (SEQ ID NO: 135), non-native NKG2D.YA (SEQ ID NO: 137), or non-native NKG2D.AF (SEQ ID NO: 139) extracellular domains (SEQ ID NOs: 151, 153, 155). These constructs (SEQ ID NOs: 152, 154, 156) were cloned into a lentiviral vector and expressed in primary human CD8+ T cells using lentiviral transduction. HeLa cells have constitutively upregulated levels of MIC ligands (MICA, MICB, ULBP3, and ULBP2 / 5 / 6 (the antibody used to confirm this could not distinguish between these three ULBP; Human ULBP-2 / 5 / 6 Antibody, R&D Systems, Minneapolis, MN)) on their surface. HeLa cells were transfected to overexpress either native ULBP1 or a variant ULBP2.R selected for NKG2D.AF on their surface, and these cells were used as targets for in vitro killing assays. HeLa target cells were pre-loaded 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 into the supernatant was quantified and normalized to the total calcein released upon detergent treatment (Figure 11). Due to the elevated levels of MIC ligands naturally expressed on the surface of HeLa cells, CD8 cells expressing native NKG2D (NKG2D.wt) as the CAR engaged HeLa cells via this overexpressed native ligand and caused 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 equivalent to non-transduced CD8 T cells). When ULBP1 was overexpressed on the surface of HeLa cells, only NKG2D.wt-CAR CD8 T cells lysed it significantly. There was some additional killing at high E:T ratios for NKG2D.YA-CAR cells, but this was not present for NKG2D.AF-CAR cells, indicating that the double mutation Y152A / Y199F renders NKG2D even more inactive than the single Y152A mutation. In HeLa cells overexpressing the non-native ULBP2.R selected by NKG2D.AF, NKG2D.wt-CAR cells directed lysis (due to recognition of endogenous MIC ligands), while 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 can be directed only by the appropriate cognate-targeting MicAbody, Ramos cells were used as targets for cell lysis in combination with a rituximab-based MicAbody conjugated to either the non-native ULBP2.S3 or ULBP2.R orthogonal ligand. As demonstrated in Figure 12A, the rituximab-ULBP2.S3 MicAbody was able to direct the cell killing activity of NKG2D.YA-CAR CD8 cells but not that of NKG2D.AF-CAR cells, while the rituximab-ULBP2.R MicAbody was able to direct the activity of NKG2D.AF-CAR cells but not that of NKG2D.YA-CAR cells. This further demonstrates the selectivity of the two non-native ULBP2 variants for their cognate non-native 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 using NKG2D.AF-CAR CD8 T cells pre-incubated with either the rituximab-ULBP2.R, trastuzumab-ULBP2.R (SEQ ID NOs: 95 and 133, heavy and light chains, respectively), or an equimolar combination of the two, at the saturating total concentration of the MicAbody. After removing unbound MicAbody by washing, CD8 cells were provided to either calcein-pre-loaded Ramos cells (expressing CD20, the target of rituximab) or CT26-Her2 (a mouse cell line transfected to express human Her2). 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 pre-incubated with the rituximab-MicAbody, only Ramos cells were lysed, while the trastuzumab-MicAbody directed cell lysis activity only against CT26-Her2 cells.However, when both rituximab and trastuzumab-ULBP2.R MicAbody were pre-administered to NKG2D.AF-CAR CD8 cells simultaneously, both target cell lines were lysed, demonstrating that these CAR cells can be readily multiplexed (by engineered selective and privileged partnering between receptor and ligand) to thereby engage different tumor targets simultaneously.

[0054] Example 7: Orthogonal α1-α2 domain as a means for selectively delivering cytokines to engineered non-natural NKG2D-expressing T cells Bispecific MicAbodies that utilize the privileged interaction between an antigen targeting the Fv domain of an antibody and an orthogonal α1-α2 domain and engineered non-natural NKG2D.YA or NKG2D.AF (SEQ ID NOs: 137 and 139) (Figure 13B)) can effectively direct the cytolytic capacity of T cells bearing NKG2D.YA or NKG2D.AF-CAR (SEQ ID NOs: 153 and 155, respectively) to eliminate antigen-expressing target cells. Furthermore, by exploiting the highly selective interaction between a cognate orthogonal ligand and a non-natural modified (also referred to as "engineered") NKG2D (eNKG2D), molecules can be selectively delivered to cells bearing the eNKG2D-extracellular domain (eNKG2D-ecd). If the heterologous atom or molecule (payload / cargo) to which the orthogonal ligand is fused has an inherent biological activity that is potentially undesirable when delivered to non-eNKG2D-expressing cells, mutations in bioactive molecules that reduce interactions with native receptors or targets can be explored. Thus, when fused to an orthogonal ligand, in the presence of cells bearing the cognate eNKG2D receptor except forThe final molecules are effectively inert to all biological functions. Only when a high-affinity interaction occurs between the orthogonal ligand and the eNKG2D receptor, the interaction between the bioactive molecule, a mutant with reduced affinity, and its natural target or receptor is promoted, and the residual function of the bioactive molecule becomes effective. All orthogonal ligand fusions to entities for directing them to eNKG2D-expressing cells are collectively called 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, with or without a linker or tag (e.g., His-tag) for assay or purification (Figure 13C). Further, if improved serum stability is desired, the antibody Fc domain can be included in either the form of just the CH2-CH3 domain or in the form of a complete antibody, depending on the desired number of distinct payloads, the valence of either the orthogonal ligand or the heterologous cargo, and the general, experimentally determined architecture that promotes the maximal function of all components (Figure 13D).

[0055] To determine whether cytokines can be selectively delivered to NKG2D.YA-CAR-expressing cells (SEQ ID NO: 153), the cognate orthologous ligand ULBP2.S3 (U2S3, SEQ ID NO: 127) was expressed by fusing it to the N-terminus of Fc1, which was modified at two residues to express a negatively charged aspartic acid residue at the Fc homodimerization boundary (SEQ ID NO: 189). A mutant form of human IL2 (mutIL2) containing two mutations, R38A / F42K, that significantly reduce the affinity of the cytokine for the IL2R-alpha complex (K.M. Heaton, G. Ju, and E.A.G. Grimm, 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 was modified at two residues to express a positively charged lysine residue at the Fc homodimerization boundary (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 charge of Fc2 provided an electrostatic steering effect that promoted the heterodimeric assembly of molecules that were 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). Furthermore, MicAdaptors composed of other combinations of fusions of ULBP2 orthogonal ligands and cytokines were explored as either heterodimeric Fc1 / Fc2 fusions or as single polypeptides with both components directly fused, their DNAs were 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) were included as assay controls as needed.

[0056] Transduce CD8 human T cells to express either the NKG2D.wt-CAR construct (SEQ ID NO: 151) or the NKG2D.YA-CAR construct (SEQ ID NO: 153), expose them to 30 IUe / mL of control cytokine or various MicAdaptors for 3 days, and quantify the level of cell proliferation with the WST-1 Cell Proliferation Reagent (Millipore Sigma). Control rhIL2 promoted the proliferation of both CAR-expressing cell types, but mutIL2 alone did not, as expected from the reduced ability to engage IL2R-alpha and thus the reduced signaling ability via IL2R-beta / gamma-C (Figure 15A). When the MicAdaptor was composed of the non-selective U2R80W ligand (U2R80W-mutIL2, SEQ ID NO: 177) that engages both the wild-type receptor and the modified NKG2D.YA receptor with high affinity, cells expressing either CAR responded by proliferation. However, when mutIL2 was fused to an orthogonal ligand such as ULBP2.S2 (SEQ ID NO: 179) that selectively engages only NKG2D.YA (a MicAdaptor that engages only NKG2D.YA, U2S2-mutIL2), only NKG2DYA-CAR cells responded by proliferation. Similar results were obtained when the cytokine fused to U2S2 was a mutant form of IL15 with the V49D mutation that has reduced 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-α Chemistry, 279:24313, PMID: 15039446) (Figure 15A).When NKG2D.YA-CAR-T cells were co-cultured with these reagents for 7 days, %GFP+ (indicating the percentage of CAR-expressing cells present compared to GFP-negative cells that were not transfected) increased, but only in the presence of the orthogonal ULBP2.S2 ligand (U2S2-mutIL2 or U2S2-Fc1 / Fc2-mutIL2, for sequence numbers see Figure 24), and not with the non-selective U2R80W variant that binds to the natural wild-type NKG2D receptor constitutively present on human CD8 cells (Figure 15B). IL21 was explored as either a wild-type fusion (IL21.wt) with a single mutation (D18A or E109R) that affects IL21R-alpha binding, or a form that includes 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-transfected CD8 cells or NKG2D.YA-CAR-expressing cells, only NKG2D.YA-bearing cells responded to all forms of the IL21 fusion that included the IL21.wt fusion (for sequence numbers see Figure 24) (Figure 15C). Interestingly, both non-transfected cells and NKG2D.YA-CAR cells amplified in response to rhIL2 compared to cytokine-free controls, but the amount of proliferation was higher in NKG2D.YA-CAR cells. U2S3-Fc1 / Fc2 (a heterodimeric Fc molecule with a single U2S3 domain and no attached cytokines or cytokine variants) and Rituximab-MicAbody (which is bivalent for the U2S3 domain but lacks any cytokine components, Figure 13Bb) did not induce proliferation over the course of 3 days of culture, even at IUe / mL concentrations much higher than the rhIL2 control (Figure 16C), so the proliferative response of NKG2D.YA-CAR-expressing cells was independent of generalized binding or engagement of the NKG2D.YA domain.

[0057] Example 8: The presence of the intracellular co-stimulatory domain in either cis or trans promotes the responsiveness of non-naturally modified NKG2D-bearing cells to cytokines and cytokine MicAdaptors We sought to determine whether the NKG2D.YA receptor is not only required but also sufficient for targeted delivery of cytokines (which may then act on recipient cells) when demonstrating that the modified NKG2D.YA domain actually functions as a highly selective docking site for delivery of heterologous cargo conjugated to an orthogonal ligand in the context of a chimeric antigen receptor construct. The NKG2D.YA extracellular domain (NKG2D.YA-ecd) was expressed as a transmembrane domain with all intracellular components removed except for retention of an intracellular eGFP tag (Figure 14, SEQ ID NO: 157). CD8 cells were transduced to express this “silent CAR” and, as expected, were unable to direct killing of Ramos target cells in the presence of rituximab-ULBP2.S3 MicAbody (SEQ ID NO: 98 and 129) without a co-stimulatory domain (Figure 16A). Importantly, cells expressing this completely inactive or silent CAR did not proliferate when exposed to U2S3-Fc1 / Fc2-mutIL2 (SEQ ID NO: 189 and 193) but responded at levels comparable to untransduced cells (Figure 16B). This observation - (a) in addition to the consistent observation of higher levels of NKG2D.YA-CAR proliferation by rhIL2 compared to untransduced cells (Figure 15C, 16B, 16C), and (b) the observation that only NKG2D.YA-CAR cells respond to all forms of IL21-MicAdaptor containing IL21.wt led us to speculate that the intracellular domain present in the NKG2D.YA-CAR construct enhanced responsiveness to these cytokines and cytokine MicAdaptors.

[0058] To verify this, a series of CAR constructs were generated in which the signaling motifs of the intracellular domain of the CAR were mutated—the two TRAF2 consensus binding sites of 4-1BB (SEQ ID NO: 161), the ITAM motifs in three pairs of 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, 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 proliferation response equivalent to 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) carried mutations in both intracellular domains of NKG2D.YA-BB ΔTRAF2 -CD3 ΔITAM -GFP (SEQ ID NO: 165) receptor, like the NKG2D.YA-BB-CD3

[0059] To further verify how the 4-1BB domain contributes to cytokine and cytokine-MicAdaptor responsiveness, a construct (SEQ ID NO: 173) was generated that contains a functional 4-1BB domain and a complete complement of the CD3-zeta domain in a CD19scFv-CAR (based on the FMC63 Fv). CD19scFv-CAR was co-expressed with NKG2D.YA-ecd (Figure 14). Both components were expressed as a single polypeptide with a T2A self-cleaving peptide motif that separates the upstream CD19scFv-CAR construct from the downstream NKG2D.YA-ecd having an independent GMCSFR alpha chain signal sequence, CD8a hinge, and CD8a transmembrane domain. This 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 the GFP signal and phycoerythrin-conjugated MicAbody staining, respectively. These 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, while control cells containing only CD19scFv-CAR (SEQ ID NO: 171) proliferated with rhIL2 and to a lesser extent with the highest concentration (300 IUe / mL) of U2S3-Fc1 / Fc2-mutIL2 (Figure 17C). Co-expression of NKG2D.YA-ecd in CD19scFv-CAR-expressing cells (SEQ ID NO: 173) showed a greater proliferative response to the 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 demonstrate that the responsiveness of NKG2D.YA-ecd-expressing cells to cytokines and cytokine-MicAdaptors is promoted by the 4-1BB domain either in cis or in trans, and that co-expression of the NKG2D.YA-ecd domain with costimulatory 4-1BB-containing CAR cells can provide additional diverse functions to engineered cells for adoptive cell therapy strategies. This function is not limited to surface receptor engagement upon ligand / silent CAR engagement, but can be extended to intracellular delivery by incorporating cytoplasmic sequence motifs that promote the turnover of the non-native NKG2D variant such that any bound MicAdaptor is co-internalized and heterologous cargo is delivered intracellularly (K.N. Pandey, Functional roles of short sequence motifs in the endocytosis of membrane receptors, 2009 Front Biosci, 14:5339, PMID:19482617).

Claims

**Claim 1** A non-natural modified NKG2D receptor comprising an amino acid sequence of either SEQ ID NO: 18 or 25, wherein the receptor is inserted into mammalian cells, the receptor binds to a non-natural modified α1-α2 domain of an NKG2D ligand but does not bind to a natural NKG2D ligand, the modified NKG2D receptor does not contain an active CD3-zeta intracellular signaling domain and does not activate intracellular signaling in mammalian cells during the formation of an immunological synapse, and wherein the non-natural modified α1-α2 domain of the NKG2D ligand for the non-natural modified NKG2D receptor comprising the amino acid sequence of SEQ ID NO: 18 contains the amino acid sequence of SEQ ID NO: 127, and the non-natural modified α1-α2 domain of the NKG2D ligand for the non-natural modified NKG2D receptor comprising the amino acid sequence of SEQ ID NO: 25 contains an amino acid sequence of any one of SEQ ID NOs: 111, 113, 115, and 117. **Claim 2** The modified NKG2D receptor according to claim 1, wherein a heterologous atom or molecule bound to the modified α1-α2 domain of the NKG2D ligand that binds to the modified NKG2D receptor activates the cell or signals the cell via respective receptor subunits on or in the mammalian cell. **Claim 3** The modified NKG2D receptor according to claim 1, wherein a heterologous atom or molecule that binds to the modified α1-α2 domain of the NKG2D ligand that binds to the modified NKG2D receptor directly affects the mammalian cell independently of any receptor on or in the cell other than the non-natural modified NKG2D receptor. **Claim 4** The modified NKG2D receptor according to claim 1, wherein a heterologous atom or molecule bound to the modified α1-α2 domain of the NKG2D ligand that binds to the modified NKG2D receptor is internalized by the mammalian cell, and the internalized heterologous atom or molecule, whether bound or unbound to the modified α1-α2 domain, affects the mammalian cell. **Claim 5** The modified NKG2D receptor according to claim 1, wherein a heterologous atom or molecule bound to the modified α1-α2 domain of the NKG2D ligand that binds to the modified NKG2D receptor provides a marker detectable by an in vivo, ex vivo, or in vitro detection or imaging system to the mammalian cell. **Claim 6** The modified NKG2D receptor according to claim 1, wherein the modified NKG2D receptor comprises a co-stimulatory domain bound thereto that enhances the function of an effector molecule introduced into a mammalian cell, and the effector molecule is not bound to the modified α1-α2 domain of the NKG2D ligand.

7. The modified NKG2D receptor according to claim 3, wherein a heterologous atom or molecule bound to the modified α1-α2 domain of the NKG2D ligand that binds to the modified NKG2D receptor damages the cell by release of energy or disruption of the cell membrane.

8. The modified NKG2D receptor according to any one of claims 1 to 7, wherein the modified NKG2D receptor does not comprise an active CD3-zeta domain and has an intracellular co-stimulatory domain bound thereto.

9. The modified NKG2D receptor according to claim 8, wherein when a heterologous atom or molecule bound to the non-natural modified α1-α2 domain of the NKG2D ligand that binds to the modified NKG2D receptor is delivered to the mammalian cell, the heterologous atom or molecule provides the mammalian cell with a targeting or homing function that promotes delivery of the mammalian cell to a target-bearing cell or target-bearing surface without directly leading to activation of the mammalian cell when the mammalian cell reaches the target surface or environment.

10. The modified NKG2D receptor according to claim 8, wherein the co-stimulatory domain enhances the function of an effector molecule introduced into a mammalian cell, and the effector molecule is not bound to the modified α1-α2 domain of the NKG2D ligand.

11. The modified NKG2D receptor according to claim 8, wherein the co-stimulatory domain is a 4-1BB co-stimulatory domain.

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