RU2823728C2 - Modified non-natural nkg2d ligands which selectively deliver attached heterologous molecules to non-natural nkg2d receptors on car cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention discloses novel modified NKG2D ligands with attached polypeptides having specific target binding properties, for example, antibodies or variable antibody fragments selectively delivered to chimeric antigen receptors (CAR) containing modified, non-natural NKG2D receptors on modified mammalian cells.

EFFECT: invention can be used when producing CAR-cells capable of destroying HIV-infected cells.

10 cl, 14 ex, 13 tbl, 10 dwg

Description

BACKGROUND OF THE INVENTION

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0001] The present invention relates generally to modified, non-natural α1-α2 domains of NKG2D ligands with attached polypeptides having specific target binding properties, such as antibodies or antibody fragments, selectively delivered to chimeric antigen receptors (CARs) comprising modified, non-natural NKG2D receptors on modified mammalian cells.

INFORMATION ON THE STATE OF THE ART FOR THE INVENTION

[0002] An antibody (Ab), Figure 1, also known as immunoglobulin (Ig), in many mammals, including humans, is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses (Charles Janeway (2001). Immunobiology. ( ^5th ed.), Chapter 3. Garland Publishing. ISBN 0-8153-3642-X. (Full electronic text on the NCBI Bookshelf). An antibody recognizes a unique part of a foreign target, called an antigen. Each tip of the two arms of the "Y" of an antibody contains an antigen-binding site, or paratope, (the structural analog of a lock) that is specific for one particular epitope (the structural analog of a key) of the antigen, allowing precise binding of these structures. Using this binding mechanism, an antibody can tag a microorganism or infected cell for attack by other parts of the immune system or can neutralize their target directly, for example by blocking a part of the microorganism that is essential for its entry and survival. The production of antibodies is a major function of the humoral, or "adaptive," immune system. Antibodies are secreted by plasma cells. Antibodies occur naturally in two physical forms, a soluble form secreted from the cell, and a membrane-bound form attached to the surface of the B cell via the Y "stalk."

[0003] Antibodies are glycoproteins belonging to the immunoglobulin superfamily and are typically composed of basic structural units, each with two large heavy chains and two small light chains. There are several different types of antibody heavy chains, and several different types of antibodies, grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals (Market E, Papavasiliou FN (October 2003). "V(D)J recombination and the evolution of the adaptive immune system". PLoS Biol. 1 (1): E16. doi:10.1371/journal.pbio.0000016. PMC 212695. PMID 14551913). Although the overall structure of all antibodies is very similar, a small region at the tip of each arm of the Y protein is extremely variable, allowing the existence of millions of antibodies with slightly different tip structures, or antigen-binding sites. This region is known as the hypervariable or variable region . Each of these natural variants can bind a different antigen. This extraordinary diversity of antibodies allows the immune system to adapt and recognize an equivalently wide variety of antigens ( Hozumi N, Tonegawa S (1976). "Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions". Proc. Natl. Acad. Sci. USA 73 (10): 3628–3632. doi:10.1073/pnas.73.10.3628. PMC 431171. PMID 824647.)

[0004] The natural "Y"-shaped Ig molecule consists of four polypeptide chains; two identical heavy chains and two identical light chains, joined by disulfide bonds. Each heavy chain has two major regions, a constant region (CH) and a variable region (VH). The constant region is essentially identical in all antibodies of the same isotype, but differs between antibodies of different isotypes. The light chain also has two sequential domains: a smaller constant region (CL) and a variable region (VL) (Woof J, Burton D (2004). "Human antibody-Fc receptor interactions illuminated by crystal structures". Nat Rev Immunol 4 (2): 89-99. doi:10.1038/nri1266. PMID 15040582).

[0005] Some parts of an antibody have the same functions. Each of the two arms of the Y, for example, contains regions that can bind to antigens and thus recognize specific foreign objects. This region of the antibody is called the Fv (fragment, variable) region . It consists of one variable domain from the heavy chain (V _H ) and one variable region from the light chain (V _L ) of the antibody (Hochman J, Inbar D, Givol D (1973). An active antibody fragment (Fv) composed of the variable portions of heavy and light chains. Biochemistry 12 (6): 1130-1135. doi:10.1021/bi00730a018. PMID 4569769). The paratope is formed at one end of Fv and is the region for binding to antigens. It consists of variable loops of β-chains, three on each V _L and V _H , and is responsible for antigen binding. These 6 loops are referred to as complementarity-determining regions (CDRs) (North B, Lehmann A, Dunbrack RL (2010). "A new clustering of antibody CDR loop conformations". J Mol Biol 406 (2): 228–256. doi:10.1016/j.jmb.2010.10.030. PMC 3065967. PMID 21035459).

[0006] Polypeptides that can be used that have a specific antigen binding function can be derived from the CDRs of the variable regions of antibodies. These two antibody variable domains, one from the light chain (VL) and one from the heavy chain (V _H ), each with 3 CDRs, can be fused in tandem, in any order, using a single, short linker peptide of 10 to about 25 amino acids to produce a linear single-chain variable fragment (scFv) polypeptide containing one of the variable domains from each of the heavy and light chains (Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., Lee, T., Pope, S. H., Riordan, G. S., and Whitlow, M. (1988) Single-chain antigen-binding proteins, Science 242 , 423-426; Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M. S., Novotny, J. Margolies, M. N., Ridge, R., Bruccoleri, R. E., Haber, E., Crea, R., and Opperman, H. (1988). Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. PNAS 85:5879-5883).

[0007] The linker is typically rich in glycine for flexibility, as well as serine, threonine, or charged amino acids for solubility, and can link either the N-terminus of V _H to the C-terminus of V _L , or vice versa. This protein retains the specificity of the parent immunoglobulin despite the removal of the constant regions and the introduction of a single linker. This format allows one skilled in the art of recombinant DNA technology to genetically fuse a linear scFv to the N- or C-terminus of the parent protein to confer the antigen binding properties of the scFv to the parent protein. There are many other proposed or derived arrangements of polyvalent and tandem scFv regions, but, importantly, as described below, all have at least two spatially distant ends (Le Gall, F.; Kipriyanov, SM; Moldenhauer, G; Little, M (1999). “Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding”. FEBS Letters 453 (1): 164–168. doi:10.1016/S0014-5793(99)00713-9. PMID 10403395).

ESSENCE OF THE INVENTION

[0008] The present invention relates to modified α1-α2 domains of NKG2D ligands attached to heterologous polypeptides, in some embodiments, antibodies or antibody fragments. The modified ligands selectively bind to cognate non-natural NKG2D receptors, which in turn bind selectively to their cognate modified ligands. Non-natural NKG2D receptors can be expressed on the surfaces of cells of the immune system and form a chimeric receptor on the surface of this effector cell. The heterologous molecule attached to the ligand can also bind a specific molecule on the surface of a target cell, thereby delivering the immune effector cell to the target cell. Such effector cells include lymphocytes, B cells, plasma cells, monocytes, macrophages and dendritic cells.

[0009] In some embodiments, the present invention relates to a modified, non-natural ligand for a modified, non-natural NKG2D receptor, wherein the ligand has an attached heterologous molecule that selectively binds an HIV protein present on the surface of an HIV-infected cell, wherein the modified ligand with its heterologous molecule can selectively bind to the modified, non-natural NKG2D receptor of a CAR cell and cause destruction of the HIV-infected cell.

[0010] In further embodiments, the HIV protein to which the heterologous molecule selectively binds is an HIV envelope protein.

[0011] In further embodiments, the epitope of the envelope protein to which the heterologous molecule selectively binds comprises SEQ ID NO: 169 or SEQ ID NO: 170.

[0012] In some embodiments of the present invention, the modified, non-natural ligand comprises SEQ ID NO: 68, 69, 70, 71, or 72.

[0013] In further embodiments, the modified, non-natural NKG2D receptor comprises SEQ ID NO: 54 or 154.

[0014] In some embodiments of the present invention, the HIV protein is expressed on an HIV-infected cell that has been shocked or activated by a mechanism or agent, or a post-latency reactivating agent, known to induce expression of the HIV protein on a latent HIV-infected cell.

[0015] In some embodiments of the present invention, a CAR cell has a plurality of modified, non-natural ligands linked to different, separate heterologous molecules that bind different epitopes, proteins, or other molecules on the surface of an HIV-infected cell.

[0016] In some embodiments of the present invention, a modified, non-natural NKG2D receptor comprising SEQ ID NO: 54 or 154 is present on a CAR cell, wherein the modified NKG2D receptor binds a modified, non-natural ligand consisting of SEQ ID NO: 68, 69, 70, 71, or 72 to which is attached a heterologous molecule or atom that does not bind an HIV protein.

[0017] In further embodiments, the heterologous molecule or atom modulates the function of a CAR cell. In further embodiments, the cellular function comprises proliferation, differentiation, ablation, imaging, antagonism of immunosuppression, homing, or cytolysis of a cell not infected with HIV.

BRIEF DESCRIPTION OF DRAWINGS

[0018] The patent or application file contains at least one drawing in color. Copies of this patent publication or patent application with the color drawing(s) may be furnished by the office upon request and payment of the required fee.

[0019] Figure 1. Drawing of a typical mammalian antibody showing its Y-shaped structure and structural components.

[0020] Figure 2. Structure of a typical CAR (Gill & June, 2015, op.cit.).

[0021] Figure 3. Structure-directed mutagenesis of the MICA α1-α2 domain to enhance affinity for NKG2D. The structure of the MICA α1-α2 domain (PDB 1HYR) with its NKG2D binding surface colored dark gray, where 57 specific amino acid sites were extensively mutated.

[0022] Figure 4. Tyrosine residues Y152 and Y199 within the native NKG2D homodimer.

[0023] Figure 5. Protein sequence alignment of the α1-α2 domains of MICA and ULBPs 1-6. Amino acids highlighted in gray were selected for NNK mutagenesis in ULBP2 (60 amino acids) and ULBP3 (36 amino acids). Residues highlighted in black were identified as key positions for selection and were identified as mutations modulating binding affinity to NKG2D (Tables 6 and 7).

[0024] Figure 6.Results ELISA of R3 antibody-fused proteins with non-native α1-α2 domains selected for binding to Y152A NKG2D-Fc. (A) R3 HC25 antibody-fused protein is not selective for Y152A NKG2D. (B) R3 HC25.17 (SEQ ID NO.: 73) antibody-fused protein is selective for Y152A NKG2D, more than for native NKG2D-Fc. (C) R3 HC.U2RW antibody-fused protein is not selective for Y152A NKG2D, more than for native NKG2D-Fc. (D) The R3 antibody-fused HC.U2S3 (SEQ ID NO.: 74) protein is selective for Y152A NKG2D over native NKG2D-Fc.

[0025] Figure 7. Evaluation of effector:target (E:T) cell ratios for killing of HIV-infected primary CD4 T cells by CAR-T cells using different concentrations of specific HIV-targeting MicAbodies. One million primary tonsil-derived cells infected with Bal-GFP R5 virus (~10% infection; ^1X104 infected cells) were incubated with ^1X105 untransduced CD8 (0:1) or with ^1X104 (1:1) or ^2X105 (20:1) CAR-T cells in the presence of different concentrations of four different broadly HIV-neutralizing MicAbodies. Cells were stained after 24 h and assessed by flow cytometry. Cells were gated on single/live/CD3+/CD8- cells, either GFP expressing or not. Results averaged over 3 studies are shown.

[0026] Figure 8. Specific killing of R5 virus-infected primary CD4 cells by CAR-T in combination with an HIV-specific MicAbody. One million primary tonsil-derived cells infected with the Bal-GFP R5 virus (~ ^1X104 infected cells) were incubated with ^1X105 CAR-T cells in the presence of varying concentrations of HIV-specific MicAbodies or a B cell-specific CD20-targeting MicAbody or a HER2-targeting MicAbody (Her2). Cells were stained 24 h later and analyzed by flow cytometry. Cells were gated as single cells/live/CD3+/CD8- and either GFP+ or GFP-. Results averaged over 4 studies are shown.

[0027] Figure 9. Specific killing of F4 founder/transplant virus-infected primary CD4 cells by CAR-T in combination with an HIV-specific MicAbody. One million primary tonsil-derived cells infected with F4-GFP (T/F) virus (~ ^1X104 infected cells) were incubated with ^1X105 convertible CAR-T cells in the presence of varying concentrations of four different HIV-specific MicAbodies, a CD20-targeting MicAbody (Ritux) or a HER2-targeting MicAbody (Her2). Cells were stained after 24 h and followed by flow cytometry. Cells were gated for single cells/live/CD3+/CD8- and either GFP+ or GFP-.

[0028] Figure 10. CAR-T and MicAbody depletion of reactivated latently infected reservoir cells from aviremic patients chronically infected with HIV and undergoing ART. CD4+ T cells were isolated by non-contact negative selection from PBMCs collected from known HIV-infected patients undergoing ART and reactivated for 72 h using 100 nM phorbol myristate acetate (PMA)+1 μM ionomycin. Cells were then washed twice and incubated for 48 h with convertible CAR-T cells or untransduced CD8 T cells in the presence of 0.1 or 1 nM of an equal concentration mixture of HIV bNAb-based MicAbodies (3BNC60, 3BNC117, PGT121, and 10-1074), designated MIX. Cells were then centrifuged and RNA was isolated from the cell pellets. Cell-associated HIV RNA was measured by ddPCR.

DETAILED DESCRIPTION OF THE INVENTION

[0029]Cells Natural killer (NK) cells, monocyte-macrophage lineage cells, and specific (CD8+ αβ and γδ) T cells of the immune system play important roles in humans and other mammals as first-line innate defenses against neoplastic and virus-infected cells (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 surfaces NKG2D, a prominent, homodimeric, surface immunoreceptor responsible for target cell recognition and activation of innate defenses against the abnormal cell (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 domain that binds to its cognate ligands that are 84% identical or homologous in sequence to monomeric MICA and MICB, polymorphic analogs of the cognate chains of class I major histocompatibility complex (MHC) 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). Non-pathological expression of MIC proteins is typically restricted to the intestinal epithelium, keratinocytes, endothelial cells, and monocytes, however, altered surface expression of these MIC proteins occurs in response to many types of cellular stress such as proliferation, oxidation, viral infection, and heat shock and marks the cell 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 γδT 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). Abnormal expression of MIC proteins also appears to be involved in some 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 the polymorphic MICA and MICB, is important to provide the immune system with the means to identify and respond to a wide range of signs of a critical situation, 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).

[0030] Viral infection is a common inducer of MIC protein expression and identifies the virus-infected cell 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 LL Lanier. 2001). In fact, to avoid such an attack on its host cell, CMV and other viruses have evolved mechanisms to prevent expression of MIC proteins on the surface of the cell they infect in order to avoid triggering the innate immune system (Lodoen, M., K. Ogasawara, JA Hamerman, H. Arase, JP Houchins, ES Mocarski, and LL 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, IA Busche, M Babic, M Mitrovic, I Gašparovic, Đ 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).

[0031] Despite their stress, many malignant cells, such as lung cancer cells and glioblastoma brain tumor cells, also escape expression of MIC proteins and, as a result, may be particularly aggressive because they too evade the innate immune system (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, M. B., M. V. Girart, L. L. Molinero, C. I. Domaica, L. E. Rossi, M. M. Barrio, J Mordoh, G. A. Rabinovich and N. W. 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).

[0032] A high-resolution structure of human MICA bound to NKG2D has been resolved and shows that the α3 domain of MICA does not directly interact 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 the MICB domain, is connected to the α1-α2 platform domain via a short, flexible linker peptide, and is itself naturally positioned as a “spacer” between the platform and the surface of the MIC-expressing cell. The 3-dimensional structures of the α3 domains of human MICA and MICB are nearly identical (root mean square distance <1 Å at 94 C-αα) 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).

[0033]Described specific non-natural α1-α2 domains of NKG2D ligands modified to bind natural human NKG2D receptors with higher affinity than the natural α1-α2 domains (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. Herein, the inventors describe non-natural α1-α2 domains of NKG2D ligands modified to bind non-natural NKG2D receptors that themselves have been mutated in regions that subsequently result in impaired or lost binding to the 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 Patent Application 14/562,534; USPTO Provisional Patent Application 62/088,456)). The present invention provides bispecific molecules consisting of specifically modified non-natural α1-α2 domains and specific targeting heterologous molecules, including, but not limited to, heterologous peptides or polypeptides that bind chimeric antigen receptors (CARs), wherein the CAR receptor consists of the ectodomain of the non-natural NKG2D receptor that binds modified α1-α2 domains with greater affinity than it binds natural α1-α2 domains. Genetically modified immune system cells, such as B cells, T cells, NK cells, and macrophages, containing such CARs can then overcome many of the disadvantages, including known severe systemic toxicities and antigen escape, of current CAR-T and CAR-NK cell-based therapeutics, as described below (Kalos M, Levine, BL, Porter, DL, Katz, S, Grupp, SA, Bagg, A and June, C. T Cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011;3:95ra73; Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 2010, 18:843-851; Gill and June 2015).

[0034] T cells, NK cells, and macrophages can be modified using gene transfer technologies to directly and stably express antibody binding domains on their surface that confer novel antigen specificities (Saar Gill & Carl H. June. Going viral: chimeric antigen receptor 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 method that combines the antigen recognition domain of a specific antibody with the intracellular domain of the CD3-ζ chain, which is the primary signaling transducer of endogenous T cell receptors (TCRs), into a single chimeric protein together with a costimulatory molecule such as CD27, CD28, ICOS, 4-1BB, or OX40, Figure 2. The thus engineered CARs can trigger T cell activation upon binding of a target antigen, in a manner similar to the endogenous T cell receptor but independent of the major histocompatibility complex (MHC).

[0035] As used herein, "soluble MIC protein,""solubleMICA," and "soluble MICB" refer to a MIC protein comprising an α1-α2 domain in the presence or absence of the α3 domains of the MIC protein, but without the membrane anchoring motif, transmembrane, or intracellular domains. The NKG2D ligands, ULBP1-6, naturally lack the 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 NKG2D ligand refers to the protein domain of the ligand that binds the NKG2D receptor.

[0036] In some embodiments, the α1-α2 domains of the non-natural NKG2D ligand proteins of the invention are at least 80% identical or homologous to a native or natural α1-α2 domain of NKG2D ligand, SEQ ID NOs: 1-19. In other embodiments, the modified α1-α2 domain is 85% identical to a native or natural α1-α2 domain of NKG2D ligand. In other embodiments, the modified α1-α2 domain is 90% identical to a native or natural α1-α2 domain of a natural NKG2D ligand protein and binds non-natural NKG2D.

[0037] The α1-α2 platform domain of a soluble MIC protein is diffusible in the extracellular or intravascular space of a mammal. Preferably, the α1-α2 platform domains of the non-naturally occurring MIC proteins of the invention are at least 80% identical or homologous to a native or naturally occurring α1-α2 domain of a human MICA or MICB protein and bind natural NKG2D or, in specific examples, bind modified, non-naturally occurring NKG2D receptors. In some embodiments, the α1-α2 platform domain is 85% identical to a native or naturally occurring α1-α2 platform domain of a human MICA, human MICB, or human ULBP1-6 protein and binds natural NKG2D or modified, non-naturally occurring NKG2D. In other embodiments, the α1-α2 platform domain is 90%, 95%, 96%, 97%, 98%, or 99% identical to a native or naturally occurring α1-α2 platform domain of human MICA, human MICB, or human ULBP1-6 and binds a natural NKG2D receptor or a modified, non-natural NKG2D receptor.

[0038] In some embodiments, a heterologous peptide tag can be fused to the N-terminus or C-terminus of the α1-α2 domain or other soluble MIC protein to facilitate purification of the soluble MIC protein. Tag sequences include peptides such as polyhistidine, myc peptide, or FLAG tag. Such tags can be removed after isolation of the MIC molecule by methods known to those skilled in the art.

[0039] In other embodiments of the invention, specific mutations in the α1-α2 domains of NKG2D ligands can be made to produce non-natural α1-α2 domains that bind non-natural NKG2D receptors that are themselves engineered to have reduced affinity for natural NKG2D ligands. This can be accomplished, for example, by genetic engineering. A non-natural NKG2D receptor modified in this way can be used to produce a non-natural NKG2D-based chimeric antigen receptor (CAR) on the surface of NK cells, T cells, macrophages, or immune stem cells that can preferentially bind to and be activated by molecules consisting of the inventive non-natural α1-α2 domains. These pairs of unnatural NKG2D receptors and their inventive cognate unnatural NKG2D ligands may provide important safety, efficacy, and manufacturing advantages for the treatment of cancer and viral infections compared to current CAR-T cells and CAR NK cells, as described below.

[0040] CAR-modified T cells have emerged as a promising approach for adoptive T cell therapy of cancer, and CARs targeting a variety of different molecules have been tested in CAR T cells as therapeutics against cancer (Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 365:725–733.). While remarkable clinical efficacy has been observed in hundreds of patients undergoing adoptive transfer of T cells expressing CD19-specific chimeric antigen receptors, the methods of custom-designing a CAR to target a specific antigen, isolating autologous T cells from the patient, genetically modifying the autologous T cells to express the customized CAR, expanding the modified cells in vitro, and quality-controlling their production have all been labor-intensive and expensive. Currently, this is only feasible in the context of large academic centers with extensive expertise and resources (Gill & June, 2015).

[0041]How only autologous CAR-T cells are infused back into the donor patient, their proliferationin vivocannot be controlled---"living cell therapy" and there is no dose-response relationship with efficacy (Gill & June, 2015). Additionally, CAR T cell escape can occur via antigen-loss escape (Stephan A. Grupp, M.D., Ph.D., Michael Kalos, Ph.D., David Barrett, M.D., Ph.D., Richard Aplenc, M.D., Ph.D., David L. Porter, M.D., Susan R. Rheingold, M.D., David T. Teachey, M.D., Anne Chew, Ph.D., Bernd Hauck, Ph.D., J. Fraser Wright, Ph.D., Michael C. Milone, M.D., Ph.D., Bruce L. Levine, Ph.D., and Carl H. June, M.D. Chimeric Antigen Receptor-Modified T Cells for Acute Lymphoid Leukemia. N Engl J Med 2013;368:1509–1518), and this pathway Escapes can be most rapidly targeted through sequential therapy using a differentially targeted CAR T cell or through an initial infusion of a T cell product containing multiple CARs of two or more specificities, which further complicates manufacturing processes and quality control.

[0042] In addition to CAR-T cells targeting tumors using single-chain antibody binding domains (scFv), CAR-T cells using the ligand binding domain of the NKG2D receptor have been studied in animals and recently in humans (Sentman CL, Meehan KR. NKG2D CARs as cell therapy for cancer. Cancer J. 2014 Mar-Apr;20(2):156-9. doi: 10.1097/PPO.0000000000000029; Manfred Lehner, Gabriel Götz, Julia Proff, Niels Schaft, Jan Dörrie, Florian Full, Armin Ensser, Yves A. Muller, Adelheid Cerwenka, Hinrich Abken, Ornella Parolini, Peter F. Ambros, Heinrich Kovar, Wolfgang Holter. Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection Research Article | published 15 Feb 2012 | PLOS ONE 10.1371/journal.pone.0031210; www.clinicaltrials.gov NCT02203825). Because NKG2D ligand expression is increased on the surface of stressed cells such as tumor cells and virus-infected cells, this family of natural NKG2D ligands is of considerable interest as targets for immunotherapies against viral infections and malignancies (Spear P, Wu MR, Sentman ML, Sentman CL. NKG2D ligands as therapeutic targets. Cancer Immun. 2013 May 1;13:8.; Song DG, Ye Q, Santoro S, Fang C, Best A, Powell DJ Jr., Chimeric NKG2D CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by histone deacetylase inhibition. Hum Gene Ther. 2013 Mar;24(3):295-305). One NKG2D CAR was a fusion protein of the full-length NKG2D receptor and CD3ζ (NKG2Dζ); the other was only the NKG2D ectodomain fused in the reverse orientation to a second-generation CAR scaffold consisting of the transmembrane and intracellular domains of CD28 and the signaling domain of CD3ζ (NKG2D28ζ). Since NKG2D activation depends on the presence of DAP10, a CAR T cell was also constructed where DAP10 was coexpressed with NKG2Dζ (NKG2Dζ10). T cells expressing any of the above NKG2D CARs produced IFNɣ and TNFα in response to NKG2D ligand stimulation and efficiently killed target tumors expressing NKG2D ligands in vitro (Heather VanSeggelen, Joanne A. Hammill, Anna Dvorkin-Gheva, Daniela GM Tantalo, Jacek M. Kwiecien, Galina F. Denisova, Brian Rabinovich, Yonghong Wan, Jonathan L. Bramson, T cells engineered with chimeric antigen receptors targeting NKG2D ligands display lethal toxicity in mice, Molecular Therapy accepted article preview online 30 June 2015; doi:10.1038/mt.2015.119). The cytotoxic potential of NK cells against a wide range of tumor subtypes could also be markedly enhanced by expression of NKG2D-DAP10-CD3ζ-based CAR (Yu-Hsiang Chang, John Connolly, Noriko Shimasaki, Kousaku Mimura, Koji Kono, and Dario Campana. Chimeric Receptor with NKG2D Specificity Enhances Natural Killer Cell Activation and Killing of Tumor Cells. Cancer Res; 73(6) March 15, 2013).

[0043] However, significant toxicity occurred following infusion into syngeneic mouse hosts using these CAR-T constructs that bind to and are activated by natural ligands of the natural NKG2D receptor. Signs of toxicity including poor physical condition, hunched posture, labored breathing, and decreased core body temperature were observed in tumor-bearing and tumor-free mice treated with NKG2D-based CAR-T cells compared to untreated control mice. The severity of NKG2D-CAR-T cell toxicity varied, with NKG2Dζ10 being severely toxic, NKG2D28ζ showing intermediate toxicity, and NKG2Dζ being tolerable. Clinical toxicity and mortality rates were exacerbated when mice were treated with chemotherapy prior to adoptive transfer of T cells expressing either NKG2D-CAR (VanSeggelen et al. 2015). Chemotherapy and radiation are known to induce NKG2D ligands in otherwise healthy tissues (Xiulong Xu, Geetha S Rao, Veronika Groh, Thomas Spies, Paolo Gattuso, Howard L Kaufman, Janet Plate and Richard A Prinz. Major histocompatibility complex class I-related chain A/B (MICA/B) expression in tumor tissue and serum of pancreatic cancer: Role of uric acid accumulation in gemcitabine-induced MICA/B expression. BMC Cancer 2011, 11:194 doi:10.1186/1471-2407-11-194; Gannagé M, Buzyn A, Bogiatzi SI, Lambert M, Soumelis V, Dal Cortivo L, Cavazzana-Calvo M, Brousse N, Caillat-Zucman Induction of NKG2D ligands by gamma radiation and tumor necrosis factor-alpha may participate in the tissue damage during acute graft-versus-host disease. Transplantation. 2008 Mar 27;85(6):911-5. doi: 10.1097/TP.0b013e31816691ef.). Further characterization revealed that toxicity was consistent with a systemic cytokine storm and lethal levels of inflammation in the lungs. These data warn that extreme caution should be exercised when using natural NKG2D ligands for targeted immunotherapy and indicate that upregulation of T cell expression of potent activating CARs may be detrimental in vivo ( VanSeggelen et al. 2015).

[0044] CAR T cells, CAR NK cells, and macrophages containing ectodomains of non-natural NKG2D receptors that do not bind or only weakly bind natural NKG2D ligands do not undergo the above form of activation and thus may not be as toxigenic as a cell expressing a CAR based on the natural NKG2D receptor. Furthermore, the ectodomains of non-natural NKG2D receptors on cells are not down-regulated by natural NKG2D ligands in soluble format or 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 bearing ectodomains of non-natural NKG2D receptors are attracted by the bispecific molecules with cognate non-natural α1-α2 domains of the present invention and its heterologous targeting motif, which has found and bound its intended target, the CAR is activated and the effector functions of the CAR cell are exerted. The effector functions of the CAR-T cell, CAR NK cell and CAR macrophage cell can terminate or impair the viability or function of the target cell. The target cell can include a malignant cell, an immunosuppressive tumor cell, a cell contributing to an autoimmune disease, a cell infected with a virus, such as, but not limited to, HIV, hepatitis virus, HTLV-1, CMV, EBV and other herpes viruses.

[0045] Since CAR-T cells or CAR-NK cells containing ectodomains of the non-natural NKG2D receptor are not activated except in the presence of a recruited bispecific molecule containing the related non-natural α1-α2 domains, their activation can be controlled by administering bispecific molecules that, as biopharmaceuticals, can exhibit pharmacokinetics and pharmacodynamics well known in the art. In the event that an adverse event occurs, the clinician can simply modify the dosing regimen of the administered bispecific molecule, rather than being forced to deploy an induced 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 target the emergence of resistance and escape in tumor or virus-infected cells following loss of the target antigen, without the need to derive, expand, and infuse multiple different autologous CAR cells (Gill & June, 2015) . Since all CAR constructs can be identical for all CAR cells and the targeting specificity is determined simply by the targeting motif of the resulting bispecific molecule of the present invention, manufacturing processes can be simplified and less expensive.

[0046] Numerous viruses have evolved with mechanisms to evade killing of their host cells by the natural immune surveillance system, particularly NKG2D-dependent components. For example, adenovirus, cytomegalovirus (CMV), herpes viruses, HIV, human T-cell lymphoma virus-1 (HTLV-1), and papilloma viruses all have one or more mechanisms. Such viruses can express viral antigens on the surface of host cells they infect, epitopes of which can serve as virus-specific molecular targets for binding by antibodies, antibody fragments, or other molecular targeting motifs. These cell-surface-exposed molecular targets are attractive as targets for antibodies or adoptive cellular therapies (ACT) to prevent the spread of viral infection or to treat viral infection by killing virus-infected cells.

[0047]Latency HIV-1 is produced early during acute infection and is initially found in memory CD4+ T cells. This reservoir, although nearly transcriptionally silent, is fully capable of producing infectious virus when the host cell is reactivated by antigen or cytokine stimulation or when antiretroviral therapy (ART) is interrupted. The latent HIV reservoir is primarily found in lymphoid tissues, where >98% of CD4+ T cells reside. Although ART is able to suppress viral replication, it is unable to eradicate latent reservoirs (Ruelas, D.S. and W.C. Greene,An integrated overview of HIV-1 latency.Cell, 2013.155(3): p. 519–29.). Efforts to clear latent HIV-1 initially focused on reactivating latent proviruses using cytokines or T-cell receptor activating agents. However, these approaches resulted in severe side effects and had low efficacy. The so-called “shock and kill” approach instead involves reactivating transcriptionally silent proviruses through the administration of latency reversal agents (LRAs), which are chemical compounds that can induce HIV-1 transcription (Cary, D.C., K. Fujinaga, and B.M. Peterlin,Molecular mechanisms of HIV latency.J Clin Invest, 2016.126(2): p. 448-54.). Following reactivation of latent viruses, the HIV envelope glycoprotein, gp160, is expressed on the surface of the activated cell, processed to gp120 and gp41. The V1, V2, V3, C1, C2 domains and the N segment of gp120 provide attractive targets for attacking HIV-infected cells using neutralizing antibodies and, as described herein, CAR-T cells.

[0048] Following reactivation of latently infected cells, the prediction was that these cells would produce viruses (which could be stopped by ART) and that these cells would die via apoptosis due to viral cytopathic effects, thus reducing the size of the latent reservoir. Testing of this hypothesis showed that reactivated cells do not die and the size of the latent reservoir is not reduced (Shan L, Deng K, Shroff NS, Durand CM, Rabi SA, Yang HC, Zhang H, Margolick JB, Blankson JN, Siliciano RF, Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation . Immunity. 2012; 36 (3) p.491-501.). Two major problems still persist following reservoir reactivation. The first concerns the emergence of viruses that are resistant to killing by CTLs (Deng K, Pertea M, Rongvaux A, Wang L, Durand CM, Ghiaur G, Lai J, McHugh HL, Hao H, Zhang H, JB, Gurer C, Murphy AJ, Valenzuela DM, Yancopoulos GD, Deeks SG, Strowig T, Kumar P, Siliciano JD, Salzberg SL, Flavell RA, Shan L, Siliciano RF Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations. Nature. 2015 Jan 15; 517 (7534) p. 381-5). This is a common problem in chronically infected individuals who were not treated with ART during the first 6 months of infection (the majority of chronically infected individuals). The second problem arises from the impact of HIV-associated chronic inflammation on CTLs, leading to CTL exhaustion (Cella M, Presti R, Vermi W, Lavender K, Turnbull E, Ochsenbauer-Jambor C, Kappes JC, Ferrari G, Kessels L, Williams I; CHAVI Clinical Core B, McMichael AJ, Haynes BF, Borrow P, Colonna M; NIAID Center for HIV/AIDS Vaccine Immunology. Loss of DNAM-1 contributes to CD8+ T-cell exhaustion in chronic HIV- 1 infection . Eur J Immunol. 2010 Apr; 40 (4): p. 949-54). It seems likely that new ways of killing reactivated reservoir cells that avoid the problems of viral persistence and cellular exhaustion are needed. The present inventors have proposed the engineering of convertible CAR-T cells that take advantage of broadly neutralizing HIV antibodies to target CTLs to destroy the reactivated cell reservoir.

[0049] Thus, the present invention increases the diversity and utility of this remarkable, highly promising immunological method for controlling cancer or viral infections using CAR-T cells, CAR NK cells and/or macrophages, while overcoming many of these current, known ACT difficulties.

[0050] As used herein, "peptide,""polypeptide," and "protein" are used interchangeably; and a "heterologous molecule,""heterologouspeptide,""heterologoussequence," or "heterologous atom" is a molecule, peptide, nucleic acid, or amino acid sequence or atom, respectively, that is not naturally occurring or is not normally found in physical association with the molecule in question. As used herein, "non-naturally occurring" and "modified" are used interchangeably. As used herein, "natural" and "native" are used interchangeably, and "NKG2D" and "NKG2D receptor" are used interchangeably. The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they have the desired biological activity. "Antibody fragments" comprise a portion of an intact antibody, preferably comprising its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment(s).

[0051] The term "comprising," when used interchangeably with "including,""containing," or "characterized by," is an inclusive or open-ended expression and does not exclude additional, unrecited elements or method steps. The phrase "consisting of" excludes any element, step, or ingredient not recited in a claim. The phrase "consisting essentially of" limits the scope of a claim to the recited materials or steps and to materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. The present disclosure includes embodiments of the inventive compositions and methods that fall within the scope of each of these phrases. Thus, a composition or method comprising the recited elements or steps includes particular embodiments in which the composition or method consists essentially of or consists of those elements or steps.

[0052] The entire contents of all references cited in this specification are hereby incorporated by reference, whether specifically incorporated beforehand or not. As used herein, each singular term is intended to include both singular and plural forms.

[0053] Having now fully described the invention, it will be apparent to those skilled in the art 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. While this invention has been described in connection with specific embodiments thereof, it will be understood that further modifications thereof are possible. This description is intended to cover any variations, uses, or adaptations of the invention, following generally the principles of the invention and including such departures from the present description as are within the known or customary practice in the art to which the invention pertains and can be applied to the essential features previously indicated herein.

[0054] Example 1 (Modified α1-α2 domains of NKG2D ligands.) Examples exist of polypeptides tethered to NKG2DLs that have been modified to greatly enhance their binding affinity to the human NKG2D receptor. The α1-α2 domain of MIC proteins is NKG2DL for the NKG2D receptor. This affinity is sufficient to physiologically activate NK cells and to stimulate lysis of cells expressing native full-length MIC proteins irreversibly tethered to the two-dimensional surface of the target cell plasma membrane (Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, Spies T, Science. 1999 Jul 30;285(5428):727-9.). However, since the engineered soluble MIC proteins of the present invention reversibly bind specific target antigens on the surface of target cells, the binding affinity of the engineered soluble MIC protein to NKG2D can directly affect the stability of the soluble MIC-dependent complex formed between NK cells and cells expressing target antigens. Especially, if the affinity between sMICA and NKG2D is increased by significantly slower dissociation rate or detachment rate of the modified sMICA from NKG2D, it is expected that the NK cell-based killing will be greater at lower densities of soluble MIC molecules bound to the target cell. Prior to the present invention, no α1-α2 mutations have been identified that alter the killing activity of soluble MIC proteins or significantly reduce the binding off rate to increase the affinity of MIC proteins for NKG2D. Computational design efforts have shown that three mutations in the α1-α2 domain of wild-type MICA: N69W, K152E, and K154D (WED-MICA) in combination can modestly affect NKG2D binding affinity by affecting the stability of unbound MICA and thus its rate of association or rate of onset of binding to NKG2D (Lengyel CS, Willis LJ, Mann P, Baker D, Kortemme T, Strong RK, McFarland BJ. J Biol Chem. 2007 Oct 19;282(42):30658-66. Epub 2007 Aug 8). A subsequent extensive computational design effort by the same group, scanning through iterative computations the 22 amino acid positions of MICA theoretically in contact with NKG2D, consistent with published structural descriptions (Li P, Morris DL, Willcox BE, Steinle A, Spies T, Strong RK., Nat Immunol. 2001 May;2(5):443-451), showed experimentally that, when combined with the previously designed 3 changes, additional rational, iterative computational design of MICA quantitatively altered its affinity for NKG2D from weak (Kd ~2.5 μM) to moderately tight (Kd=51 nM) using only seven combined mutations (Henager, Samuel H., 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, 2102, Combining different design strategies for rational affinity maturation of the MICA-NKG2D interface. Protein Science 21:1396–1402). In contrast, the experimental method of the present invention experimentally selected amino acid modifications of MICA that slowed the rate of dissociation between the α1-α2 domain of MICA and NKG2D, starting with MICA stabilized by the 3 WED substitutions from Lengyel et al (Lengyel CS, Willis LJ, Mann P, Baker D, Kortemme T, Strong RK, McFarland BJ., J Biol Chem. 2007 Oct 19;282(42):30658–66. Epub 2007 Aug 8).

[0055] This example relates to the modification of the binding affinity of NKG2D for soluble MIC proteins by engineering specific mutations at selected amino acid positions within the α1-α2 domain that affect the kinetics of the binding off rate and thus alter the NK cell-mediated killing activity of inventive non-natural, target MIC molecules.

[0056] To construct soluble non-natural α1-α2 domains with altered affinity for NKG2D, 57 residues in the α1-α2 domain were selected for extensive mutagenesis (Figure 12). Synthetic DNA libraries encoding the α1-α2 domain and containing mutagenic NNK codons at each of the 57 amino acid positions were synthesized, individually cloned as fusion proteins with the minor coat protein pIII of phage M13, and phage particles displaying mutagenized α1-α2 variants were produced in E. coli SS320 cells according to standard methods (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, CF, 3rd. (2011) Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, Cold Spring Harbor protocols 2011 ). α1-α2 phage libraries were screened for increased binding affinity using recombinant biotinylated NKG2D as the target antigen and subjected to cyclic repeated rounds of targeted extended binding, extended washing, and elution of phage clones to select high-affinity variants enriched for slow off-rates or binding switch-off. A group of specific amino acid mutations occurred at high frequency at 6 positions in α1-α2 and were selected as preferred amino acid substitutions with enhanced NKG2D binding affinity (Figure 3, Table 1).

[0057] Table 1. Selected affinity mutations at the indicated 6 amino acid positions of the α1-α2 MIC domain. The amino acids from SEQ ID NO.: 20 at each of the 6 positions are shown in bold in the first row of the table. Identified affinity mutations are listed in decreasing frequency from top to bottom. All amino acids are represented by their single-letter IUPAC abbreviated names.

S20 G68 K125 E152 H161 Q166 P L L T R F T F R V S S D S F G A H A A T F K Y L Y A Y G W N I N A L V E V Q F L T Y D Y M W I I S N S H M P

[0058] The present inventors synthesized DNA polynucleotides (SEQ ID NO. 21-24) encoding the α1-α2 domains from 4 representative variants 15, 16, 17, 18, containing various combinations of specific open mutations (Table 2).

[0059] Table 2. Sequences of specific α1-α2 domain variants. Specific amino acid substitutions for variants 15, 16, 17, and 18 (SEQ ID NOs.: 25-28, respectively) are listed relative to the amino acids of SEQ ID NO.: 20 shown in bold. All amino acids are represented by their single-letter IUPAC abbreviated names.

Option SEQ ID NO.: S20 G68 K125 N161 15 31 S G N R 16 32 S G L R 17 33 S L L R 18 34 P L L R

[0060] For the NKG2DL in the above example, the present inventors directly attached heterologous molecules, such as a polypeptide, to each of the 4 modified α1-α2 NKG2DLs using a linker peptide. Four His-tagged proteins (SEQ ID NOs.: 25-28) consisting of the modified NKG2DLs with attached heterologous molecules were expressed in insect cells and purified to characterize their NKG2D binding affinity and binding kinetic parameters. Using a competitive binding ELISA, the present inventors determined the relative NKG2D binding affinity of the 4 modified α1-α2 variants. Soluble wild-type (WT) NKG2DL, sMICA protein, was coated onto all wells of a maxisorp ELISA plate to provide a binding partner for the human NKG2D-Fc reagent. Solutions of the four α1-α2 variants, as well as the WT and WED α1-α2 domains (SEQ ID NO.: 20), were titrated in ELISA wells and allowed to competitively inhibit the binding of 2 nM human NKG2D-Fc to WT sMICA coated on the plate. The level of human NKG2D-Fc bound to WT NKG2DL on the plate was detected using an anti-Fc-HRP antibody. Figure 13, panel A, shows variants 16, 17 and 18 having IC ₅₀ values of 0.7, 0.6, 0.5 nM, while variant 15 had an IC ₅₀ value of 1.7 nM, all had significantly better binding to NKG2D, 27, 32, 38 and 11 times better than NKG2DL WT, respectively, as well as significantly better than WED-MICA (Table 3).

[0061] Table 3. Equilibrium and kinetic parameters of binding for variants α1-α2. IC ₅₀ values were derived from 4-parameter curve fits to competitive binding titrations (Figure 12), and kinetic binding parameters were derived from single exponential curve fits to binding kinetics (Figure 13). Equilibrium binding constants (K _d ) were derived from the kinetic binding parameters using the equation K _d = k _OFF /k _ON .

Kinetic parameters of binding Option α1-α2 IC ₅₀ (nM) k _ON (M ^-1 s ^-1 ) k _OFF (s ^-1 ) K _d (nM) WT 19.4 1.3×10 ⁵ 1.8×10 ^-3 13.8 WED 4.4 2.9×10 ⁵ 1.7×10 ^-3 5.9 15 1.7 0.7×10 ⁵ 1.1×10 ^-4 1.5 18 0.7 2.0×10 ⁵ 0.9×10 ^-4 0.5 1? 0.6 2.0×10 ⁵ 0.7×10 ^-4 0.4 18 0.5 2.3×10 ⁵ 0.9×10 ^-4 0.4

[0062] Importantly, the relative IC ₅₀ differences also resulted in improved binding to murine NKG2D-Fc and demonstrated the ability to improve binding of soluble, modified α1-α2 domains across human and non-human NKG2D receptors, an important property for preclinical drug development.

[0063] To understand the kinetic basis for the altered affinity, both the binding and dissociation rates for the binding of the α1-α2 NKG2DL variants to a surface coated with biotinylated human NKG2D were measured using biolayer interferometry (Octet) at 100 nM of each of the modified α1-α2 proteins. Consistent with the IC ₅₀ ELISA results, variants 16, 17, and 18 each showed significant decreases in dissociation rates (18-fold relative to WT), which was largely responsible for the increase in affinity (~30-fold relative to WT a1-a2; Table 3). Although variant 15 showed a similar slow off-rate as 16, 17, and 18, its binding rate was reduced, resulting in an affinity stronger than WT but weaker than variants 16, 17, and 18. Since the only difference between variant 15 (SEQ ID NO.:25) and 16 (SEQ ID NO.:26) was K125N versus K125L, the mutation at position 125 clearly altered the binding rate, while the reduced off-rate was attributed to the H161R mutation. Thus, while a selected group of NKG2DL mutations (Table 1) utilized a proportion of the increase in α1-α2 affinity for NKG2D via a significant decrease in the off-rate, specific substitutions also altered the binding rate, resulting in a range of gradual increases in affinity that we have shown to have differential activity in NK cell-mediated killing assays, as described below.

[0064] The ability of affinity variants α1-α2 to redirect NK cell-mediated lysis of FGFR3-expressing target cells was demonstrated in vitro in a calcein release assay. The human natural killer (NK) cell line, NKL, was co-cultured with calcein-loaded P815 target cells ectopically expressing FGFR3 and titrated using soluble engineered MIC proteins. The results in Figure 15 showed that the killing potency of the FGFR3-specific soluble MIC variants correlated with the affinity of their engineered α1-α2. Specifically, variants 16, 17, and 18 showed ~15-fold greater killing than WT at 0.78 nM. WED-MICA (SEQ ID NO.:20) was only slightly better than WT. Thus, the present invention describes amino acid substitutions in the α1-α2 domain that increased NKG2D binding affinity by decreasing the rate of binding switch-off of soluble MIC protein to human NKG2D and hence resulted in predictably increased killing activity. WED-MICA, which showed slightly greater affinity than WT MICA for NKG2D due to an increase in binding rate rather than a decrease in off-rate, did not show significant improvement in target cell killing. Furthermore, WED-MICA showed significantly weaker binding to mouse NKG2D even than WT MICA, while variants 15, 16, 17 and 18 each showed greater affinity for both human and mouse NKG2D.

[0065] These affinity variants α1-α2 NKG2DL 15, 16, 17 and 18 enhanced the binding affinity of the attached polypeptide to the NKG2D receptor and thus enhanced NK cell-mediated lysis of target cells.

[0066] Example 2. (Non-natural α1-α2 domains of NKG2D ligands and the related non-natural NKG2D receptors to which they bind)

[0067] The α1-α2 domain of MICA and other NKG2D ligands bind the NKG2D receptor at a known specific site (Li et al 2001; Benjamin J. McFarland, Tanja Kortemme, Shuyuarn F. Yu, David Baker, and Roland K. Strong. Symmetry Recognizing Asymmetry: Analysis of the Interactions between the C-Type Lectin-like Immunoreceptor NKG2D and MHC Class I-like Ligands. Structure, Vol. 11, 411-422, April, 2003) and drive the activation of the NKG2D receptor-bearing immune cell, which subsequently kills target cells displaying MICA or other ligands. The present inventors used phage display to construct unnatural α1-α2 MICA domains through extensive mutagenesis at 57 specific sites likely involved in NKG2D binding (Figure 16). Synthetic DNA libraries encoding the α1-α2 domain and containing mutagenic NNK codons at each of their 57 amino acid positions were synthesized, individually cloned as fusion proteins with the minor coat protein pIII of phage M13, and phage particles displaying mutagenized α1-α2 variants were produced in E. coli SS320 cells according to standard methods (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, CF, 3 ^rd , 2011. Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, Cold Spring Harbor protocols 2011 ). Libraries of α1-α2 phage were screened for increased binding affinity using recombinant biotinylated NKG2D as the target antigen and subjected to cyclic repeated rounds of targeted extended binding, extended washing, and elution of phage clones to select high-affinity variants enriched for slow off-rates or binding switch-off. A panel of specific amino acid mutations at 9 positions in the α1-α2 domain were selected as preferred sites of amino acid substitutions with enhanced NKG2D binding affinity. We synthesized DNA polynucleotides encoding the α1-α2 domains of 8 representative variants (SEQ ID NOs: 29-36) containing different combinations of the specific mutations (Table 4).

[0068] Table 4. Variants of the non-natural α1-α2 domain selected for increased affinity for the natural NKG2D receptor and the MICwed variant described previously (McFarland et al., 2003). The positions of the indicated amino acid substitutions are given with reference to the residue positions in SEQ ID NO.: 7, and the conventional names of the variants and their SEQ ID NOs are provided.

TABLE 4 ak # in wt MICA 20 68 69 125 152 154 158 161 166 option a1a2 SEQ ID NO. wt MICA 42 S G N TO TO TO H H Q MICwed 55 S G W TO E D H H Q DSM20 56 S A W L Q D R H F DSM25 57 S G W L E D H R S DSM27 58 S G W L TO TO H R S DSM28 59 S G N L TO TO H R S DSM42 60 S G W L E D H R Q DSM48 61 S G W L A D I R A DSM49 62 T Q W TO F D R T T

[0069] DNA polynucleotides encoding the 8 α1-α2 domain variants were amplified using PCR primers (SEQ ID NOs.: 37-38). Using the restriction enzymes Blp1 and Sap1, each was subcloned into a His-tagged α1-α2-α3-Fv fusion protein expression construct (SEQ ID NO.: 39) to replace the sequence encoding the native (wt) α1-α2 sequences with the mutant α1-α2 sequences. The 9 fusion proteins (SEQ ID NOs.: 40-48) were expressed in 293 cells (Expi293™ Expression System, Life Technologies, Thermo Fisher, Inc.) and affinity purified using Ni affinity chromatography (HisTrap HP, GE Healthcare Life Sciences).

[0070] To construct NKG2D receptor proteins, we synthesized DNA encoding the extracellular domain ("ectodomain") of the wild-type receptor (SEQ ID NO: 49) and used PCR primers (SEQ ID NOs: 50-51) and XbaI and BamHI sites to clone the synthetic DNA into an expression vector with an N-terminal His-avitag tag (SEQ ID NO: 78). His-avitag-native NKG2D (SEQ ID NO: 52) was transiently expressed in 293 cells and purified using Ni affinity chromatography. After purification, NKG2D proteins were site-specifically biotinylated using BirA to attach a biotin moiety to the avitag sequence (BirA Biotin Protein Ligase Standard Reaction Kit, Avidity, LLC, Aurora, CO).

[0071] To characterize and compare the kinetic parameters of binding of the native and 8 variant α1-α2 domains to native NKG2D, we measured their binding to a surface coated with biotinylated native NKG2D ectodomain using biolayer interferometry (Octet) at 100 nM of each α1-α2-α3-Fv fusion protein. The results are shown in Table 5.

[0072] Table 5: Kinetic parameters for binding of α3-Fv fusion proteins with the wild-type (wt or native) domain and 8 α1-α2 domain variants to native NKG2D. MICwed-Fv was tested in this case in 2 separate Octet assays, one comparing with the α3-Fv fusion protein with the wt α1-α2 domain and the other comparing with α3-Fv fusion proteins with 7 other non-native α1-α2 domains. The common names of each of the α1-α2 domain variants and the SEQ ID NO. The α3-Fv proteins fused to them are presented along with their affinity (Kd) values in moles (M), binding rates (kon) in inverse mole-seconds (1/Ms), and dissociation or debinding rates (kdis) in inverse seconds.

TABLE 5 option a1a2 SEQ ID NO.: Kd (M) kon (1/Ms) kdis (1/s) wt MICA-Fv 66 1,38E-08 1.30E+05 1,80E-03 MICwed-Fv, assay A 67 5,90E-09 2.90E+05 1,70E-03 MICwed-Fv, assay B 67 1,55E-08 2.01E+05 3,12E-03 MICv20-Fv 68 8.51E-11 3.59E+05 3.05E-05 MICv25-Fv 69 6,16E-11 4.67E+05 2,88E-05 MICv27-Fv 70 4.11E-10 2.08E+05 8.54E-05 MICv28-Fv 71 3.30E-10 2.46E+05 7.03E-05 MICv42-Fv 72 1.09E-10 3.47E+05 3.78E-05 MICv48-Fv 73 2,44E-10 5.95E+05 1,45E-04 MICv49-Fv 74 7.46E-10 3.70E+04 2,76E-05

[0073] As shown in Table 5, the selected α1-α2 domain mutations as fusion proteins with the heterologous α3-Fv polypeptides of SEQ ID NOs.: 42-48 increased the affinity of the α1-α2 domain for native NKG2D by significantly decreasing the off-rate. The off-rate ranged from 20 to more than 100 times slower than that of wt (SEQ ID NO.: 40) and the previously described MICwed α1-α2 domain variant (SEQ ID NO.: 41).

[0074] In this example of the present invention, the inventors further showed, as described below, that the non-natural α1-α2 domain (DSM25, SEQ ID NO.:31, Table 4), which in the form of the fusion protein α1-α2-α3-Fv had a high affinity for and a very slow off-rate from natural NKG2D (Table 2; SEQ ID NO.:43), had a tight binding affinity to a non-natural NKG2D receptor containing a specific mutation that abolished its binding to natural NKG2D ligands. Other authors have shown that mutations at tyrosine 152 and tyrosine 199 in human NKG2D, the equivalent of positions 73 and 120 of the NKG2D ectodomain (SEQ ID NO.:49 and Figure 4), abolish binding to the natural ligand, MICA (David J. Culpepper, Michael K. Maddox1, 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).

[0075] To construct non-natural NKG2D receptor proteins, the present inventors used PCR primers (SEQ ID NOs.:50-51) to clone DNA encoding the natural NKG2D ectodomain (SEQ ID NO.:49) and insert it into an expression vector with an N-terminal His-avitag tag SEQ ID NO.:52 to generate His-avitag-NKG2D (SEQ ID NO.:53). Site-directed mutagenesis was performed on the natural NKG2D ectodomain DNA construct to introduce Y152A, Y199A, or Y152A plus Y199A mutations, and three non-natural human NKG2D variants were generated (SEQ ID NOs.:54-56, respectively). Native NKG2D and three non-native NKG2D mutants with His-avitag tags were transiently expressed in 293 cells and purified using Ni-affinity chromatography. After purification, NKG2D proteins were subjected to site-specific biotinylation using BirA to attach a biotin moiety to the avitag sequence (BirA Biotin-Protein Ligase Standard Reaction Kit, Avidity, LLC, Aurora, CO.).

[0076] To generate fusion proteins of heterologous α3-Fc polypeptides with the MICwed α1-α2 domain (SEQ ID NO.: 29) and the DSM25 α1-α2 domain (SEQ ID NO.: 31), DNA polynucleotides encoding the α1-α2 domains were amplified using PCR primers (SEQ ID NO.: 37-38). Using restriction enzymes XbaI and NcoI, each was subcloned into the α1-α2-α3-Fc fusion protein expression construct (SEQ ID NO.: 57) to replace the sequence encoding the native (wt) α1-α2 sequences with the mutant α1-α2 sequences. Three fusion proteins, MICA-Fc (SEQ ID NO.: 58), MICwed-Fc (SEQ ID NO.: 59), and MICv25-Fc (SEQ ID NO.: 60) were expressed in 293 cells (Expi293™ Expression System, Life Technologies, Thermo Fisher, Inc.) and affinity purified using protein A affinity chromatography (cat. no. 20334, Pierce Biotechnology, Rockford, IL).

[0077] In addition to the purification of the above three Fc fusion proteins, NKG2D-Fc ligand, MICB-Fc, ULBP1-Fc, ULBP2-Fc, ULBP3-Fc, and ULBP4-Fc fusion proteins were purchased from R&D Systems, Inc. (Minneapolis, MN). Binding of the various α1-α2-Fc domain fusion proteins to proteins with both the native and non-native NKG2D ectodomain was analyzed using a plate-based ELISA method. All native and non-native domain α1-α2-Fc fusion proteins were coated overnight at 4°C onto individual wells of 96-well Maxisorp plates using a coating concentration of 2 μg/mL in phosphate-buffered saline (PBS). Plates were washed 3 times in PBS/0.05% Tween20 at 20-22°C and blocked with 0.5% bovine serum albumin for 2 hours. Biotinylated natural and non-natural NKG2D receptor proteins were titrated against plate-bound NKG2D ligands for 2 hours at 20-22°C, washed 3 times with PBS/0.05% Tween20 at 20-22°C, and bound NKG2D proteins were subsequently detected using a streptavidin-HRP secondary detection step and developed using 1-Step Ultra TMB Elisa. The native form of the NKG2D ectodomain (SEQ ID NO.:49) was able to bind all α1-α2-Fc domain fusion proteins tested. The ligand with the non-natural α1-α2 domain MIC-v25 bound with the highest affinity (EC _{50 =} 14 nM), which was 8-fold better than MICwed and more than 100-fold better than all tested ligands with the natural α1-α2 domain. All tested ligands, both with natural and non-natural α1-α2 domains, lost binding to the Y199A mutant NKG2D receptor (SEQ ID NO.:55; Figure 18, panel B) and to the Y152A plus Y199A double mutant receptor (SEQ ID NO.:56). However, of all ligands tested with both natural and non-natural α1-α2 domains, only the non-natural α1-α2 domain (SEQ ID NO.:31) of MICv25-Fc (SEQ ID NO.:60) retained binding to the Y152A mutant of the NKG2D ectodomain (SEQ ID NO.:54) with an EC ₅₀ of 50 nM.

[0078] While the binding specificity of native NKG2D has been shown to favor high-affinity non-natural ligands, its strong binding to natural NKG2D ligands that are present on specific healthy tissues and many stressed tissues poses an unusual risk of toxicity when using current NKG2D CAR assays (VanSeggelen et al. 2015). The non-natural NKG2D receptor Y152A specifically bound to a single protein consisting of a high-affinity non-natural α1-α2 domain engineered for a markedly reduced off-rate. This prototypical example highlights the ability of the unnatural α1-α2 domains to bind unnatural NKG2D receptors, thus enabling selective control of unnatural NKG2D CARs using bispecific proteins containing the inventive unnatural α1-α2 domain of NKG2D ligands.

[0079] Example 3 (Modified α1-α2 domains of NKG2D ligands).

[0080] This embodiment relates to additional affinity variants of α1-α2 NKG2DL obtained by engineering the α1-α2 domains of ULBP proteins. The ULBP proteins contain α1-α2 domains that are NKG2D ligands capable of binding to the NKG2D receptor (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). This NKG2D binding affinity is sufficient to physiologically activate NK cells and to stimulate lysis of cells expressing native full-length ULBP proteins naturally and irreversibly tethered to two-dimensional surfaces of the target cell plasma membrane (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). However, since engineered soluble α1-α2 domains fused to heterologous polypeptides, in particular embodiments of the present invention, reversibly bind specific target antigens on the surface of a target cell, the binding affinity of engineered α1-α2 ULBP domains to NKG2D may directly affect the stability of the artificial synapse formed between NK cells and cells expressing target antigens, as already shown by engineered soluble MIC proteins (Examples 21-2). To diversify the repertoire of engineered non-natural α1-α2 domains as NKG2D ligands, ULBP proteins were used as a substrate or starting point for phage display-based modification of their binding affinity to NKG2D. Despite the structural homology observed between ULBP and MICA (Radaev, S., Rostro, B., Brooks, AG., Colonna, M., Sun, PD. (2001) Conformational plasticity revealed by the cocrystal structure of NKG2D and its class I MHC-like Ligand ULBP3. Immunity 15, 1039-49.), the sequence homology is <50% for the α1-α2 domains of ULBP, relative to MICA. Therefore, we looked for codon position identities in the α1-α2 domains of ULBP that improve NKG2D binding affinity.

[0081] To construct soluble, non-natural α1-α2 domains, ULBP, ULBP2, and ULBP3 proteins were selected for phage display and selection of mutants with high affinity NKG2D binding. Sixty amino acid positions in the α1-α2 domain of ULBP2 (SEQ ID NO: 61) and thirty-six amino acid positions in the α1-α2 domain of ULBP3 (SEQ ID NO: 62) were selected for extensive mutagenesis. In addition, conserved cysteine to serine mutations were made at C8S in ULBP2 (SEQ ID NO: 61) and C103S in ULBP3 (SEQ ID NO: 62), eliminating unpaired free cysteine residues to increase the stability and function of NKG2D ligands with attached polypeptides, as well as to improve phage panning processes. Synthetic DNA libraries encoding these α1-α2 domains with cysteine to serine modification and containing mutagenic NNK codons at each of the selected amino acid positions were synthesized individually; cloned as fusion proteins with the minor coat protein pIII of phage M13; and phage particles displaying mutagenized α1-α2 variants of ULBP2 or ULBP3 were produced in E. coli SS320 cells according to standard methods (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, CF, 3rd. (2011). Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, Cold Spring Harbor protocols 2011 ). α1-α2 phage display libraries were screened for increased binding affinity to NKG2D using human NKG2D-Fc as the target protein, and subjected to cyclic repeated rounds of targeted extended binding, extended washing, and elution of phage clones to select high-affinity variants enriched for slow off-rates or off-rates of binding. For ULBP2, specific amino acid mutations were found at high frequencies at positions R80, V151, V152, and A153 in α1-α2, and identified as preferred amino acid substitutions with enhanced NKG2D binding affinity (Figure 19, panel A; and Table 6).

[0082] Table 6. Selected affinity mutations at the indicated 4 amino acid positions of the ULBP2 α1-α2 domain. The amino acids from SEQ ID NO: 61 at each of the 4 positions are shown in bold in the first row of the table. Identified affinity mutations are listed in decreasing frequency from top to bottom. All amino acids are represented by their single-letter IUPAC abbreviated names.

R80 V151 V152 A153 L D L E W E W TO V Q G F TO P I N S R A T E P T

[0083] For ULBP3, specific amino acid mutations were found at various locations relative to ULBP2. Positions R162 and K165 in the α1-α2 domain of ULBP3 contained specific mutations that were identified as preferred amino acid substitutions with enhanced NKG2D binding affinity (Table 7). These modified non-natural α1-α2 domains derived from ULBP2 and ULBP3 can be used to enhance NKG2D binding in a variety of therapeutic formats, in the form of single proteins or fusion proteins with heterologous peptides or polypeptides.

[0084] Table 7. Selected affinity mutations at the indicated 2 amino acid positions of the ULBP3 α1-α2 domain. The amino acids from SEQ ID NO: 62 at each of the 2 positions are shown in bold in the first row of the table. Identified affinity mutations are listed with decreasing frequency from top to bottom. All amino acids are represented by their single-letter IUPAC abbreviated names.

K162 K165 G S A P Y A T H N Q G

[0085] Example 4 (Binding and cytolysis via modified α1-α2 ULBP domains fused to antibody peptides)

[0086] The following example relates to the coupling of anti-NKG2DL antibody polypeptides that have been modified to significantly enhance their binding affinity to the human and murine NKG2D receptor. The α1-α2 domain of each ULBP protein is the natural ligand for the NKG2D receptor, i.e., NKG2DL. The antibodies are highly stable glycoproteins consisting of two large heavy chains and two small light chains (Figure 1). There was no IgG antibody format in the art that could directly activate immunocytes using the non-natural α1-α2 domains of ULBPs, which bind more tightly than the native ULBP domains to the NKG2D receptor. In addition, the α1-α2 domains of ULBP provide alternative NKG2DLs for the construction of antibody fusion proteins that may have differential properties in vivo relative to the α1-α2 domains of MICA. For example, an in vivo anti-drug antibody response to the α1-α2 domains of MICA within an antibody fusion protein is unlikely to be reactive with or interfere with modified α1-α2 domains of ULBP, due to the low sequence homology between the α1-α2 domains of ULBP and MICA (Figure 5). This example shows that fusion proteins between engineered NKG2D ULBP α1-α2 ligands (Table 6 and 7) and the heavy chain of an IgG molecule have enhanced NKG2D binding and target cell killing relative to natural NKG2D ULBP α1-α2 ligands. This further demonstrates the utility of fusion proteins of modified α1-α2 domains with heterologous proteins or peptides.

[0087] To generate engineered α1-α2 domain antibody fusion proteins, DNA sequences encoding the modified C8S α1-α2 domains of ULBP2 (SEQ ID NO.: 61), variants R80W and V151D (SEQ ID NO.: 63 and 64, respectively) and the modified C103S α1-α2 domain of ULBP3 (SEQ ID NO.: 62), variant R162G (SEQ ID NO.: 65), were synthesized and cloned into C-terminal fusion proteins with the heavy chain sequence from a Her2-specific antibody (Carter, P., Presta, L., Gorman, CM., Ridgway, JB., Henner, D., Wong, WL., Rowland, AM., Kotts, C., Carver, ME., Shepard, HM. (1992) Proc Natl Acad Sci 15, 4285-9.). The resulting fusion proteins were cloned into the mammalian expression vector pD2509 and expressed with the light chain of the parent antibody to form paired full-length IgG antibodies. Transient expression was performed in HEK293 cells using the Expi293 expression system according to the manufacturer's protocol (Life Technologies) and purified using standard protein A affinity chromatography. Binding ELISAs performed on ULBP2 and ULBP3 heavy chain fusion proteins showed that modified ULBP2 fusion proteins (HC_R80W and HC_V151D) and UBLP3 fusion protein (HC_R162G) bound with higher affinity to human NKG2D relative to their corresponding native α1-α2 domains fused to the same heavy chain.

[0088] To characterize the target cell killing properties of the engineered ULBP-antibody fusion proteins, a human natural killer (NK) cell line, NKL, was co-cultured with calcein-loaded Her2-expressing SKBR3 target cells and titrated with the engineered antibody fusion proteins. The results showed that the enhanced cytolytic (killing) activity of the Her2-specific non-natural ULBP2 and non-natural ULBP3 α1-α2-antibody fusion proteins reflected the increased affinity of their engineered α1-α2 domains for NKG2D. Specifically, ULBP2 fusion variants HC_R80W and HC_V151D, and ULBP3 fusion variant HC_R162G, killed SKBR3 cells more efficiently than antibody fusion proteins containing either native α1-α2 domain. These data further demonstrated that modified α1-α2 variant-antibody fusion proteins represent a versatile platform for mediating tight binding of IgG molecules to NKG2D and for directing antigen-specific cell lysis.

[0089] Example 5 (Construction of orthogonal non-natural α1-α2 domains with selective binding to non-natural Y152A NKG2D)

[0090] Tools for selective control of CAR-T cell therapies are highly sought after to mitigate toxicity and improve tumor efficacy (Gill and June, op.cit.). Previous efforts have been made to develop CARs using the CD16 ectodomain, which can then be recruited via the Fc domain of therapeutic monoclonal antibodies, providing antibody-based control of CAR-T targeting (Chang et al., op.cit.). However, CD16-based CAR-T cells can recognize all endogenous antibody molecules in blood and tissues, and therapeutic antibodies used to control these cells may encounter interference due to endogenous CD16 receptors on NK cells. Both of these features pose problems with non-tumor-specific toxicity and poor pharmacokinetics, respectively.

[0091] To address these issues, the present inventors engineered non-natural NKG2D CAR-T cells that lack binding to all natural NKG2D ligands and can be controlled by binding to high-affinity non-natural α1-α2 domains, as described in Example 2. An additional requirement for the non-natural α1-α2 domains is to maintain high affinity for non-natural NKG2D and avoid binding to natural NKG2D domains. Thus, engineered α1-α2 domains with strong selectivity for unnatural NKG2D receptors over natural NKG2D represent an ideal system for selective targeting of unnatural NKG2D-CAR receptors, or any receptor or protein fused to unnatural NKG2D ectodomains that can be selectively recruited by unnatural α1-α2 domains.

[0092] The present inventors used phage display to construct orthogonal non-natural α1-α2 domains that exhibit selective binding to the NKG2D receptor Y152A. As a starting point, three non-natural α1-α2 domains with high affinity for native NKG2D were selected as seed domains for further mutagenesis and screening by phage display. Synthetic DNA libraries were generated for the individual α1-α2 domain variants DSM25, ULBP2 R80W, and ULBP3 R162G (SEQ ID NOs.: 31, 63, and 65), whereby codons for amino acid residues that, when bound, are located in close proximity to the Y152 position on the NKG2D receptor were replaced with NNK codons. The DSM 25 libraries consisted of NNK positions at residues 71-75 and 155-159, ULBP2 R80W libraries with NNK codons at positions 154-159, and ULBP3 R162G libraries with NNK codons at positions 155-159. The libraries were cloned as fusion proteins with the minor coat protein pIII of phage M13; and phage particles displaying mutagenized α1-α2 domain variants were produced in E. coli SS320 cells according to standard methods (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, CF, 3rd. (2011). Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, Cold Spring Harbor protocols 2011 ). The α1-α2 phage display libraries were sorted for high-affinity binding to the unnatural NKG2D receptor Y152A by selectively capturing phage clones bound to biotinylated Y152A NKG2D-Fc protein in the presence of non-biotinylated natural competitor NKG2D-Fc protein. Selective clones were enriched by subjecting them to multiple cyclic rounds of competitive selection using increasing concentrations of non-biotinylated native NKG2D-Fc.

[0093] After four rounds of selection, phage clones were sequenced to identify specific mutations in the NNK mutagenesis regions. Tables 8, 9, and 10 show the selected amino acid residues found to be prevalent for each α1-α2 domain obtained from the Y152A NKG2D selective screen.

[0094] Table 8. Selected mutations in DSM25 that resulted in Y152A-specific phage clones.

K71 D72 L73 R74 M75 T155 H156 Y157 H158 A159 T T L L R I G G G L L F L R S S S I D R H R L L R W

[0095] Table 9. Selected mutations in ULBP2 R80W that resulted in Y152A-specific phage clones.

M154 S155 F156 H157 YI58 F159 T M L E L W K M T V I W S I L Q T T Y R

[0096] Table 10. Selected mutations in ULBP3 R162G that resulted in Y152A-specific phage clones.

F155 F156 K157 M 158 V159 D L I R R W M Y L I R V T W Y L K L

[0097] To confirm phage clones having the proper selective binding, phages were produced for individual clones: MICA25.17, MICA25.18, ULBP2.S1, ULBP2.S2, ULBP2.S3, ULBP3.S1, and ULBP3.S2 (SEQ ID NOS: 66, 67, 68, 69, 70, 71, and 72, respectively) and titrated against Y152A or native NKG2D in a binding ELISA. Figure 23, panel AC, shows that all 7 phage clones had greater than 10-fold selective binding to non-native Y152A NKG2D compared to native or wild-type NKG2D.

[0098] To confirm that the Y152A-selective α1-α2 domain variants retain specific binding properties in the context of antibody fusion proteins, we cloned MICA25.17 and ULBP2.S3 as C-terminal fusion proteins with the heavy chain of an FGFR3-specific antibody described previously (Qing et al, 2009. op. cit.; SEQ ID NOs: 73 and 74, respectively). The resulting fusion proteins were cloned into the mammalian expression vector pD2509 and co-expressed with the light chain of the parent antibody as paired full-length IgG antibodies (R3 HC25.17 and R3 HC.U2S3). Transient expression was performed in HEK293 cells using the Expi293 expression system according to the manufacturer's protocol (Life Technologies) and purified using standard protein A affinity chromatography. ELISAs measuring the binding of the R3 HC25.17 and R3 HC.U2S3 α1-α2 heavy chain antibody fusion proteins to unnatural Y152A NKG2D and to natural NKG2D showed their significantly higher binding affinity for Y152A NKG2D relative to natural NKG2D (Figure 6, panels B and D). In contrast, the DSM25 and ULBP2 R80W antibody fusion proteins had preferential binding to natural NKG2D-Fc (Figure 6, panels A and C). Together, these data demonstrate the invention of non-natural α1-α2 orthogonal domains that have high affinity binding to non-natural NKG2D receptors and significantly reduced binding affinity to the natural NKG2D receptor. Furthermore, fused α1-α2 orthogonal domains to antibody polypeptides retained their selective binding properties and could be used to retarget non-natural NKG2D receptors to new antigens, such as in the context of CAR-T cells.

[0099] Example 6 (CAR-T cell targeting and killing activity using a non-natural NKG2D ectodomain was controlled using orthogonal α1-α2 domains fused to targeting antibodies).

[0100] To demonstrate the selective control of CAR-T cells engineered with a chimeric receptor with an introduced non-natural NKG2D ectodomain, we engineered CARs with either natural NKG2D or non-natural Y152A NKG2D ectodomains, based on previous work using 4-1BB/CD3-zeta CAR constructs (Campana Patent 8399645), fusing the corresponding NKG2D ectodomains to the CD8 hinge region (Figure 2) of the CAR. These constructs were cloned into a lentiviral vector and expressed in primary human CD8 positive T cells using lentiviral transduction. The resulting native NKG2D CAR T cells had specific cell killing activity in vitro , consistent with recognition of native MICA ligand expressed on target cells. Specifically, the results showed that while native NKG2D CAR T cells killed P1 cells expressing native MICA ligands, non-native Y152A NKG2D CAR T cells were significantly inactivated and had significantly reduced killing of MICA-expressing P1 cells. Furthermore, orthogonal α1-α2 antibody heavy chain fusion proteins, R3 HC25.17 and R3 HC.U2S3, selectively activated non-native Y152A CAR T cells to kill FGFR3-expressing P1 target cells but were unable to redirect the killing activity of native NKG2D CAR T cells. This was in contrast to the R3 HC25 and R3 HC.U2R80W α1-α2 heavy chain antibody fusion proteins, which were not selective for unnatural Y152A NKG2D and activated both natural and unnatural CAR-T cells to kill P1 target cells. These data showed that unnatural α1-α2 orthogonal domains engineered to selectively bind unnatural Y152A NKG2D specifically activated unnatural Y152A NKG2D CAR-T cells while avoiding natural NKG2D receptors.

[0101] Example 7 (Construction of orthogonal unnatural α1-α2 domains with selective binding to unnatural Y152A/Y199F NKG2D)

[0102] Other authors have shown that mutations at tyrosine 152 or tyrosine 199 in human NKG2D, equivalent to positions 73 and 120 of the NKG2D ectodomain (SEQ ID NO.:49), 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). The present inventors concluded that while mutation of either tyrosine residue strongly affected the ability of NKG2D to bind its natural ligands, simultaneous mutation of both tyrosine 152 (Y152) and tyrosine 199 (Y199) could virtually abolish the ability of the receptor to be attracted by all native ligands. The present inventors therefore decided to examine individual and combined Y152 and Y199 substitutions and characterize them for their biochemical behavior with the goal of identifying both single- and double-mutated variants that were unable to attract any natural ligands. These variants, which were also well expressed and assembled, were of particular interest because they represented inert ligands that could be more easily recovered for analysis.

[0103] The native (wild-type) NKG2D ectodomain (NKG2D.wt, SEQ ID NO: 49) and candidate variants of non-native NKG2D ectodomains (SEQ ID NOs: 75-92) - also termed "engineered NKG2D" or "eNKG2D", were cloned as fusion proteins with the C-terminus of human IgG1 Fc (Fab-less domains), via a short factor Xa recognition linker Ile-Glu-Gly-Arg (SEQ ID NO: 93) and interchangeably designated as Fc-NKG2D.wt or NKG2D.wt and Fc-eNKG2D or eNKG2D (SEQ ID NOs: 94-112). ^gBlocks® DNA fragments (Integrated DNA Technologies, San Diego, CA) corresponding to the MHCI signal sequence (SEQ ID NOs: 113 and 114), human IgG1 Fc with linker (SEQ ID NO: 115), and NKG2D ectodomain variants (SEQ ID NOs: 116-124) were synthesized and inserted into pD2610-V12 (ATUM, Newark, CA). DNA constructs to examine substitutions at Y152, Y199, or the Y152/Y199 combination mutations (Table 1) were transiently expressed in Expi293 ^™ cells (ThermoFisher Scientific, Waltham, MA), and secreted protein was purified by protein A affinity chromatography (cat. no. 20334, Pierce Biotechnology, Rockford, IL). Eluted material was characterized by size exclusion chromatography (SEC) on Akta Pur Superdex columns, and correctly collected, size-appropriate material was fractionated and separated from aggregate peaks prior to inclusion in analyses.

[0104]Characterization by means of SEC of the purified NKG2D.Y199A-Fc fusion protein revealed a composition of predominantly aggregated material (Figure 2). In contrast, both native Fc-NKG2D and Fc-NKG2D.Y152A fusion protein material were resolvable via a discrete, non-aggregated peak that was easily distinguished from the faster migrating aggregate. The effect of the Y199A mutation on aggregation was also evident for the Y152A/Y199A double mutant Fc-NKG2D fusion protein variant, showing that it had a predominant effect on protein misfolding (Figure 2). This aspect of incorporating Y199A with any combination of Y152 mutations into NKG2D variants thus presented a challenge to obtaining the material needed for further engineering attempts and raised concerns regarding assembly and cell surface presentation. Therefore, attempts were made to explore other substitutions at Y152 and Y199 that could be combined to produce a more robust molecule. Candidates with combined Y152 and Y199 mutations of eNKG2D were tested in the form of Fc fusion proteins and are characterized in detail (Table 1). In addition, all purified and expressed Fc-eNKG2D fusion protein candidates were profiled by SEC and their chromatograms revealed varying levels of aggregate formation (Figures 2 and 3, Table 1). Of the single amino acid substitutions tested at residue 152, alanine, serine, threonine, and valine all did not affect the assembly of the Fc-NKG2D molecule, although Y152-leucine (Y152L) resulted in highly aggregated material. Like alanine, neither glutamate nor aspartate were tolerated at position 199, although phenylalanine only moderately increased aggregate formation. Of the mutation combinations examined, Y152A/Y199F, Y152S/Y199F, Y152T/Y199F, and Y152F/Y199F had no negative effect on formation of the desired dimer, while other combinations resulted in increased aggregation.

[0105] Example 8: (Preparation of bispecific antibody-based molecules, “MicAbodies”, using variants of the non-natural NKG2D ligand)

[0106] To generate variants of non-natural MicA fused to human IgG1, DNA polynucleotides encoding the α1-α2 domains, such as those from MICwed (SEQ ID NO: 7) and MIC25 (SEQ ID NO: 31), were amplified by PCR using primers that also introduced a polynucleotide encoding either an APTSSSGGGGS linker for fusion to the C-terminal kappa light chain (SEQ ID NO: 135) or a GGGS linker for fusion to the C-terminal heavy chain of human IgG1 (SEQ ID NO: 136). In addition, two mutations were introduced into the CH2 domain of the heavy chain - D265A/N297A (Kabat numbering) - which reduce binding to all FcγR receptors, thus abolishing 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 (ULBP2.wt) without its GPI-linkage (SEQ ID NO: 61) was similarly cloned and fused to DNA polynucleotides encoding linkers and the heavy chain or light chain of IgG1. These bispecific antibodies - termed " ^MicAbodyTM " in the singular, "MicAbodies" in the plural - are bivalent for the fusion α1-α2 domain. Examples of antibodies used to generate MicAbodies for the purposes of the eNKG2D engineering study include, but are not limited to, trastuzumab (SEQ ID NOs: 137 and 138) and rituximab (SEQ ID NOs: 139 and 140) and are therefore named “trastuzumab-MicAbody” and “rituximab-MicAbody,” 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 recognizing 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, 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, IL), the eluted material was characterized by size exclusion chromatography (SEC) on Akta Pur Superdex columns, and fractionation was performed as needed. Additionally, SDS-PAGE analysis was performed on purified samples to confirm the expected molecular weights of the heavy chain fused and light chain fused molecules.

[0107] Example 9: (Identification of modified NK2GD variants that are unable to bind either to natural NKG2D binding ligands or to non-natural ligands that have enhanced binding to wild-type NKG2D)

[0108] The binding affinity of the α1-α2 variants to the extracellular domains of wild-type NKG2D and non-native eNKG2D proteins was analyzed using a plate-based ELISA. Each of the SEC-fractionated native Fc-NKG2D and non-native Fc-eNKG2D fusion proteins was coated overnight at 4°C onto individual wells of Nunc Maxisorp 96-well plates (Thermo Fisher Scientific, Waltham, MA) using a coating concentration of 1 μg/mL in phosphate-buffered saline (PBS). 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 natural or non-natural Fc-NKG2D fusion proteins for 60 min at 20-22°C in PBS/0.5% bovine serum albumin (BSA)/0.05% Tween-20 (PBS-BT), washed 3 times with PBS-T at 20-22°C, and bound bispecific proteins were detected using HRP-conjugated anti-human kappa antibody in PBS-BT (Abcam, Cambridge MA) and developed using 1- ^StepTM Ultra TMB ELISA Substrate Solution (Thermo Fisher Scientific, Waltham, MA). Binding of ULBP2.wt rituximab-MicAbody (SEQ ID NOS: 139 and 141) discriminated between wild-type NKG2D and eNKG2D variants with reduced binding to the latter, and the variant ligands - MICwed (SEQ ID NOS: 20 and 78) and MIC25 (SEQ ID NOS: 138 and 80) - were more stringent in identifying eNKG2D variants with abolished ligand binding. Binding behavior for each eNKG2D variant against all three bispecific ligands revealed combinations of NKG2D modifications that resulted in the greatest reduction in wild-type and variant ligand binding and allowed selection of leading inert NKG2D variants.

[0109] Additional biophysical analysis of eNKG2D variant binding to ligands was also performed using biolayer interferometry (BLI) using the FortéBio Octet System (all FortéBio 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). The ligands in MicAbody format were coupled to anti-human IgG Fc binding biosensor (AHC) tips. After establishing background values, the tips were exposed to a titration series of Fc-eNKG2D fusion proteins ranging from 300 nM to 0.41 nM and the binding/dissociation kinetics were monitored, with all steps performed in PBS-BT. The Fc-eNKG2D fusion proteins were then bound to AHC tips and MicAbodies were titrated to characterize the binding kinetics.

[0110] To determine the maximal response, as determined by binding of native NKG2D to either MICwed or MIC25, native Fc-NKG2D fusion proteins were bound to the AHC biosensors and 20 nM trastuzumab-MICwed or 20 nM trastuzumab-MIC25 MicAbodies were incubated for two minutes and then dissociation kinetics were monitored for 30 seconds. Binding assays were then performed under the same conditions using the Fc-eNKG2D fusion receptors as the binder, and the binding level for each eNKG2D was ranked as a percentage of the maximal binding response obtained by Fc-NKG2D.wt (Table 2). For MICwed, the responses for all single mutation Fc-eNKG2D variants except Y199F were reduced to 50%. Y199F retained 100% binding response. However, all double-mutant Fc-eNKG2D variants had completely abolished binding to MICwed. For MIC25, all single-mutant Fc-eNKG2D variants and Y152V/Y199F retained 100% binding response relative to wild-type Fc-NKG2D binding. However, binding was reduced to 50% for several of the double-mutant Fc-eNKG2D variants, including Y152A/Y199F, Y152S/Y199F, and Y152T/Y199F.

[0111]Tests ELISA using Fc-eNKG2D fusion proteins as binders was performed for ULBP2.wt, MICwed, MIC25 MicAbodies titrated starting from 300 nM. EC values₅₀were calculated, when possible, using GraphPad Prism (Table 11).

Table 11: EC ₅₀ values (nM) for the Fc-eNKG2D ELISA. nt=not tested; nb=no binding or very low binding even at 300 nM, thus EC ₅₀ value was not calculated

MicAbody Fc-eNKG2D ULBP2.wt MICwed MIC25 wt NKG2D.wt Y|Y 1.41 0,0067 ~0.0039 Y152 eNKG2D A|Y 27.86 4.30 0.0057 eNKG2D2 S|Y 34.78 4.16 0.0056 eNKG2D3 T|Y 31.14 4.33 0.0056 eNKG2D4 V|Y 35.78 4.84 ~0.0043 eNKG2D14 L|Y 87.63 9.39 0,010 Y199 eNKG2D1 Y|F 23.08 0.32 0.0048 eNKG2D10 Y|D n.t. n.t. n.t. eNKG2D11 Y|E n.t. n.t. n.t. Y152|Y199 eNKG2D5 A|F nb 280.5 0.79 eNKG2D6 L|F nb nb 0.37 eNKG2D7 S|F nb 347.3 20.94 eNKG2D8 T|F nb 570.6 4.51 eNKG2D9 V|F nb 90.0 0.43 eNKG2D15 F|F 57.05 31.3 0.046 eNKG2D12 D|D nb nb nb eNKG2D13 E|E nb nb nb

[0112] 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. While affinity was reduced for ULBP2 and MICwed MicAbodies, MIC25 binding to eNKG2D candidates was maintained when using all single-mutant eNKG2D candidates. However, all double-mutant eNKG2D candidates had abolished or significantly reduced binding to all three ligands - ULBP2, MICwed, and MIC25 - in Micabody formats.

[0113]Options eNKG2D 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 and ELISA (Tables 2 and 3). Additionally, eNKG2D 5, 7, and 8 had the lowest levels of aggregation, suggesting more robust protein assembly when expressed in 293T (Table 1). eNKG2D5 (SEQ ID NO: 102) was further examined for binding to wild-type ligands, in the form of MicAbodies bound to Octet AHC tips. The single mutant fc-NKG2D.Y152A (SEQ ID NO: 95) had reduced binding to all natural ligands relative to natural (SEQ ID NO: 94) NKG2D (Figure 5). The response curves for binding of eNKG2D5 (Y152A/Y199F) were even further reduced relative to Y152A eNKG2D. eNKG2D5 (Y152A/Y199F, hereafter referred to as “AF” or “NKG2D.AF”) was selected as the lead NKG2D variant for which cognate selective, orthogonal, non-natural ligands were designed.

[0114] Example 10: (Construction of orthogonal unnatural α1-α2 domains with selective binding to the unnatural ectodomain of NKG2D.AF)

[0115] The present inventors used phage display to construct orthogonal non-natural α1-α2 domains that selectively bind to the NKG2D.AF receptor (SEQ ID NO: 102). As a starting point, the non-natural α1-α2 domain of ULBP2.R80W (Figure 1B; SEQ ID NO: 142) with high affinity for the natural, wild-type NKG2D ectodomain (NKG2D.wt) was selected as the starting domain for further mutagenesis and screening by phage display. Synthetic DNA libraries were generated for the α1-α2 domain of ULBP2.R80W (SEQ ID NO: 108), which further contains a C8S mutation to eliminate the potential for disulfide bonds. Codons of amino acid residues of the ligand that, when bound, are located in close proximity to positions Y152 and Y199 on the natural NKG2D receptor were replaced with NNK codons; the libraries consisted of NNK codons at positions 154-159. Libraries were cloned as fusion proteins with the minor coat protein pIII of phage M13, and phage particles displaying mutagenized variants of the α1-α2 domain were produced in E. coli SS320 cells according to standard methods (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, CF, 3 ^rd ed. (2011). These α1-α2 phage display libraries were sorted for high-affinity binding to the unnatural receptor NKG2D.AF by selectively capturing phage clones bound to biotinylated Fc-NKG2D.AF protein in the presence of non-biotinylated natural competitor protein Fc-NKG2D.wt. Selective clones were enriched by subjecting them to multiple cyclic rounds of competitive selection using increasing concentrations of non-biotinylated natural Fc-NKG2D.

[0116] Following four rounds of selection, individual clones were arrayed in a 96-well format and spot ELISA was performed to confirm preferential binding for plate-bound non-native NKG2D.AF versus NKG2D.wt. Bound phage were detected using biotinylated E1 monoclonal antibody against the phage coat protein M13 (ThermoFisher Scientific, Waltham, MA), detected by streptavidin-HRP (R&D Systems, Minneapolis, MN), and developed by 1-Step Ultra TMB ELISA (ThermoFisher Scientific, Waltham, MA). The spot ELISA signal for each clone was expressed as the ratio of phage binding to NKG2D.AF to phage binding to NKG2D.wt. Phages with a ratio greater than or equal to 14 were sequenced to identify specific mutations within the NNK-mutated regions. In cases where multiple clones were identified representing the same sequence, the ratio of ELISA signals was plotted and the concordance of phage clones was confirmed by clustering of data points (data not shown).

[0117] Thirty variants identified in the ELISA were propagated in individual monocultures to generate high-titer phage microbatches. Purified phage concentrations were normalized to an _OD268 of 0.5, then subjected to serial 1:3 dilutions against plate-bound Fc-NKG2D.AF or Fc-NKG2D.wt, with phage detection and development ELISA performed as described above. All thirty variants analyzed in this manner consistently showed selective binding to NKG2D.AF, with little to no binding to NKG2D.wt, even at the highest phage concentrations analyzed. The selected phages also showed a shift of two or more log phage concentrations to achieve half-maximal binding between NKG2D.AF and NKG2D.wt.

[0118] To confirm that the NKG2D.AF-selective α1-α2 domain variants retained specific binding properties in the context of antibody fusion proteins, 21 variants (Table 5; e.g., SEQ ID NOs: 143-150) were cloned as C-terminal fusion proteins with an APTSSSGGGGS linker to the light chain of the rituximab antibody. The resulting fusion proteins 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 parent antibody to form paired full-length 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). ELISAs measuring the binding of each ULBP2 α1-α2 antibody fusion protein variant to non-native Fc-NKG2D.AF and to native Fc-NKG2D.wt showed that they had significantly higher binding affinity for NKG2D.AF relative to native NKG2D.wt (Table 12).

Table 12: Specificity of NKG2D.AF-selected ULBP2 variants in the rituximab-MicAbody format retained their binding to NKG2D.AF by quantitative ELISA. The specific amino acid modifications of each ULBP2 variant are shown, as well as their binding ratios to the Fc-NKG2D.wt versus Fc-NKG2D.AF fusion protein.

[0119]

ULBP2 residue ELISA EC ₅₀ - binding of rituximab-MicAbody (fusion protein light chain - ULBP2) to NKG2D.wt or NKG2D.AF 8 80 154 155 156 157 158 159 ULBP2 variant C R M S F H Y F wt ec ₅₀ nM AFEC ₅₀ nM wt/AF EC ratio ₅₀ AF/wt EC ratio ₅₀ A S W T T T W Q I 28.95 0.061 471.12 0.00212 B S W T M L R Q W 34.18 0.025 1373.49 0,00073 C S W T I L W Q T 129.83 0.029 4414.23 0.00023 D S W T L L W Q A 10.02 0,020 489.80 0.00204 E S W T L L W S W 51.45 0.031 1650,77 0.00061 F S W T V L W Q A 37.58 0.023 1639.79 0.00061 G S W T V L W S A 40.54 0.024 1664.58 0,00060 I S W T N I W Q Y 1.04 0,010 99.11 0.01009 J S W T H L W G W 5.77 0.062 93.61 0.01068 L S W T L F W Q S 25.33 0.053 479.31 0.00209 O S W T S L W Q S 17.04 0.026 652.71 0,00153 P S W T M L R Q F 2.37 0.069 34.45 0.02903 R S W T L L W G W 104.45 0.031 3398,27 0.00029 T S W T L L W Q W 4.37 0.029 151.52 0,00660 U S W T M L W K W 19.58 0.033 595.39 0,00168 W S W T M F R Q W 27.09 0,020 1322,15 0,00076 Y S W T S L W S W 83.49 0,090 927.71 0.00108 Z S W T N L W S A 98.80 0.025 3892,50 0.00026 AA S W T M F W S W 654.83 0.033 20092.30 0,00005 A.B. S W T L M W Q W 389.34 0.036 10801,65 0,00009 AD S W T T L W Q V 57.33 0.038 1504.95 0,00066

[0120]

[0121] Collectively, these data demonstrated the invention of non-natural, orthogonal α1-α2 domains that had high affinity binding to the non-natural NKG2D.AF receptor and significantly reduced binding affinity to the natural NKG2D receptor. Furthermore, fusion proteins of these orthogonal α1-α2 domains with antibody polypeptides retained their selective binding properties and were used, for example, in the context of chimeric antigen receptor (CAR) T cells, to retarget non-natural NKG2D.AF receptors to specific antigens.

[0122] Example 11: (Identification of non-natural NKG2D ligands that can discriminate between non-natural NKG2D receptor variants by selectively binding one or the other)

[0123] Phage display to construct orthogonal non-natural α1-α2 domains with selective binding to the NKG2D.Y152A receptor (hereafter referred to as NKG2D.YA) was performed using the non-natural α1-α2 domain of ULBP2.R80W (SEQ ID NO: 142) as a starting point, as described above. The α1-α2 phage display libraries were panned for high-affinity binding to the non-natural receptor Fc-NKG2D.YA by selectively capturing phage clones bound to biotinylated Fc-NKG2D.YA protein (SEQ ID NO: 95) in the presence of a competitive non-biotinylated natural Fc-NKG2D.wt protein (SEQ ID NO: 94). Additional phage clone validation work led to the identification of variants with preferential binding to Fc-NKG2D.YA versus Fc-NKG2D.wt (Table 13).

[0124] Table 13: Selected mutations at the indicated amino acid locations of ULBP2.R80W (SEQ ID NO: 132) resulted in Y152A-specific phage clones.

M154 S155 F156 H157 Y158 F159 T M L E L W TO M T V I W S I L Q T T V R

[0125]For ULBP2.S3 (SEQ ID NO: 151), for example, consistently showed selective binding by ELISA and Octet assay (both in the monomeric His-tagged and bispecific fusion antibody format) to non-natural NKG2D.YA, relative to natural NKG2D.wt. This represented a distinct form of the invention of non-natural α1-α2 orthogonal domains having high affinity binding to non-natural NKG2D receptors (in this case, NKG2D.YA, as opposed to NKG2D.AF as in Example 2). Furthermore, fusion proteins of the α1-α2 orthogonal domains with antibody polypeptides retained their selective binding properties and were used to selectively retarget non-natural NKG2D receptors to specific molecules, defined by fused heterologous peptides, such as antibodies.

[0126] To determine whether the non-natural α1-α2 domain with selective binding to NKG2D.YA (ULBP2.S3, SEQ ID NO: 151) and the non-natural α1-α2 domains with selective binding to NKG2D.AF could discriminate between the two non-natural receptor variants, titration ELISAs were performed. All 21 of the selected α1-α2 variants that bound NKG2D.AF were directly compared for binding to NKG2D.AF versus NKG2D.YA. Of these, four showed the properties of failing to bind NKG2D.wt, strong affinity for NKG2D.AF, and greatly reduced (15-20-fold) or abolished binding to NKG2D.YA relative to NKG2D.AF. These four non-naturally occurring α1-α2 ULBP2 variants, ULBP2.C, ULBP2.R, ULBP2.AA, and ULBP2.AB (SEQ ID NOs: 143, 145, 147, and 149), were also screened for changes in the predicted immunogenicity profile, relative to the wild-type ULBP2 peptide sequence (SEQ ID NO: 61), using the NetMHC4.0 server (to query MHC class I peptide binding against all HLA supertype members using 9-mer peptide assay; http://www.cbs.dtu.dk/services/NetMHC/) and the NetMHCII 2.3 server (to query MHC class II peptide binding against HLA-DR, HLA-DQ, HLA-DP haplotypes using 15-mer peptide assay; http://www.cbs.dtu.dk/services/NetMHCII/), both algorithms were developed at 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). Mutations included in ULBP2.C, ULBP2.R, and ULBP2.AB did not increase the predicted immunogenicity, while the immunogenicity of ULPB2.AA was weakly increased for several haplotypes (Figures 8 and 9). As a consequence of the specificity of ULBP2.R for NKG2D.AF and its lack of predicted immunogenicity, ULBP2.R was selected for additional ELISA analysis to directly compare its binding behavior with that of ULBP2.S3 (selected via NKG2D.YA, a non-natural, orthogonal ligand), ULBP2.R80W (a non-natural ligand with enhanced affinity for wild-type NKG2D), and wild-type ULBP2 (ULBP2.wt). Binding of four rituximab-MicAbody reagents (SEQ ID NOs: 139 and 151, 139 and 152, 153 and 140, and 139 and 141 as the heavy chain and light chain for ULBP2.R, ULBP2.S3, ULBP2.R80W, and ULBP2.wt, respectively) was analyzed against wild-type NKG2D (NKG2D.wt) and two inert, non-native variants NKG2D.YA and NKG2D.AF. The data showed that the NKG2D.YA-selected ULBP2.S3 MicAbody variant bound with high affinity to NKG2D.YA but did not recruit NKG2D.AF or native NKG2D. Furthermore, the NKG2D.AF-selected MicAbody variant of ULBP2.R bound with high affinity to NKG2D.AF but did not recruit NKG2D.YA or native NKG2D. These results demonstrate the enormous potential for exploring the NKG2D-MIC ligand axis and developing unique pairs of novel, selective non-natural NKG2D receptors and their corresponding, cognate non-natural MIC ligand binding partners.

[0127] Example 12: (Targeting and killing activity of CAR-T cells expressing the non-native NKG2D.AF ectodomain is controlled by orthogonal α1-α2 domains fused to heterologous targeting polypeptides)

[0128] Tools for selective control of CAR-T cell therapies are highly sought after to mitigate toxicity and improve tumor efficacy (Gill and June, op.cit.). Previous efforts have been made to develop CARs using the CD16 ectodomain, which could then be recruited via the Fc domain of therapeutic monoclonal antibodies, allowing antibody-based control of CAR-T targeting (Chang et al., op.cit.). However, CD16-based CAR-T cells can recognize almost all endogenous antibody molecules in blood and tissues, and therapeutic antibodies used to control these cells may face competition from endogenous CD16 receptors on NK cells, PMNs, monocytes, and macrophages. Both of these features contribute to the problems of non-tumor-specific toxicity and poor pharmacokinetics, respectively.

[0129] Natural NKG2D ligands are present on specific healthy tissues and many stressed tissues, creating an unusual risk of toxicity when using current NKG2D-CAR approaches (VanSeggelen et al., 2015). The non-natural NKG2D receptor Y152A was shown to specifically bind to the non-natural α1-α2 domain of NKG2D ligands, providing an example of the means by which non-natural NKG2D-CAR activity can be selectively controlled using bispecific proteins containing the inventive non-natural α1-α2 domain of NKG2D ligands.

[0130] The present inventors engineered CAR-T cells with a receptor comprising a modified Y152A/Y199F ("AF") NKG2D ectodomain that lacks binding to all natural NKG2D ligands, or previously described non-natural α1-α2 domains orthogonal and related to the modified Y152A NKG2D (NKG2D.YA). The inventive related non-natural α1-α2 domains bound the non-natural NKG2D.AF ectodomain with high affinity and avoided binding to the natural NKG2D ectodomains and to the NKG2D.YA ectodomain. Thus, the engineered α1-α2 domains having strong selectivity for the unnatural NKG2D.AF ectodomain over natural NKG2D and unnatural NKG2D.YA provide an ideal system for selectively controlling the unnatural NKG2D CAR receptors, or any receptor or protein fused to the unnatural NKG2D ectodomains, that can be selectively recruited via the unnatural α1-α2 domains of the present invention. The present invention further provides the possibility of individual cells expressing two separate CARs, one comprising NKG2D.YA and the other NKG2D.AF, each signaling using entirely different intracellular domains. These different CARs can have independent, dual control of cellular activity through the extracellular action of the corresponding, cognate orthogonal MicAbody or other non-antibody fusion polypeptide.

[0131] To demonstrate the selective control of CAR-T cells modified with a chimeric receptor with an introduced non-natural NKG2D.AF ectodomain, the present inventors constructed CARs with native NKG2D.wt (SEQ ID NO: 49), non-natural NKG2D.YA (SEQ ID NO: 54), or non-natural NKG2D.AF (SEQ ID NO: 154) ectodomains based on prior work using 4-1BB/CD3zeta CAR constructs (Campana, Patent 8,399,645) fusing the corresponding NKG2D ectodomains to the CD8 hinge region of the CAR (SEQ ID NOs: 155, 157, 159). These constructs (SEQ ID NOS: 156, 158, 160) were cloned into a lentiviral vector and expressed in primary human CD8 positive T cells using lentiviral transduction. HeLa cells have up-regulated levels of MIC ligands on their surface, including MICA, MICB, ULBP3, and ULBP2/5/6 (the antibody used to detect this cannot distinguish between the three ULBPs; anti-human ULBP-2/5/6 antibody, R&D Systems, Minneapolis, MN). HeLa cells were transfected to also express either native ULBP1 or the NKG2D.AF selected ULBP2.R variant on their surface, and these cells were used as a target for in vitro killing assays. Target HeLa 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 five hours, after which the amount of calcein released into the supernatant was quantified and normalized to the total amount of calcein released upon detergent treatment. Due to the increased levels of MIC ligands naturally expressed on the surface of HeLa cells, CD8 cells expressing native NKG2D (NKG2D.wt) as a CAR recruited HeLa cells via this overexpressed natural ligand and cytolyzed them. However, both NKG2D.YA and NKG2D.AF-CAR-CD8 cells transduced showed very little lysis of native HeLa cells even at high E:T ratios, with activity levels on par with untransduced CD8 T cells. When ULBP1 was overexpressed on the surface of HeLa cells, only NKG2D.wt-CAR-CD8 T cells significantly lysed them. There was some additional killing at high E:T ratios using NKG2D.YA-CAR cells, but none with NKG2D.AF-CAR cells, indicating that the Y152A/Y199F double mutation renders NKG2D even more inert than the Y152A single mutation. For HeLa cells overexpressing the NKG2D.AF-selective non-native ULBP2.R, NKG2D.wt-CAR cells induce lysis (due to recognition of endogenous MIC ligands), whereas NKG2D.AF-CAR cells induce significant levels of lysis, consistent with engagement of the receptor and its selective ligand.

[0132] To demonstrate that lysis of either NKG2D.YA or NKG2D.AF-CAR cells can be induced by the respective cognate targeting MicAbody alone, Ramos cells were used as a target for killing in combination with rituximab-based MicAbodies linked to non-natural ULBP2.S3 or ULBP2.R orthogonal ligands. The rituximab-ULBP2.S3 MicAbody could drive the cell killing activity of NKG2D.YA-CAR-CD8 cells but not NKG2D.AF-CAR cells, while the rituximab-ULBP2.R MicAbody could drive the activity of NKG2D.AF-CAR but not NKG2D.YA-CAR cells. This further demonstrates the selectivity of the two unnatural ULBP2 variants for the related unnatural NKG2D variants for which they were engineered as preferred partners. To demonstrate the specificity of the antibody portion of the MicAbody, in vitro killing assays were performed using NKG2D.AF-CAR-CD8 cells that were pre-armed by incubation with rituximab-ULBP2.R, trastuzumab-ULPB2.R (SEQ ID NOs: 95 and 133, heavy and light chain, respectively), or an equimolar combination of the two at a saturating total concentration of MicAbody. After removal of unbound MicAbody by washing, CD8 cells were introduced to either Ramos cells (expressing CD20, the target of rituximab) or CT26-Her2 (a mouse cell line transfected to express human Her2) pre-loaded with calcein. After two hours of incubation in the two different E:T ratios, the amount of released calcein was quantified. When cells were pre-armed with rituximab-MicAbody, only Ramos cells were lysed, while trastuzumab-MicAbody directed cytolytic activity only against CT26-Her2 cells. However, when NKG2D.AF-CAR-CD8 cells were simultaneously pre-armed with both rituximab- and trastuzumab-ULBP2.R MicAbodies, both target cell lines were lysed, indicating that these CAR cells - through a selective, privileged partnership engineered between receptor and ligand - were readily multiplexed and thus targeted to engage different tumor targets simultaneously.

[0133] Example 13: (Depletion of Human Tonsillar CD4 T Cells Productively Infected with HIV) CD8+ T cells were isolated from PBMCs of healthy donors, activated with anti-CD3/CD28 beads, and transduced with a CAR consisting of inert NKG2D, the hinge and transmembrane domain of CD8, the 4-1BB costimulatory domain, and CD3ξ. These CAR-T cells are designated convertible CAR cells. These convertible CAR-T cells were capable of only indirect binding to broadly neutralizing HIV antibodies fused to a modified, non-natural ligand related to the inert NKG2D receptor of the convertible CAR. Untransduced CD8 T cells from the same donor were also prepared in parallel as a negative control. Four HIV-specific MicAbodies were generated based on the sequence of the broadly neutralizing antibodies 3BNC60, 3BNC117, PGT121, and 10-1074 (SEQ ID NOs. 161 and 162 (3BNC60), MicAbody heavy and light chains, respectively; 163 and 164 (3BNC117), MicAbody heavy and light chains, respectively; 165 and 166 (PGT121), MicAbody heavy and light chains, respectively; 167 and 168 (10-1074), MicAbody heavy and light chains, respectively). These MicAbodies bind to specific epitopes of the HIV envelope gp160 molecules. The target epitope bound by 3BNC60 and 3BNC117 is SEQ ID NO.:169; via PGF12 and 10-1074 is SEQ ID NO.: 170; (Deng K, Pertea M, Rongvaux A, Wang L, Durand CM, Ghiaur G, Lai J, McHugh HL, Hao H, Zhang H, JB, Gurer C, Murphy AJ, Valenzuela DM, Yancopoulos GD, Deeks SG, Strowig T, Kumar P, Siliciano JD, Salzberg SL, Flavell RA, Shan L, Siliciano RF Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations. Nature. 2015 Jan 15; 517 (7534) p. 381-5.). MicAbodies targeting CD20 or HER2 were also used as negative controls.

[0134] Human tonsillar cells from 4 healthy donors were processed to generate human lymphoid aggregate culture (HLAC). HLAC cells were overlaid on 293T cells pre-transfected with DNA corresponding to R5-tropic HIV-1 and a GFP reporter gene. After 24 h, HLAC cells were removed and HIV infection was allowed to continue for an additional 4 days. GFP-positive infected HLAC cells were then exposed to untransduced CD8 T cells or convertible CAR-T cells armed with the indicated MicAbodies and cultured for 48 h in the presence of 5 μM saquinavir to prevent viral spread. Cells were then collected by centrifugation, washed, and stained to assess the viability of infected and uninfected cells using an LSRII flow cytometer.

[0135] Assessment of effector:target (E:T) cell ratios for killing of HIV-infected primary CD4 T cells by CAR-T cells using different concentrations of specific HIV-targeting MicAbodies. As described above, one million primary tonsil-derived cells infected with Bal-GFP R5 virus (~10% infection; ^1X104 infected cells) were incubated with ^1X105 untransduced CD8 (0:1) or with ^1X104 (1:1) or ^2X105 (20:1) CAR-T cells in the presence of different concentrations of four different broadly HIV-neutralizing MicAbodies. Cells were stained after 24 h and assessed by flow cytometry. Cells were gated on a cell/live/CD3+/CD8- basis, either GFP-expressing or non-GFP-expressing basis. Results averaged over 3 studies are shown in Figure 7. In these studies, the combination of HIV-specific MicAbodies and convertible CAR-T cells resulted in specific killing of tonsillar cells infected with the R5 HIV virus. The optimal effector:target ratio for killing ranged between 1:1 and 10:1, without reducing the viability of uninfected cells. Killing was highly restricted to infected cells, i.e., GFP-expressing cells. GFP-cells present in the same culture showed little or no reduction in cell numbers (Figures B and C; GFP+ vs. GFP-). Furthermore, there was no killing of uninfected cells and no killing of infected cells when donor-matched non-transduced CD8 T cells or non-HIV-targeting MicAbodies (e.g., CD20-targeting MicAbody or Her2-targeting MicAbody) were used.

[0136] Specific killing of R5 virus-infected primary CD4 cells by CAR-T in combination with an HIV-specific MicAbody. One million primary tonsil-derived cells infected with Bal-GFP R5 virus (~1X10 ⁴ infected cells) were incubated with 1X10 ⁵ CAR-T cells in the presence of varying concentrations of HIV-specific MicAbodies or a B-cell-specific CD20-targeting MicAbody or a HER2-targeting MicAbody (Her2). Cells were stained 24 h later and analyzed by flow cytometry. Cells were gated for single cells/live/CD3+/CD8- and either GFP+ or GFP-. Results averaged over 4 studies are shown in Figure 8.

[0137] Specific killing of F4 founder/transplant virus-infected primary CD4 cells by CAR-T in combination with an HIV-specific MicAbody. One million primary tonsil-derived cells infected with F4-GFP virus (T/F) (~1X10 ⁴ infected cells) were incubated with 1X10 ⁵ convertible CAR-T cells in the presence of varying concentrations of four different HIV-specific MicAbodies, a CD20-targeting MicAbody (Ritux) or a HER2-targeting MicAbody (Her2). Cells were stained 24 h later and then processed for flow cytometry. Cells were gated for single cells/live/CD3+/CD8- and either GFP+ or GFP-. The results are shown in Figure 9. Efficient killing was observed when cells were infected with R5 virus or infected with the F4 HIV founder/transfer virus, which is a viral strain that is successfully transmitted horizontally from one individual to another.

[0138] Example 14 (CAR-T and MicAbody Destruction of Reactivated Latently Infected Reservoir Cells from Aviremic Patients Chronically Infected with HIV and Undergoing ART)

Peripheral blood mononuclear cells (PBMCs) from 6 HIV-positive, aviremic individuals undergoing ART were obtained by continuous flow leukapheresis centrifugation followed by centrifugation of the cells on Ficoll-Hypaque density gradients. Resting CD4+ T lymphocytes were then isolated by “no-touch” antibody-based negative selection. Cells were cultured in RPMI medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. 10 million resting CD4+ lymphocytes were stimulated with 80 nM PMA+1 μM ionomycin for 72 h. Following reactivation, cells were incubated with CAR-T or donor-matched untransduced CD8 cells with different MicAbodies in the presence of 5 μM saquinavir for 48 h. Cells were harvested by centrifugation at 300 g for 10 min. Cell pellets were then lysed and RNA was isolated using the RNeasy kit (Qiagen). The Superscript III one-step RT-PCR system was used to generate cDNA and simultaneously preamplify viral mRNA (i.e., 10 cycles of preamplification) before analysis and quantification by droplet digital PCR (ddPCR). CD4+ T cells were isolated by contactless negative selection from PBMCs collected from known HIV-infected patients undergoing ART and reactivated for 72 h with 100 nM phorbol myristate acetate (PMA) + 1 μM ionomycin. Cells were then washed twice and incubated for 48 h with convertible CAR-T cells or untransduced CD8 T cells in the presence of 0.1 or 1 nM of an equal concentration mixture of HIV bNAb-based MicAbodies (3BNC60, 3BNC117, PGT121, and 10-1074), designated MIX in the figure. Cells were then centrifuged and RNA was isolated from the cell pellets. Cell-associated HIV RNA was measured by ddPCR. The results are shown in Figure 10.

In this example of reactivation of latent reservoir cells (3 days with PMA+ionomycin) from infected aviremic individuals undergoing ART (n=6), we observed that CAR-T cells were able to effectively deplete these reactivated reservoir cells by approximately 50% compared to donor-matched untransduced CD8 T cells+MicAbodies cocktail. The size of the inducible reservoir was assessed by quantifying cell-associated HIV RNA in the presence and absence of inducer and effector cells using ddPCR.

These findings from Examples 13 and 14 together provide ex vivo proof of concept that convertible CAR-T cells plus cognate MicAbodies engineered using broadly neutralizing human IgG1 antibodies can be used as a novel, efficient, and highly selective killing method to kill successfully reactivated HIV-infected cells within the latent HIV-1 reservoir.

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LIST OF SEQUENCES

<110> XYPHOS BIOSCIENCES INC.

<120> MODIFIED UNNATURAL NKG2D LIGANDS THAT SELECTIVELY

DELIVER ATTACHED HETEROLOGOUS MOLECULES TO UNNATURAL

NKG2D RECEPTORS ON CAR CELLS

<130> F252863

<150> US 62/797644

<151> 2019-01-28

<160> 170

<170> PatentIn version 3.5

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Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

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Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

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His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

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Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

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Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

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His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

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Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

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Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

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Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

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Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

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Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

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Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

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Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

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Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

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Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr

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Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Ile Arg Asn

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Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

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His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

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Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

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Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

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Phe Leu Arg Tyr Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

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Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

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Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

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Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

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Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

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Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr

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Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

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Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

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His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

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Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

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Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

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Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

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His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

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Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

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Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

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Pro Ser

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Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

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Ser Val Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Tyr Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

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Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

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Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr

115 120 125

Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

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Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

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His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Glu Ser Gly Val

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Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

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Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr

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Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser

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His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

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Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

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Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

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Pro Ser

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Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

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Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

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Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

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Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr

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Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

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Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

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His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

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Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

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Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

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Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

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His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

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Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

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Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

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Pro Ser

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Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

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Ser Val Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

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Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

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Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

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Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr

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Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

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Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

145 150 155 160

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

180 185 190

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

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Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

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His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

225 230 235 240

Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

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Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

260 265 270

Pro Ser

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Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

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Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

145 150 155 160

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

180 185 190

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

195 200 205

Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

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His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

225 230 235 240

Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg

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Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

260 265 270

Pro Ser Gly Lys

275

<210> 8

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Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Ala Lys Thr Trp Asp Thr Glu Thr Glu

50 55 60

Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile

65 70 75 80

Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr

115 120 125

Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn

130 135 140

Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met

145 150 155 160

Gln Ala Asp Cys Leu Gln Lys Leu Gln Leu Pro Pro Met Val Asn Val

165 170 175

Ile Cys Ser Glu Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala

180 185 190

Ser Ser Phe Tyr Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly

195 200 205

Val Ser Leu Ser His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp

210 215 220

Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly

225 230 235 240

Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly

245 250 255

Thr His Pro Val Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg

260 265 270

Thr Asp Phe Pro Tyr Val Ser Ala Ala Met Pro Cys Phe Val Ile Ile

275 280 285

Ile Ile Leu Cys Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu

290 295 300

Gly Pro

305

<210> 9

<211> 318

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICB

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Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Ala Glu Thr Trp Asp Thr Glu Thr Glu

50 55 60

Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile

65 70 75 80

Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Met His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr

100 105 110

Asn Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr

115 120 125

Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn

130 135 140

Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met

145 150 155 160

Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val

165 170 175

Ala Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Cys Ser Glu

180 185 190

Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr

195 200 205

Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser

210 215 220

His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

225 230 235 240

Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Arg

245 250 255

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val

260 265 270

Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro

275 280 285

Tyr Val Ser Ala Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys

290 295 300

Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro

305 310 315

<210> 10

<211> 318

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICB

<400> 10

Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Ala Lys Thr Trp Asp Thr Glu Thr Glu

50 55 60

Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile

65 70 75 80

Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr

115 120 125

Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn

130 135 140

Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met

145 150 155 160

Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val

165 170 175

Ala Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Ile Cys Ser Glu

180 185 190

Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr

195 200 205

Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser

210 215 220

His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

225 230 235 240

Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Arg

245 250 255

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val

260 265 270

Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro

275 280 285

Tyr Val Ser Ala Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys

290 295 300

Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro

305 310 315

<210> 11

<211> 318

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICB

<400> 11

Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asn Val Leu Gly Ala Lys Thr Trp Asp Thr Glu Thr Glu

50 55 60

Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile

65 70 75 80

Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr

115 120 125

Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn

130 135 140

Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met

145 150 155 160

Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val

165 170 175

Ala Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Cys Ser Glu

180 185 190

Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr

195 200 205

Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser

210 215 220

His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

225 230 235 240

Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Arg

245 250 255

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val

260 265 270

Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro

275 280 285

Tyr Val Ser Ala Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys

290 295 300

Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro

305 310 315

<210> 12

<211> 318

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICB

<400> 12

Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Ala Glu Thr Trp Asp Thr Glu Thr Glu

50 55 60

Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile

65 70 75 80

Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr

100 105 110

Asn Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr

115 120 125

Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn

130 135 140

Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met

145 150 155 160

Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val

165 170 175

Ala Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Cys Ser Glu

180 185 190

Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr

195 200 205

Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser

210 215 220

His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

225 230 235 240

Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Lys

245 250 255

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val

260 265 270

Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro

275 280 285

Tyr Val Ser Ala Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys

290 295 300

Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro

305 310 315

<210> 13

<211> 318

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICB

<400> 13

Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Ala Glu Thr Trp Asp Thr Glu Thr Glu

50 55 60

Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile

65 70 75 80

Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr

100 105 110

Asn Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr

115 120 125

Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn

130 135 140

Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met

145 150 155 160

Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val

165 170 175

Ala Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Cys Ser Glu

180 185 190

Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr

195 200 205

Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser

210 215 220

His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

225 230 235 240

Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Arg

245 250 255

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val

260 265 270

Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro

275 280 285

Tyr Val Ser Ala Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys

290 295 300

Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro

305 310 315

<210> 14

<211> 244

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP-1 (NO ACCESS Q9BZM6)

<400> 14

Met Ala Ala Ala Ala Ser Pro Ala Phe Leu Leu Cys Leu Pro Leu Leu

1 5 10 15

His Leu Leu Ser Gly Trp Ser Arg Ala Gly Trp Val Asp Thr His Cys

20 25 30

Leu Cys Tyr Asp Phe Ile Ile Thr Pro Lys Ser Arg Pro Glu Pro Gln

35 40 45

Trp Cys Glu Val Gln Gly Leu Val Asp Glu Arg Pro Phe Leu His Tyr

50 55 60

Asp Cys Val Asn His Lys Ala Lys Ala Phe Ala Ser Leu Gly Lys Lys

65 70 75 80

Val Asn Val Thr Lys Thr Trp Glu Glu Gln Thr Glu Thr Leu Arg Asp

85 90 95

Val Val Asp Phe Leu Lys Gly Gln Leu Leu Asp Ile Gln Val Glu Asn

100 105 110

Leu Ile Pro Ile Glu Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu

115 120 125

His Glu Ala His Gly His Gly Arg Gly Ser Trp Gln Phe Leu Phe Asn

130 135 140

Gly Gln Lys Phe Leu Leu Phe Asp Ser Asn Asn Arg Lys Trp Thr Ala

145 150 155 160

Leu His Pro Gly Ala Lys Lys Met Thr Glu Lys Trp Glu Lys Asn Arg

165 170 175

Asp Val Thr Met Phe Phe Gln Lys Ile Ser Leu Gly Asp Cys Lys Met

180 185 190

Trp Leu Glu Glu Phe Leu Met Tyr Trp Glu Gln Met Leu Asp Pro Thr

195 200 205

Lys Pro Pro Ser Leu Ala Pro Gly Thr Thr Gln Pro Lys Ala Met Ala

210 215 220

Thr Thr Leu Ser Pro Trp Ser Leu Leu Ile Ile Phe Leu Cys Phe Ile

225 230 235 240

Leu Ala Gly Arg

<210> 15

<211> 246

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP-2 (NO ACCESS Q9BZM5)

<400> 15

Met Ala Ala Ala Ala Ala Thr Lys Ile Leu Leu Cys Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Ser Gly Trp Ser Arg Ala Gly Arg Ala Asp Pro His Ser

20 25 30

Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg Pro Gly Pro Arg

35 40 45

Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu His Tyr

50 55 60

Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro Leu Gly Lys Lys

65 70 75 80

Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro Val Leu Arg Glu

85 90 95

Val Val Asp Ile Leu Thr Glu Gln Leu Arg Asp Ile Gln Leu Glu Asn

100 105 110

Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu

115 120 125

Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln Phe Ser Phe Asp

130 135 140

Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg Met Trp Thr Thr

145 150 155 160

Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn Asp Lys

165 170 175

Val Val Ala Met Ser Phe His Tyr Phe Ser Met Gly Asp Cys Ile Gly

180 185 190

Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr Leu Glu Pro Ser

195 200 205

Ala Gly Ala Pro Leu Ala Met Ser Ser Gly Thr Thr Gln Leu Arg Ala

210 215 220

Thr Ala Thr Thr Leu Ile Leu Cys Cys Leu Leu Ile Ile Leu Pro Cys

225 230 235 240

Phe Ile Leu Pro Gly Ile

245

<210> 16

<211> 244

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP-3 (ACCESS NO NP_078794)

<400> 16

Met Ala Ala Ala Ala Ser Pro Ala Ile Leu Pro Arg Leu Ala Ile Leu

1 5 10 15

Pro Tyr Leu Leu Phe Asp Trp Ser Gly Thr Gly Arg Ala Asp Ala His

20 25 30

Ser Leu Trp Tyr Asn Phe Thr Ile Ile His Leu Pro Arg His Gly Gln

35 40 45

Gln Trp Cys Glu Val Gln Ser Gln Val Asp Gln Lys Asn Phe Leu Ser

50 55 60

Tyr Asp Cys Gly Ser Asp Lys Val Leu Ser Met Gly His Leu Glu Glu

65 70 75 80

Gln Leu Tyr Ala Thr Asp Ala Trp Gly Lys Gln Leu Glu Met Leu Arg

85 90 95

Glu Val Gly Gln Arg Leu Arg Leu Glu Leu Ala Asp Thr Glu Leu Glu

100 105 110

Asp Phe Thr Pro Ser Gly Pro Leu Thr Leu Gln Val Arg Met Ser Cys

115 120 125

Glu Cys Glu Ala Asp Gly Tyr Ile Arg Gly Ser Trp Gln Phe Ser Phe

130 135 140

Asp Gly Arg Lys Phe Leu Leu Phe Asp Ser Asn Asn Arg Lys Trp Thr

145 150 155 160

Val Val His Ala Gly Ala Arg Arg Met Lys Glu Lys Trp Glu Lys Asp

165 170 175

Ser Gly Leu Thr Thr Phe Phe Lys Met Val Ser Met Arg Asp Cys Lys

180 185 190

Ser Trp Leu Arg Asp Phe Leu Met His Arg Lys Lys Arg Leu Glu Pro

195 200 205

Thr Ala Pro Pro Thr Met Ala Pro Gly Leu Ala Gln Pro Lys Ala Ile

210 215 220

Ala Thr Thr Leu Ser Pro Trp Ser Phe Leu Ile Ile Leu Cys Phe Ile

225 230 235 240

Leu Pro Gly Ile

<210> 17

<211> 263

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP-4/RAET1E (ACCESS NO Q8TD07)

<400> 17

Met Arg Arg Ile Ser Leu Thr Ser Ser Pro Val Arg Leu Leu Leu Phe

1 5 10 15

Leu Leu Leu Leu Leu Ile Ala Leu Glu Ile Met Val Gly Gly His Ser

20 25 30

Leu Cys Phe Asn Phe Thr Ile Lys Ser Leu Ser Arg Pro Gly Gln Pro

35 40 45

Trp Cys Glu Ala Gln Val Phe Leu Asn Lys Asn Leu Phe Leu Gln Tyr

50 55 60

Asn Ser Asp Asn Asn Met Val Lys Pro Leu Gly Leu Leu Gly Lys Lys

65 70 75 80

Val Tyr Ala Thr Ser Thr Trp Gly Glu Leu Thr Gln Thr Leu Gly Glu

85 90 95

Val Gly Arg Asp Leu Arg Met Leu Leu Cys Asp Ile Lys Pro Gln Ile

100 105 110

Lys Thr Ser Asp Pro Ser Thr Leu Gln Val Glu Met Phe Cys Gln Arg

115 120 125

Glu Ala Glu Arg Cys Thr Gly Ala Ser Trp Gln Phe Ala Thr Asn Gly

130 135 140

Glu Lys Ser Leu Leu Phe Asp Ala Met Asn Met Thr Trp Thr Val Ile

145 150 155 160

Asn His Glu Ala Ser Lys Ile Lys Glu Thr Trp Lys Lys Asp Arg Gly

165 170 175

Leu Glu Lys Tyr Phe Arg Lys Leu Ser Lys Gly Asp Cys Asp His Trp

180 185 190

Leu Arg Glu Phe Leu Gly His Trp Glu Ala Met Pro Glu Pro Thr Val

195 200 205

Ser Pro Val Asn Ala Ser Asp Ile His Trp Ser Ser Ser Ser Leu Pro

210 215 220

Asp Arg Trp Ile Ile Leu Gly Ala Phe Ile Leu Leu Val Leu Met Gly

225 230 235 240

Ile Val Leu Ile Cys Val Trp Trp Gln Asn Gly Glu Trp Gln Ala Gly

245 250 255

Leu Trp Pro Leu Arg Thr Ser

260

<210> 18

<211> 332

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP-5 (NO ACCESS Q6H3X3)

<400> 18

Met Ala Ala Ala Ala Ser Pro Ala Phe Leu Leu Arg Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Ser Ser Trp Cys Arg Thr Gly Leu Ala Asp Pro His Ser

20 25 30

Leu Cys Tyr Asp Ile Thr Val Pro Lys Phe Arg Pro Gly Pro Arg Trp

35 40 45

Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu His Tyr Asp

50 55 60

Cys Gly Ser Lys Thr Val Thr Pro Val Ser Pro Leu Gly Lys Lys Leu

65 70 75 80

Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro Val Leu Arg Glu Val

85 90 95

Val Asp Leu Thr Glu Gln Leu Leu Asp Ile Gln Leu Glu Asn Tyr Ile

100 105 110

Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu Gln Lys

115 120 125

Ala Glu Gly His Gly Ser Gly Ser Trp Gln Leu Ser Phe Asp Gly Gln

130 135 140

Ile Phe Leu Leu Phe Asp Ser Glu Asn Arg Met Trp Thr Thr Val His

145 150 155 160

Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn Asp Lys Asp Met

165 170 175

Thr Met Ser Phe His Tyr Ile Ser Met Gly Asp Cys Thr Gly Trp Leu

180 185 190

Glu Asp Phe Leu Met Gly Met Asp Ser Thr Leu Glu Pro Ser Ala Gly

195 200 205

Ala Pro Pro Thr Met Ser Ser Gly Thr Ala Gln Pro Arg Ala Thr Ala

210 215 220

Thr Thr Leu Ile Leu Cys Cys Leu Leu Ile Met Cys Leu Leu Ile Cys

225 230 235 240

Ser Arg His Ser Leu Thr Gln Ser His Gly His His Pro Gln Ser Leu

245 250 255

Gln Pro Pro Pro His Pro Pro Leu Leu His Pro Thr Trp Leu Leu Arg

260 265 270

Arg Val Leu Trp Ser Asp Ser Tyr Gln Ile Ala Lys Arg Pro Leu Ser

275 280 285

Gly Gly His Val Thr Arg Val Thr Leu Pro Ile Ile Gly Asp Asp Ser

290 295 300

His Ser Leu Pro Cys Pro Leu Ala Leu Tyr Thr Ile Asn Asn Gly Ala

305 310 315 320

Ala Arg Tyr Ser Glu Pro Leu Gln Val Ser Ile Ser

325 330

<210> 19

<211> 246

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP-6 (ACCESS NO NP_570970)

<400> 19

Met Ala Ala Ala Ala Ile Pro Ala Leu Leu Leu Cys Leu Pro Leu Leu

1 5 10 15

Phe Leu Leu Phe Gly Trp Ser Arg Ala Arg Arg Asp Asp Pro His Ser

20 25 30

Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg Pro Gly Pro Arg

35 40 45

Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu His Tyr

50 55 60

Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro Leu Gly Lys Lys

65 70 75 80

Leu Asn Val Thr Met Ala Trp Lys Ala Gln Asn Pro Val Leu Arg Glu

85 90 95

Val Val Asp Ile Leu Thr Glu Gln Leu Leu Asp Ile Gln Leu Glu Asn

100 105 110

Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu

115 120 125

Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln Phe Ser Ile Asp

130 135 140

Gly Gln Thr Phe Leu Leu Phe Asp Ser Glu Lys Arg Met Trp Thr Thr

145 150 155 160

Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn Asp Lys

165 170 175

Asp Val Ala Met Ser Phe His Tyr Ile Ser Met Gly Asp Cys Ile Gly

180 185 190

Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr Leu Glu Pro Ser

195 200 205

Ala Gly Ala Pro Leu Ala Met Ser Ser Gly Thr Thr Gln Leu Arg Ala

210 215 220

Thr Ala Thr Thr Leu Ile Leu Cys Cys Leu Leu Ile Ile Leu Pro Cys

225 230 235 240

Phe Ile Leu Pro Gly Ile

245

<210> 20

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICA-WED

<400> 20

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 21

<211> 507

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding variant 15 alpha1-alpha2

<400> 21

gctgctgagc cacacagtct ccgctacaac cttacggtgt tgagctggga cggctctgtc 60

cagagtggct ttctgactga ggtacatctc gatggtcagc ccttcctccg atgcgacaga 120

caaaagtgca gggccaagcc acagggccaa tgggccgaag atgtacttgg caataagact 180

tgggacagag aaaccagaga tctgactggc tggggtaagg acttacgcat gactctcgca 240

cacattaaag accagaagga aggtcttcat tcgctccagg aaattagagt ctgtgaaatc 300

catgaagaca acagcacaag aagttcccaa catttctact acgacggcga gctgttctta 360

tcacagaatt tagagaccaa cgagtggaca atgccccaaa gctcgagggc ccagaccctc 420

gctatgaatg tgaggaattt ccttaaggag gacgctatgg aaactgacac ccactaccat 480

gcgatgcgcg ccgattgcct gcaggaa 507

<210> 22

<211> 507

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding variant 16 alpha1-alpha2

<400> 22

gctgctgagc cacacagtct ccgctacaac cttacggtgt tgagctggga cggctctgtc 60

cagagtggct ttctgactga ggtacatctc gatggtcagc ccttcctccg atgcgacaga 120

caaaagtgca gggccaagcc acagggccaa tgggccgaag atgtacttgg caataagact 180

tgggacagag aaaccagaga tctgactggc tggggtaagg acttacgcat gactctcgca 240

cacattaaag accagaagga aggtcttcat tcgctccagg aaattagagt ctgtgaaatc 300

catgaagaca acagcacaag aagttcccaa catttctact acgacggcga gctgttctta 360

tcacagaatt tagagaccct cgagtggaca atgccccaaa gctcgagggc ccagaccctc 420

gctatgaatg tgaggaattt ccttaaggag gacgctatgg aaactgacac ccactaccat 480

gcgatgcgcg ccgattgcct gcaggaa 507

<210> 23

<211> 507

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding variant 17 alpha1-alpha2

<400> 23

gctgctgagc cacacagtct ccgctacaac cttacggtgt tgagctggga cggctctgtc 60

cagagtggct ttctgactga ggtacatctc gatggtcagc ccttcctccg atgcgacaga 120

caaaagtgca gggccaagcc acagggccaa tgggccgaag atgtacttgg caataagact 180

tgggacagag aaaccagaga tctgactctc tggggtaagg acttacgcat gactctcgca 240

cacattaaag accagaagga aggtcttcat tcgctccagg aaattagagt ctgtgaaatc 300

catgaagaca acagcacaag aagttcccaa catttctact acgacggcga gctgttctta 360

tcacagaatt tagagaccct cgagtggaca atgccccaaa gctcgagggc ccagaccctc 420

gctatgaatg tgaggaattt ccttaaggag gacgctatgg aaactgacac ccactaccat 480

gcgatgcgcg ccgattgcct gcaggaa 507

<210> 24

<211> 507

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding variant 18 alpha1-alpha2

<400> 24

gctgctgagc cacacagtct ccgctacaac cttacggtgt tgagctggga cggctctgtc 60

cagcccggct ttctgactga ggtacatctc gatggtcagc ccttcctccg atgcgacaga 120

caaaagtgca gggccaagcc acagggccaa tgggccgaag atgtacttgg caataagact 180

tgggacagag aaaccagaga tctgactctc tggggtaagg acttacgcat gactctcgca 240

cacattaaag accagaagga aggtcttcat tcgctccagg aaattagagt ctgtgaaatc 300

catgaagaca acagcacaag aagttcccaa catttctact acgacggcga gctgttctta 360

tcacagaatt tagagaccct cgagtggaca atgccccaaa gctcgagggc ccagaccctc 420

gctatgaatg tgaggaattt ccttaaggag gacgctatgg aaactgacac ccactaccat 480

gcgatgcgcg ccgattgcct gcaggaa 507

<210> 25

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICA, variant 15 alpha1-alpha2

<400> 25

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Asn Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 26

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICA, variant 16 alpha1-alpha2

<400> 26

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 27

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICA, variant 17 alpha1-alpha2

<400> 27

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Leu Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 28

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICA, variant 18 alpha1-alpha2

<400> 28

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Pro Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Leu Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 29

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICwed alpha1-alpha2

<400> 29

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val

180

<210> 30

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide DSM20 alpha1-alpha2

<400> 30

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Ala Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Gln Thr Asp Thr His Tyr Arg Ala Met

145 150 155 160

His Ala Asp Cys Leu Phe Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val

180

<210> 31

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide DSM25 alpha1-alpha2

<400> 31

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val

180

<210> 32

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide DSM27 alpha1-alpha2

<400> 32

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val

180

<210> 33

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide DSM28 alpha1-alpha2

<400> 33

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val

180

<210> 34

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide DSM42 alpha1-alpha2

<400> 34

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val

180

<210> 35

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide DSM48 alpha1-alpha2

<400> 35

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Ala Thr Asp Thr His Tyr Ile Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Ala Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val

180

<210> 36

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide DSM49 alpha1-alpha2

<400> 36

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Thr Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gln Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Phe Thr Asp Thr His Tyr Arg Ala Met

145 150 155 160

Thr Ala Asp Cys Leu Thr Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val

180

<210> 37

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide primer

<400> 37

atctataatg ctgagcccca cagtcttcg 29

<210> 38

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide primer

<400> 38

cttgctcttc agatatcgcc gtagttc 27

<210> 39

<211> 7556

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic DNA construct expressing the wt domain of a1a2-Fv

<400> 39

ggagagacca cacccaagct gtctagagcc gccacgatgg ggctgggccc ggtcttcctg 60

cttctggctg gcatcttccc ttttgcacct ccgggagctg ctgctgagcc ccacagtctt 120

cgttataacc tcacggtgct gtcctgggat ggatctgtgc agtcagggtt tctcactgag 180

gtacatctgg atggtcagcc cttcctgcgc tgtgacaggc agaaatgcag ggcaaagccc 240

cagggacagt gggcagaaga tgtcctggga aataagacat gggacagaga gaccagagac 300

ttgacaggga atggaaagga cctcaggatg accctggctc atatcaagga ccagaaagaa 360

ggcttgcatt ccctccagga gattagggtc tgtgagatcc atgaagacaa cagcaccagg 420

agctcccagc atttctacta cgatggggag ctctttctct cccaaaacct ggagactaag 480

gaatggacaa tgccccagtc ctccagagct cagaccttgg ccatgaacgt caggaatttc 540

ttgaaggaag atgcaatgaa gaccaagaca cactatcacg ctatgcatgc agactgcctg 600

caggaactac ggcgatatct aaaatccggc gtagtcctga ggagaacagt gccccccatg 660

gtgcaggtga ctcgctctga ggcctctggc ggatctgggg accgtgtgac aatcacctgc 720

agagcctccc aggacgtctc cactgccgtg gcgtggtacc aacagaagcc cgggaaggca 780

cccaaactgc tcatttacag cgcatccttt ctctactctg gcgtgccgtc tcgctttagc 840

gggtccggca gcggtacaga ctttactctg accatctcct ctctgcaacc ggaggatttt 900

gcaacctatt attgccagca atcctacaca acccccccca cctttggcca gggcaccaag 960

gtggagatca agggaggttc tagccgctcc agcagctctg gaggtggagg ctctggcgga 1020

ggaggcgagg tgcaactggt ggagtctggg ggcggcctgg tccagcccgg cggaagcttg 1080

cgcctgagct gtgccgcctc cggttttacc ttcaccagca ctggaatctc ctgggtgcgc 1140

caagctcccg gcaaagggct cgaatgggtg ggccgtatct accccaccaa cggaagcacc 1200

aactatgcag acagcgtgaa ggggcgcttc actatctccg ccgacaccag caaaaacacc 1260

gcgtacctgc agatgaattc tttgagggca gaggatactg ccgtgtacta ctgcgcgagg 1320

acatacggca tttacgatct gtatgtggat tacaccgaat acgtgatgga ctattggggc 1380

cagggcactc tggtcacagt gtctagcggt ggcagctccc gcagctccag cagcggtggt 1440

ggcggtagcg gaggcggagg cgatatccag atgactcaga gtccctcttc tctgagtgct 1500

tctggcggaa gtgggcagat caccgtcaca tgtcgcgcaa gcggctttta tccttggaac 1560

atcaccctga gctggcggca ggacggcgtc agcctgtccc atgataccca acagtgggga 1620

gatgtgctcc cggacggtca gggaacttac cagacctggg ttgcaactcg catctcccag 1680

ggggaggagc agcgtttcac atgttatatg gagcactctg gccagcacag cactcatccg 1740

gtgccgtccg gaaagggatc tcatcaccat caccaccact aggatccgtt gaggtctcta 1800

aaagcgtctt cctgttctca tcacatcata tcaaggttat ataccatcaa tattgccaca 1860

gatgttactt agccttttaa tatttctcta atttagtgta tatgcaatga tagttctctg 1920

atttctgaga ttgagtttct catgtgtaat gattatttag agtttctctt tcatctgttc 1980

aaatttttgt ctagttttat tttttactga tttgtaagac ttctttttat aatctgcata 2040

ttacaattct ctttactggg gtgttgcaaa tattttctgt cattctatgg cctgactttt 2100

cttaatggtt ttttaatttt aaaaataagt cttaatattc atgcaatcta attaacaatc 2160

ttttctttgt ggttaggact ttgagtcata agaaattttt ctctacactg aagtcatgat 2220

ggcatgcttc tatattattt tctaaaagat ttaaagtttt gccttctcca tttagactta 2280

taattcactg gaattttttt gtgtgtatgg tatgacatat gggttccctt ttatttttta 2340

catataaata tatttccctg tttttctaaa aaagaaaaag atcatcattt tcccattgta 2400

aaatgccata tttttttcat aggtcactta catatatcaa tgggtctgtt tctgagctct 2460

actctatttt atcagcctca ctgtctatcc ccacacatct catgctttgc tctaaatctt 2520

gatatttagt ggaacattct ttcccatttt gttctacaag aatatttttg ttattgtctt 2580

tgggctttct atatacattt tgaaatgagg ttgacaagtt aataatcaac ctctggatta 2640

caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg 2700

atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc 2760

ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca 2820

acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac 2880

cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact 2940

catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc 3000

cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg 3060

gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc 3120

ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcct cttcgccttc gccctcagac 3180

gagtcggatc tccctttggg ccgcctcccc gcatctgtgc cttctagttg ccagccatct 3240

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 3300

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 3360

ggtggggtgg ggcaggacag caagggggag gattggcaag acaatagcag gctttgcatt 3420

tttagacatt tagaagccta tatcttgtta cagaattgga attacacaaa aattctacca 3480

tattttgaaa gcttaggttg ttctgaaaaa aacaatatat tgttttcctg ggtaaactaa 3540

aagtcccctc gaggaaaggc ccctaaagtg aaacagtgca aaacgttcaa aaactgtctg 3600

gcaatacaag ttccactttg accaaaacgg ctggcagtaa aagggttaag aagactgtca 3660

gccttgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 3720

taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 3780

cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 3840

ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 3900

aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 3960

tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 4020

gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 4080

cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 4140

ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 4200

cttgaagtgg tgggctaact acggctacac tagaagaaca gtatttggta tctgcgctct 4260

gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 4320

cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 4380

tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg acgcgcgcgt 4440

aactcacgtt aagggatttt ggtcatgagt tagaaaaact catcgagcat caaatgaaac 4500

tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat 4560

gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg 4620

attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta 4680

tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc 4740

atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca 4800

tcaaccaaac cgttattcat tcgtgattgc gcctgagcga ggcgaaatac gcgatcgctg 4860

ttaaaaggac aattacaaac aggaatcgag tgcaaccggc gcaggaacac tgccagcgca 4920

tcaacaatat tttcacctga atcaggatat tcttctaata cctggaacgc tgtttttccg 4980

gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc 5040

ggaagtggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg 5100

gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaag 5160

cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa 5220

tcagcatcca tgttggaatt taatcgcggc ctcgacgttt cccgttggat atggctcatt 5280

ttttacttcc tcaccttgtc gtattatact atgccgatat actatgccga tgattaattg 5340

tcgacactgc gggggctctg tgtggtaagc aggtcttaac ctttttactg ccaatgacgc 5400

atgggatacg tcgtggcagt aaaagggctt aaatgccaac gacgcgtccc atacgttgtt 5460

ggcattttaa ttcttctctc tgcagcggca gcatgtgccg ccgctgcaga gagtttctag 5520

cgatgacagc ccctctgggc aacgagccgg gggggctgtc tttctttatg ttttaaatgc 5580

actgacctcc cacattccct ttttagtaaa atattcagaa ataatttaaa tacatcattg 5640

caatgaaaat aaatgttttt tattaggcag aatccagatg ctcaaggccc ttcataatat 5700

cccccagttt agtagttgga cttagggaac aaaggaacct ttaatagaaa ttggacagca 5760

agaaagcgag tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca 5820

cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta gaaggggagg ttgcggggcg 5880

cggatgtctc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact ccggggagca 5940

cgacggcgct gcccagaccc ttgccctggt ggtcgggcga cacgccgacg gtggccagga 6000

accacgcggg ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg 6060

ccagccggga accgctcaac tcggccatgc gcgggccgat ctcggcgaac accgcccccg 6120

cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca 6180

ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct 6240

cgatgtggcg gtccggatcg acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg 6300

cgagggtgcg tacggccctg gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt 6360

actcggtcat ggtggcggac gaaaggcccg gagatgagga agaggagaac agcgcggcag 6420

acgtgcgctt ttgaagcgtg cagaatgccg ggcctccgga ggaccttcgg gcgcccgccc 6480

cgcccctgag cccgcccctg agcccgcccc cggacccacc ccttcccagc ctctgagccc 6540

agaaagcgaa ggagcaaagc tgctattggc cgctgcccca aaggcctacc cgcttccatt 6600

gctcagcggt gctgtccatc tgcacgagac tagtgagtcg tgctacttcc atttgtcacg 6660

tcctgcacga cgcgagctgc ggggcggggg ggaacttcct gactagggga ggagtagaag 6720

gtggcgcgaa ggggccacca aagaacggag ccggttggcg cctaccggtg gatgtggaat 6780

gtgtgcgagg ccagaggcca cttgtgtagc gccaagtgcc cagcggggct gctaaagcgc 6840

atgctccaga ctgccttggg aaaagcgcct cccctacccg gtagagaaac ttgatctgtc 6900

gccgcaattc aaacttcgtg aggctccggt gcccgtcagt gacctgctat actctggaga 6960

cgacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca 7020

ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg 7080

gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtccg 7140

ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc 7200

ttacgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatgctg 7260

atgcggtttt ggcagtacac caatgggcgt ggatagcggt ttgactcacg gggatttcca 7320

agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt 7380

ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg 7440

gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag aggccatcca 7500

cgctgttttg acctccatag tggacaccgg gaccgatcca gcctccgcgt ctcagg 7556

<210> 40

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide wt MICA-Fv

<400> 40

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

145 150 155 160

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 41

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICwed-Fv

<400> 41

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 42

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICv20-Fv

<400> 42

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Ala Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Gln Thr Asp Thr His Tyr Arg Ala Met

145 150 155 160

His Ala Asp Cys Leu Phe Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 43

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICv25-Fv

<400> 43

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 44

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICv27-Fv

<400> 44

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 45

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICv28-Fv

<400> 45

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 46

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICv42-Fv

<400> 46

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 47

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICv48-Fc

<400> 47

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Ala Thr Asp Thr His Tyr Ile Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Ala Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 48

<211> 558

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide MICv49-Fv

<400> 48

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Thr Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gln Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Phe Thr Asp Thr His Tyr Arg Ala Met

145 150 155 160

Thr Ala Asp Cys Leu Thr Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu

180 185 190

Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

195 200 205

Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

210 215 220

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val

225 230 235 240

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

245 250 255

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

260 265 270

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

275 280 285

Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

305 310 315 320

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

325 330 335

Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

340 345 350

Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala

355 360 365

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn

370 375 380

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

385 390 395 400

Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr

405 410 415

Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

420 425 430

Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser

435 440 445

Gly Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

450 455 460

Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly

465 470 475 480

Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser

485 490 495

Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln

500 505 510

Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu

515 520 525

Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His

530 535 540

Pro Val Pro Ser Gly Lys Gly Ser His His His His His

545 550 555

<210> 49

<211> 137

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic ectodomain of NKG2D

<400> 49

Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr

1 5 10 15

Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr

20 25 30

Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys

35 40 45

Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln

50 55 60

Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His

65 70 75 80

Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser

85 90 95

Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu

100 105 110

Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn

115 120 125

Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 50

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 50

ctgtctagag ccgccaacat ggggctgggc ccggtcttcc 40

<210> 51

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 51

aacggatcct acacagtcct ttgcatgcag 30

<210> 52

<211> 6362

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide, pD2509-CMV-Avi-His-natural

NKG2D ectodomain

<400> 52

ggagagacca cacccaagct gtctagagcc gccaacatgg ggctgggccc ggtcttcctg 60

cttctggctg gcatcttccc ttttgcacct ccgggagctg ctgctgagcc cccacatcat 120

caccaccatg gccttaacga catcttcgaa gctcaaaaga tcgaatggca tgaaaactca 180

ttattcaacc aagaagttca aattcccttg accgaaagtt actgtggccc atgtcctaaa 240

aactggatat gttacaaaaa taactgctac caattttttg atgagagtaa aaactggtat 300

gagagccagg cttcttgtat gtctcaaaat gccagccttc tgaaagtata cagcaaagag 360

gaccaggatt tacttaaact ggtgaagtca tatcattgga tgggactagt acacattcca 420

acaaatggat cttggcagtg ggaagatggc tccattctct cacccaacct actaacaata 480

attgaaatgc agaagggaga ctgtgcactc tatgcctcga gctttaaagg ctatatagaa 540

aactgttcaa ctccaaatac atacatctgc atgcaaagga ctgtgtagga tccgttgagg 600

tctctaaaag cgtcttcctg ttctcatcac atcatatcaa ggttatatac catcaatatt 660

gccacagatg ttacttagcc ttttaatatt tctctaattt agtgtatatg caatgatagt 720

tctctgattt ctgagattga gtttctcatg tgtaatgatt atttagagtt tctctttcat 780

ctgttcaaat ttttgtctag ttttattttt tactgatttg taagacttct ttttataatc 840

tgcatattac aattctcttt actggggtgt tgcaaatatt ttctgtcatt ctatggcctg 900

acttttctta atggtttttt aattttaaaa ataagtctta atattcatgc aatctaatta 960

acaatctttt ctttgtggtt aggactttga gtcataagaa atttttctct acactgaagt 1020

catgatggca tgcttctata ttattttcta aaagatttaa agttttgcct tctccatta 1080

gacttataat tcactggaat ttttttgtgt gtatggtatg acatatgggt tcccttttat 1140

tttttacata taaatatatt tccctgtttt tctaaaaaag aaaaagatca tcattttccc 1200

attgtaaaat gccatatttt tttcataggt cacttacata tatcaatggg tctgtttctg 1260

agctctactc tattttatca gcctcactgt ctatccccac acatctcatg ctttgctcta 1320

aatcttgata tttagtggaa cattctttcc cattttgttc tacaagaata tttttgttat 1380

tgtctttggg ctttctatat acattttgaa atgaggttga caagttaata atcaacctct 1440

ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 1500

atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 1560

tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 1620

caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 1680

tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 1740

ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 1800

caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 1860

cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 1920

ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcctcttc gccttcgccc 1980

tcagacgagt cggatctccc tttgggccgc ctccccgcat ctgtgccttc tagttgccag 2040

ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 2100

gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 2160

ctggggggtg gggtggggca ggacagcaag ggggaggatt ggcaagacaa tagcaggctt 2220

tgcattttta gacatttaga agcctatatc ttgttacaga attggaatta cacaaaaatt 2280

ctaccatatt ttgaaagctt aggttgttct gaaaaaaaca atatattgtt ttcctgggta 2340

aactaaaagt cccctcgagg aaaggcccct aaagtgaaac agtgcaaaac gttcaaaaac 2400

tgtctggcaa tacaagttcc actttgacca aaacggctgg cagtaaaagg gttaagaaga 2460

ctgtcagcct tgagcggtat cagctcactc aaaggcggta atacggttat cccagaatc 2520

aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 2580

aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 2640

tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 2700

ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 2760

cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 2820

ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 2880

ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 2940

gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 3000

agagttcttg aagtggtggg ctaactacgg ctacactaga agaacagtat ttggtatctg 3060

cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 3120

aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 3180

aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgacgc 3240

gcgcgtaact cacgttaagg gattttggtc atgagttaga aaaactcatc gagcatcaaa 3300

tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc 3360

tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg 3420

tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata 3480

aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt 3540

ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca 3600

ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgaggcg aaatacgcga 3660

tcgctgttaa aaggacaatt acaaacagga atcgagtgca accggcgcag gaacactgcc 3720

agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaacgctgtt 3780

tttccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg 3840

atggtcggaa gtggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca 3900

tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca 3960

tacaagcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca 4020

tataaatcag catccatgtt ggaatttaat cgcggcctcg acgtttcccg ttggatatgg 4080

ctcatttttt acttcctcac cttgtcgtat tatactatgc cgatatacta tgccgatgat 4140

taattgtcga cactgcgggg gctctgtgtg gtaagcaggt cttaaccttt ttactgccaa 4200

tgacgcatgg gatacgtcgt ggcagtaaaa gggcttaaat gccaacgacg cgtcccatac 4260

gttgttggca ttttaattct tctctctgca gcggcagcat gtgccgccgc tgcagagagt 4320

ttctagcgat gacagcccct ctgggcaacg agccgggggg gctgtctttc tttatgtttt 4380

aaatgcactg acctcccaca ttcccttttt agtaaaatat tcagaaataa tttaaataca 4440

tcattgcaat gaaaataaat gttttttatt aggcagaatc cagatgctca aggcccttca 4500

taatatcccc cagtttagta gttggactta gggaacaaag gaacctttaa tagaaattgg 4560

acagcaagaa agcgagtcag gcaccgggct tgcgggtcat gcaccaggtg cgcggtcctt 4620

cgggcacctc gacgtcggcg gtgacggtga agccgagccg ctcgtagaag gggaggttgc 4680

ggggcgcgga tgtctccagg aaggcgggca ccccggcgcg ctcggccgcc tccactccgg 4740

ggagcacgac ggcgctgccc agacccttgc cctggtggtc gggcgacacg ccgacggtgg 4800

caggaacca cgcgggctcc ttgggccggt gcggcgccag gaggccttcc atctgttgct 4860

gcgcggccag ccgggaaccg ctcaactcgg ccatgcgcgg gccgatctcg gcgaacaccg 4920

cccccgcttc gacgctctcc ggcgtggtcc agaccgccac cgcggcgccg tcgtccgcga 4980

cccacacctt gccgatgtcg agcccgacgc gcgtgaggaa gagttcttgc agctcggtga 5040

cccgctcgat gtggcggtcc ggatcgacgg tgtggcgcgt ggcggggtag tcggcgaacg 5100

cggcggcgag ggtgcgtacg gccctgggga cgtcgtcgcg ggtggcgagg cgcaccgtgg 5160

gcttgtactc ggtcatggtg gcggacgaaa ggcccggaga tgaggaagag gagaacagcg 5220

cggcagacgt gcgcttttga agcgtgcaga atgccgggcc tccggaggac cttcgggcgc 5280

ccgccccgcc cctgagcccg cccctgagcc cgcccccgga cccacccctt cccagcctct 5340

gagcccagaa agcgaaggag caaagctgct attggccgct gccccaaagg cctacccgct 5400

tccattgctc agcggtgctg tccatctgca cgagactagt gagtcgtgct acttccattt 5460

gtcacgtcct gcacgacgcg agctgcgggg cgggggggaa cttcctgact aggggaggag 5520

tagaaggtgg cgcgaagggg ccaccaaaga acggagccgg ttggcgccta ccggtggatg 5580

tggaatgtgt gcgaggccag aggccacttg tgtagcgcca agtgcccagc ggggctgcta 5640

aagcgcatgc tccagactgc cttgggaaaa gcgcctcccc tacccggtag agaaacttga 5700

tctgtcgccg caattcaaac ttcgtgaggc tccggtgccc gtcagtgacc tgctatactc 5760

tggagacgac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 5820

acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 5880

tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 5940

agtccgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 6000

atgaccttac gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 6060

atgctgatgc ggttttggca gtacaccaat gggcgtggat agcggtttga ctcacgggga 6120

tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg 6180

gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg taggcgtgta 6240

cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagaggc 6300

catccacgct gttttgacct ccatagtgga caccgggacc gatccagcct ccgcgtctca 6360

gg 6362

<210> 53

<211> 160

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, His-avitag-natural ectodomain of NKG2D

<400> 53

Glu Pro His His His His His Gly Leu Asn Asp Ile Phe Glu Ala

1 5 10 15

Gln Lys Ile Glu Trp His Glu Asn Ser Leu Phe Asn Gln Glu Val Gln

20 25 30

Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile

35 40 45

Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp

50 55 60

Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys

65 70 75 80

Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr

85 90 95

His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp

100 105 110

Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met

115 120 125

Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile

130 135 140

Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

145 150 155 160

<210> 54

<211> 160

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, His-avitag-unnatural ectodomain NKG2D Y152A

<400> 54

Glu Pro His His His His His Gly Leu Asn Asp Ile Phe Glu Ala

1 5 10 15

Gln Lys Ile Glu Trp His Glu Asn Ser Leu Phe Asn Gln Glu Val Gln

20 25 30

Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile

35 40 45

Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp

50 55 60

Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys

65 70 75 80

Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala

85 90 95

His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp

100 105 110

Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met

115 120 125

Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile

130 135 140

Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

145 150 155 160

<210> 55

<211> 160

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, His-avitag-unnatural ectodomain NKG2D Y199A

<400> 55

Glu Pro His His His His His Gly Leu Asn Asp Ile Phe Glu Ala

1 5 10 15

Gln Lys Ile Glu Trp His Glu Asn Ser Leu Phe Asn Gln Glu Val Gln

20 25 30

Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile

35 40 45

Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp

50 55 60

Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys

65 70 75 80

Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr

85 90 95

His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp

100 105 110

Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met

115 120 125

Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Ala Ile

130 135 140

Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

145 150 155 160

<210> 56

<211> 160

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, His-avitag-unnatural ectodomain of NKG2D

Y152A +Y199A

<400> 56

Glu Pro His His His His His Gly Leu Asn Asp Ile Phe Glu Ala

1 5 10 15

Gln Lys Ile Glu Trp His Glu Asn Ser Leu Phe Asn Gln Glu Val Gln

20 25 30

Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile

35 40 45

Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp

50 55 60

Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys

65 70 75 80

Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala

85 90 95

His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp

100 105 110

Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met

115 120 125

Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Ala Ile

130 135 140

Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

145 150 155 160

<210> 57

<211> 7640

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide expressing vector wt MIC-Fc

<400> 57

ggagagacca cacccaagct gtctagagcc gccaacatgg ggctgggccc ggtcttcctg 60

cttctggctg gcatcttccc ttttgcacct ccgggagctg ctgctgagcc ccacagtctt 120

cgttataacc tcacggtgct gtcctgggat ggatctgtgc agtcagggtt tctcactgag 180

gtacatctgg atggtcagcc cttcctgcgc tgtgacaggc agaaatgcag ggcaaagccc 240

cagggacagt gggcagaaga tgtcctggga aataagacat gggacagaga gaccagagac 300

ttgacaggt ggggaaagga cctcaggatg accctggctc atatcaagga ccagaaagaa 360

ggcttgcatt ccctccagga gattagggtc tgtgagatcc atgaagacaa cagcaccagg 420

agctcccagc atttctacta cgatggggag ctctttctct cccaaaacct ggagactaag 480

gaatggacaa tgccccagtc ctccagagct cagaccttgg ccatgaacgt caggaatttc 540

ttgaaggaag atgcaatgga gaccgataca cactatcacg ctatgcatgc agactgcctg 600

caggaactac ggcgatatct aaaatccggc gtagtcctga ggagaacagt gccccccatg 660

gtgaatgtca cccgcagcga ggcctcagag ggcaacatta ccgtgacatg cagggcttct 720

ggcttctatc cctggaatat cacactgagc tggcgtcagg atggggtatc tttgagccac 780

gacacccagc agtgggggga tgtcctgcct gatgggaatg gaacctacca gacctgggtg 840

gccaccagga tttgccaagg agaggagcag aggttcacct gctacatgga acacagcggg 900

aatcacagca ctcaccctgt gccctctggg aaaatcgaag gacgcatgga cccaaagagt 960

tgcgacaaaa ctcacacatg cccaccgtgc ccaggtaagc cagcccaggc ctcgccctcc 1020

agctcaaggc gggacaggtg ccctagagta gcctgcatcc agggacaggc cccagccggg 1080

tgctgacacg tccacctcca tctcttcctc agcacctgaa ctcctggggg gaccgtcagt 1140

cttcctcttc cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac 1200

atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga 1260

cggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta 1320

ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa 1380

gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa 1440

aggtgggacc cgtggggtgc gagggccaca tggacagagg ccggctcggc ccaccctctg 1500

ccctgagagt gactgctgta ccaacctctg tccctacagg gcagccccga gaaccacagg 1560

tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc 1620

tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcagccgg 1680

agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc ttcctctaca 1740

gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca tgctccgtga 1800

tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct ccgggtaaat 1860

gataggatcc ggttgaggtc tctaaaagcg tcttcctgtt ctcatcacat catatcaagg 1920

ttatatacca tcaatattgc cacagatgtt acttagcctt ttaatatttc tctaatttag 1980

tgtatatgca atgatagttc tctgatttct gagattgagt ttctcatgtg taatgattat 2040

ttagagtttc tctttcatct gttcaaattt ttgtctagtt ttatttttta ctgatttgta 2100

agacttcttt ttataatctg catattacaa ttctctttac tggggtgttg caaatatttt 2160

ctgtcattct atggcctgac ttttcttaat ggttttttaa ttttaaaaat aagtcttaat 2220

attcatgcaa tctaattaac aatcttttct ttgtggttag gactttgagt cataagaaat 2280

ttttctctac actgaagtca tgatggcatg cttctatatt attttctaaa agatttaaag 2340

ttttgccttc tccatttaga cttataattc actggaattt ttttgtgtgt atggtatgac 2400

atatgggttc ccttttattt tttacatata aatatatttc cctgtttttc taaaaaagaa 2460

aaagatcatc attttcccat tgtaaaatgc catatttttt tcataggtca cttacatata 2520

tcaatgggtc tgtttctgag ctctactcta ttttatcagc ctcactgtct atccccacac 2580

atctcatgct ttgctctaaa tcttgatatt tagtggaaca ttctttccca ttttgttcta 2640

caagaatatt tttgttattg tctttgggct ttctatatac attttgaaat gaggttgaca 2700

agttaataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta 2760

tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc 2820

ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga 2880

ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac 2940

ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc 3000

cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacagggc 3060

tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg 3120

gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc 3180

ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc 3240

gcctcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatct 3300

gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3360

gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3420

agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3480

caagacaata gcaggctttg catttttaga catttagaag cctatatctt gttacagaat 3540

tggaattaca caaaaattct accatatttt gaaagcttag gttgttctga aaaaaacaat 3600

atattgtttt cctgggtaaa ctaaaagtcc cctcgaggaa aggcccctaa agtgaaacag 3660

tgcaaaacgt tcaaaaactg tctggcaata caagttccac tttgaccaaa acggctggca 3720

gtaaaagggt taagaagact gtcagccttg agcggtatca gctcactcaa aggcggtaat 3780

acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 3840

aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 3900

tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 3960

aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4020

gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 4080

acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4140

accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4200

ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4260

gtatgtaggc ggtgctacag agttcttgaa gtggtgggct aactacggct acactagaag 4320

aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4380

ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4440

gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4500

cgctcagtgg aacgacgcgc gcgtaactca cgttaaggga ttttggtcat gagttagaaa 4560

aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat 4620

ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg 4680

gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat 4740

ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc 4800

ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta 4860

cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga 4920

gcgaggcgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgagtgcaac 4980

cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct 5040

aatacctgga acgctgtttt tccggggatc gcagtggtga gtaaccatgc atcatcagga 5100

gtacggataa aatgcttgat ggtcggaagt ggcataaatt ccgtcagcca gtttagtctg 5160

accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct 5220

ggcgcatcgg gcttcccata caagcgatag attgtcgcac ctgattgccc gacattatcg 5280

cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgac 5340

gtttcccgtt ggatatggct cattttttac ttcctcacct tgtcgtatta tactatgccg 5400

atatactatg ccgatgatta attgtcgaca ctgcgggggc tctgtgtggt aagcaggtct 5460

taaccttttt actgccaatg acgcatggga tacgtcgtgg cagtaaaagg gcttaaatgc 5520

caacgacgcg tcccatacgt tgttggcatt ttaattcttc tctctgcagc ggcagcatgt 5580

gccgccgctg cagagagttt ctagcgatga cagcccctct gggcaacgag ccgggggggc 5640

tgtctttctt tatgttttaa atgcactgac ctcccacatt ccctttttag taaaatattc 5700

agaaataatt taaatacatc attgcaatga aaataaatgt tttttattag gcagaatcca 5760

gatgctcaag gcccttcata atatccccca gtttagtagt tggacttagg gaacaaagga 5820

acctttaata gaaattggac agcaagaaag cgagtcaggc accgggcttg cgggtcatgc 5880

accaggtgcg cggtccttcg ggcacctcga cgtcggcggt gacggtgaag ccgagccgct 5940

cgtagaaggg gaggttgcgg ggcgcggatg tctccaggaa ggcgggcacc ccggcgcgct 6000

cggccgcctc cactccgggg agcacgacgg cgctgcccag acccttgccc tggtggtcgg 6060

gcgacacgcc gacggtggcc aggaaccacg cgggctcctt gggccggtgc ggcgccagga 6120

ggccttccat ctgttgctgc gcggccagcc gggaaccgct caactcggcc atgcgcgggc 6180

cgatctcggc gaacaccgcc cccgcttcga cgctctccgg cgtggtccag accgccaccg 6240

cggcgccgtc gtccgcgacc cacaccttgc cgatgtcgag cccgacgcgc gtgaggaaga 6300

gttcttgcag ctcggtgacc cgctcgatgt ggcggtccgg atcgacggtg tggcgcgtgg 6360

cggggtagtc ggcgaacgcg gcggcgaggg tgcgtacggc cctggggacg tcgtcgcggg 6420

tggcgaggcg caccgtgggc ttgtactcgg tcatggtggc ggacgaaagg cccggagatg 6480

aggaagagga gaacagcgcg gcagacgtgc gcttttgaag cgtgcagaat gccgggcctc 6540

cggaggacct tcgggcgccc gccccgcccc tgagcccgcc cctgagcccg cccccggacc 6600

caccccttcc cagcctctga gcccagaaag cgaaggagca aagctgctat tggccgctgc 6660

cccaaaggcc tacccgcttc cattgctcag cggtgctgtc catctgcacg agactagtga 6720

gtcgtgctac ttccatttgt cacgtcctgc acgacgcgag ctgcggggcg ggggggaact 6780

tcctgactag gggaggagta gaaggtggcg cgaaggggcc accaaagaac ggagccggtt 6840

ggcgcctacc ggtggatgtg gaatgtgtgc gaggccagag gccacttgtg tagcgccaag 6900

tgcccagcgg ggctgctaaa gcgcatgctc cagactgcct tgggaaaagc gcctccccta 6960

cccggtagag aaacttgatc tgtcgccgca attcaaactt cgtgaggctc cggtgcccgt 7020

cagtgacctg ctatactctg gagacgactt acggtaaatg gcccgcctgg ctgaccgccc 7080

aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 7140

actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 7200

caagtgtatc atatgccaag tccgccccct attgacgtca atgacggtaa atggcccgcc 7260

tggcattatg cccagtacat gaccttacgg gactttccta cttggcagta catctacgta 7320

ttagtcatcg ctattaccat gctgatgcgg ttttggcagt acaccaatgg gcgtggatag 7380

cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 7440

tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa 7500

atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 7560

cagatcgcct ggagaggcca tccacgctgt tttgacctcc atagtggaca ccgggaccga 7620

tccagcctcc gcgtctcagg 7640

<210> 58

<211> 438

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, MICA-Fc

<400> 58

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met

145 150 155 160

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

180 185 190

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

195 200 205

Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

210 215 220

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

225 230 235 240

Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg

245 250 255

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

260 265 270

Pro Ser Gly Lys Ile Glu Gly Arg Met Asp Pro Lys Ser Cys Asp Lys

275 280 285

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

290 295 300

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

305 310 315 320

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

325 330 335

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

340 345 350

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

355 360 365

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

370 375 380

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

385 390 395 400

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

405 410 415

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

420 425 430

Cys Leu Val Lys Gly Phe

435

<210> 59

<211> 513

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, MICwed-Fc

<400> 59

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

180 185 190

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

195 200 205

Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

210 215 220

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

225 230 235 240

Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg

245 250 255

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

260 265 270

Pro Ser Gly Lys Ile Glu Gly Arg Met Asp Pro Lys Ser Cys Asp Lys

275 280 285

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

290 295 300

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

305 310 315 320

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

325 330 335

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

340 345 350

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

355 360 365

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

370 375 380

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

385 390 395 400

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

405 410 415

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

420 425 430

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

435 440 445

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

450 455 460

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

465 470 475 480

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

485 490 495

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

500 505 510

Lys

<210> 60

<211> 513

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, MICv25-Fc

<400> 60

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met

145 150 155 160

Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu

180 185 190

Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr

195 200 205

Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser

210 215 220

His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr

225 230 235 240

Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg

245 250 255

Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val

260 265 270

Pro Ser Gly Lys Ile Glu Gly Arg Met Asp Pro Lys Ser Cys Asp Lys

275 280 285

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

290 295 300

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

305 310 315 320

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

325 330 335

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

340 345 350

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

355 360 365

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

370 375 380

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

385 390 395 400

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

405 410 415

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

420 425 430

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

435 440 445

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

450 455 460

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

465 470 475 480

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

485 490 495

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

500 505 510

Lys

<210> 61

<211> 189

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP2 alpha1-alpha2

<400> 61

Ala Ala Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys

1 5 10 15

Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu

20 25 30

Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val

35 40 45

Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln

50 55 60

Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Arg

65 70 75 80

Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln

85 90 95

Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser

100 105 110

Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu

115 120 125

Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu

130 135 140

Lys Trp Glu Asn Asp Lys Val Val Ala Met Ser Phe His Tyr Phe Ser

145 150 155 160

Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp

165 170 175

Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro Pro Met Val

180 185

<210> 62

<211> 187

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP3 alpha1-alpha2

<400> 62

Ala Ala Glu Pro His Ser Leu Trp Tyr Asn Phe Thr Ile Ile His Leu

1 5 10 15

Pro Arg His Gly Gln Gln Trp Cys Glu Val Gln Ser Gln Val Asp Gln

20 25 30

Lys Asn Phe Leu Ser Tyr Asp Cys Gly Ser Asp Lys Val Leu Ser Met

35 40 45

Gly His Leu Glu Glu Gln Leu Tyr Ala Thr Asp Ala Trp Gly Lys Gln

50 55 60

Leu Glu Met Leu Arg Glu Val Gly Gln Arg Leu Arg Leu Glu Leu Ala

65 70 75 80

Asp Thr Glu Leu Glu Asp Phe Thr Pro Ser Gly Pro Leu Thr Leu Gln

85 90 95

Val Arg Met Ser Cys Glu Ser Glu Ala Asp Gly Tyr Ile Arg Gly Ser

100 105 110

Trp Gln Phe Ser Phe Asp Gly Arg Lys Phe Leu Leu Phe Asp Ser Asn

115 120 125

Asn Arg Lys Trp Thr Val Val His Ala Gly Ala Arg Arg Met Lys Glu

130 135 140

Lys Trp Glu Lys Asp Ser Gly Leu Thr Thr Phe Phe Lys Met Val Ser

145 150 155 160

Met Arg Asp Cys Lys Ser Trp Leu Arg Asp Phe Leu Met His Arg Lys

165 170 175

Lys Arg Leu Glu Pro Thr Ala Pro Pro Met Val

180 185

<210> 63

<211> 186

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP2 R80W

<400> 63

Ala Ala Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys

1 5 10 15

Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu

20 25 30

Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val

35 40 45

Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln

50 55 60

Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp

65 70 75 80

Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln

85 90 95

Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser

100 105 110

Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu

115 120 125

Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu

130 135 140

Lys Trp Glu Asn Asp Lys Val Val Ala Met Ser Phe His Tyr Phe Ser

145 150 155 160

Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp

165 170 175

Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro

180 185

<210> 64

<211> 186

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP2 V151D

<400> 64

Ala Ala Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys

1 5 10 15

Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu

20 25 30

Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val

35 40 45

Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln

50 55 60

Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Arg

65 70 75 80

Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln

85 90 95

Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser

100 105 110

Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu

115 120 125

Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu

130 135 140

Lys Trp Glu Asn Asp Lys Asp Val Ala Met Ser Phe His Tyr Phe Ser

145 150 155 160

Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp

165 170 175

Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro

180 185

<210> 65

<211> 184

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP3 R162G

<400> 65

Ala Ala Glu Pro His Ser Leu Trp Tyr Asn Phe Thr Ile Ile His Leu

1 5 10 15

Pro Arg His Gly Gln Gln Trp Cys Glu Val Gln Ser Gln Val Asp Gln

20 25 30

Lys Asn Phe Leu Ser Tyr Asp Cys Gly Ser Asp Lys Val Leu Ser Met

35 40 45

Gly His Leu Glu Glu Gln Leu Tyr Ala Thr Asp Ala Trp Gly Lys Gln

50 55 60

Leu Glu Met Leu Arg Glu Val Gly Gln Arg Leu Arg Leu Glu Leu Ala

65 70 75 80

Asp Thr Glu Leu Glu Asp Phe Thr Pro Ser Gly Pro Leu Thr Leu Gln

85 90 95

Val Arg Met Ser Cys Glu Ser Glu Ala Asp Gly Tyr Ile Arg Gly Ser

100 105 110

Trp Gln Phe Ser Phe Asp Gly Arg Lys Phe Leu Leu Phe Asp Ser Asn

115 120 125

Asn Arg Lys Trp Thr Val Val His Ala Gly Ala Arg Arg Met Lys Glu

130 135 140

Lys Trp Glu Lys Asp Ser Gly Leu Thr Thr Phe Phe Lys Met Val Ser

145 150 155 160

Met Gly Asp Cys Lys Ser Trp Leu Arg Asp Phe Leu Met His Arg Lys

165 170 175

Lys Arg Leu Glu Pro Thr Ala Pro

180

<210> 66

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, MICA25.17

<400> 66

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Thr Thr Leu Leu Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Ile Gly Tyr Arg Leu Met

145 150 155 160

Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val

180

<210> 67

<211> 182

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, MICA25.18

<400> 67

Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly

1 5 10 15

Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro

20 25 30

Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln

35 40 45

Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg

50 55 60

Asp Leu Thr Gly Trp Gly Thr Phe Leu Arg Met Thr Leu Ala His Ile

65 70 75 80

Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys

85 90 95

Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr

100 105 110

Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr

115 120 125

Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn

130 135 140

Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Arg Ser Gly Leu Leu Met

145 150 155 160

Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val

165 170 175

Val Leu Arg Arg Thr Val

180

<210> 68

<211> 186

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP2.S1

<400> 68

Ala Ala Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys

1 5 10 15

Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu

20 25 30

Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val

35 40 45

Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln

50 55 60

Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp

65 70 75 80

Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln

85 90 95

Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser

100 105 110

Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu

115 120 125

Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu

130 135 140

Lys Trp Glu Asn Asp Lys Val Val Ala Thr Thr Leu Tyr Thr Trp Ser

145 150 155 160

Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp

165 170 175

Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro

180 185

<210> 69

<211> 186

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP2.S2

<400> 69

Ala Ala Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys

1 5 10 15

Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu

20 25 30

Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val

35 40 45

Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln

50 55 60

Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp

65 70 75 80

Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln

85 90 95

Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser

100 105 110

Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu

115 120 125

Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu

130 135 140

Lys Trp Glu Asn Asp Lys Val Val Ala Thr Leu Met Arg Ile Trp Ser

145 150 155 160

Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp

165 170 175

Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro

180 185

<210> 70

<211> 186

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP2.S3

<400> 70

Ala Ala Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys

1 5 10 15

Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu

20 25 30

Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val

35 40 45

Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln

50 55 60

Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp

65 70 75 80

Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln

85 90 95

Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser

100 105 110

Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu

115 120 125

Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu

130 135 140

Lys Trp Glu Asn Asp Lys Val Val Ala Thr Lys Leu Tyr Leu Trp Ser

145 150 155 160

Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp

165 170 175

Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro

180 185

<210> 71

<211> 184

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP3.S1

<400> 71

Ala Ala Glu Pro His Ser Leu Trp Tyr Asn Phe Thr Ile Ile His Leu

1 5 10 15

Pro Arg His Gly Gln Gln Trp Cys Glu Val Gln Ser Gln Val Asp Gln

20 25 30

Lys Asn Phe Leu Ser Tyr Asp Cys Gly Ser Asp Lys Val Leu Ser Met

35 40 45

Gly His Leu Glu Glu Gln Leu Tyr Ala Thr Asp Ala Trp Gly Lys Gln

50 55 60

Leu Glu Met Leu Arg Glu Val Gly Gln Arg Leu Arg Leu Glu Leu Ala

65 70 75 80

Asp Thr Glu Leu Glu Asp Phe Thr Pro Ser Gly Pro Leu Thr Leu Gln

85 90 95

Val Arg Met Ser Cys Glu Ser Glu Ala Asp Gly Tyr Ile Arg Gly Ser

100 105 110

Trp Gln Phe Ser Phe Asp Gly Arg Lys Phe Leu Leu Phe Asp Ser Asn

115 120 125

Asn Arg Lys Trp Thr Val Val His Ala Gly Ala Arg Arg Met Lys Glu

130 135 140

Lys Trp Glu Lys Asp Ser Gly Leu Thr Thr Asp Leu Ile Arg Arg Ser

145 150 155 160

Met Gly Asp Cys Lys Ser Trp Leu Arg Asp Phe Leu Met His Arg Lys

165 170 175

Lys Arg Leu Glu Pro Thr Ala Pro

180

<210> 72

<211> 184

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP3.S2

<400> 72

Ala Ala Glu Pro His Ser Leu Trp Tyr Asn Phe Thr Ile Ile His Leu

1 5 10 15

Pro Arg His Gly Gln Gln Trp Cys Glu Val Gln Ser Gln Val Asp Gln

20 25 30

Lys Asn Phe Leu Ser Tyr Asp Cys Gly Ser Asp Lys Val Leu Ser Met

35 40 45

Gly His Leu Glu Glu Gln Leu Tyr Ala Thr Asp Ala Trp Gly Lys Gln

50 55 60

Leu Glu Met Leu Arg Glu Val Gly Gln Arg Leu Arg Leu Glu Leu Ala

65 70 75 80

Asp Thr Glu Leu Glu Asp Phe Thr Pro Ser Gly Pro Leu Thr Leu Gln

85 90 95

Val Arg Met Ser Cys Glu Ser Glu Ala Asp Gly Tyr Ile Arg Gly Ser

100 105 110

Trp Gln Phe Ser Phe Asp Gly Arg Lys Phe Leu Leu Phe Asp Ser Asn

115 120 125

Asn Arg Lys Trp Thr Val Val His Ala Gly Ala Arg Arg Met Lys Glu

130 135 140

Lys Trp Glu Lys Asp Ser Gly Leu Thr Thr Tyr Phe Tyr Leu Arg Ser

145 150 155 160

Met Gly Asp Cys Lys Ser Trp Leu Arg Asp Phe Leu Met His Arg Lys

165 170 175

Lys Arg Leu Glu Pro Thr Ala Pro

180

<210> 73

<211> 657

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, R3 HC25.17

<400> 73

Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala

1 5 10 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

20 25 30

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr

35 40 45

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

50 55 60

Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val

65 70 75 80

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

85 90 95

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

100 105 110

Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr

115 120 125

Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

130 135 140

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

145 150 155 160

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

165 170 175

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

180 185 190

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

195 200 205

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

210 215 220

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

225 230 235 240

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

245 250 255

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

260 265 270

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

275 280 285

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

290 295 300

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

305 310 315 320

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

325 330 335

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

340 345 350

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

355 360 365

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

370 375 380

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

385 390 395 400

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

405 410 415

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

420 425 430

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

435 440 445

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

450 455 460

Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Glu Pro His Ser

465 470 475 480

Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly Ser Val Gln Ser

485 490 495

Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro Phe Leu Arg Cys

500 505 510

Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln Trp Ala Glu Asp

515 520 525

Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg Asp Leu Thr Gly

530 535 540

Trp Gly Thr Thr Leu Leu Met Thr Leu Ala His Ile Lys Asp Gln Lys

545 550 555 560

Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys Glu Ile His Glu

565 570 575

Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr Asp Gly Glu Leu

580 585 590

Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr Met Pro Gln Ser

595 600 605

Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn Phe Leu Lys Glu

610 615 620

Asp Ala Met Glu Thr Asp Ile Gly Tyr Arg Leu Met Arg Ala Asp Cys

625 630 635 640

Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val Val Leu Arg Arg

645 650 655

Th

<210> 74

<211> 656

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, R3 HC.U2S3

<400> 74

Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala

1 5 10 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

20 25 30

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr

35 40 45

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

50 55 60

Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val

65 70 75 80

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

85 90 95

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

100 105 110

Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr

115 120 125

Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

130 135 140

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

145 150 155 160

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

165 170 175

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

180 185 190

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

195 200 205

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

210 215 220

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

225 230 235 240

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

245 250 255

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

260 265 270

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

275 280 285

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

290 295 300

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

305 310 315 320

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

325 330 335

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

340 345 350

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

355 360 365

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

370 375 380

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

385 390 395 400

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

405 410 415

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

420 425 430

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

435 440 445

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

450 455 460

Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Glu Pro His Ser

465 470 475 480

Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg Pro Gly Pro Arg

485 490 495

Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu His Tyr

500 505 510

Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro Leu Gly Lys Lys

515 520 525

Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro Val Leu Arg Glu

530 535 540

Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile Gln Leu Glu Asn

545 550 555 560

Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu

565 570 575

Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln Phe Ser Phe Asp

580 585 590

Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg Met Trp Thr Thr

595 600 605

Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn Asp Lys

610 615 620

Val Val Ala Thr Lys Leu Tyr Leu Trp Ser Met Gly Asp Cys Ile Gly

625 630 635 640

Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr Leu Glu Pro Ser

645 650 655

<210> 75

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D Y152A

<400> 75

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 76

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D Y199A

<400> 76

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Ala Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 77

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D Y152A/Y199A

<400> 77

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Ala Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 78

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D Y199F eNKG2D1

<400> 78

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 79

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D Y152S eNKG2D2

<400> 79

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ser His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 80

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D Y152T eNKG2D3

<400> 80

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Thr His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 81

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D Y152V eNKG2D4

<400> 81

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Val His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 82

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D

Y152A/Y199F eNKG2D5

<400> 82

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 83

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D

Y152L/Y199F eNKG2D6

<400> 83

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Leu His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 84

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D

Y152S/Y199F eNKG2D7

<400> 84

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ser His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 85

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D

Y152T/Y199F eNKG2D8

<400> 85

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Thr His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 86

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D

Y152V/Y199F eNKG2D9

<400> 86

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Val His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 87

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D Y199D eNKG2D10

<400> 87

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Asp Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 88

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D Y199E eNKG2D11

<400> 88

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Glu Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 89

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D

Y152D/Y199D eNKG2D12

<400> 89

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Asp His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Asp Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 90

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D

Y152E/Y199E eNKG2D13

<400> 90

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Glu His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Glu Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 91

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D Y152L eNKG2D14

<400> 91

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Leu His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 92

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide unnatural ectodomain NKG2D

Y152F/Y199F eNKG2D15

<400> 92

Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

1 5 10 15

Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

20 25 30

Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

35 40 45

Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

50 55 60

Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Phe His Trp Met Gly Leu

65 70 75 80

Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

85 90 95

Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys

100 105 110

Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 93

<211> 237

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic human IgG1 Fc peptide with IEGR linker

<400> 93

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg

225 230 235

<210> 94

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein human IgG1 Fc-NKG2D

<400> 94

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 95

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein human IgG1 Fc-NKG2D Y152A

<400> 95

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 96

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide Fc human IgG1-ectodomain NKG2D Y199A

<400> 96

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Ala Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 97

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide Fc human IgG1-ectodomain NKG2D Y152A/Y199A

<400> 97

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Ala Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 98

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein human IgG1 Fc-NKG2D

Y199F eNKG2D1

<400> 98

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 99

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein human IgG1 Fc-NKG2D

Y152S eNKG2D2

<400> 99

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 100

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein human IgG1 Fc-NKG2D

Y152T eNKG2D3

<400> 100

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Thr His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 101

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein human IgG1 Fc-NKG2D

Y152V eNKG2D4

<400> 101

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Val His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 102

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein Fc IgG1

human-NKG2D Y152A/Y199F eNKG2D5

<400> 102

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 103

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein Fc IgG1

human-NKG2D Y152L/Y199F eNKG2D6

<400> 103

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Leu His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 104

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein Fc IgG1

human-NKG2D Y152S/Y199F eNKG2D7

<400> 104

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Ser His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 105

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein Fc IgG1

human-NKG2D Y152T/Y199F eNKG2D8

<400> 105

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Thr His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 106

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein Fc IgG1

human-NKG2D Y152V/Y199F eNKG2D9

<400> 106

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Val His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 107

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein human IgG1 Fc-NKG2D

Y199D eNKG2D10

<400> 107

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Asp Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 108

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein human IgG1 Fc-NKG2D

Y199E eNKG2D11

<400> 108

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Glu Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 109

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein Fc IgG1

human-NKG2D Y152D/Y199D eNKG2D12

<400> 109

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Asp His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Asp Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 110

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein Fc IgG1

human-NKG2D Y152E/Y199E eNKG2D13

<400> 110

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Glu His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Glu Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 111

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein human IgG1 Fc-NKG2D

Y152L eNKG2D14

<400> 111

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Leu His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 112

<211> 376

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide fusion protein Fc IgG1

human-NKG2D Y152F/Y199F eNKG2D15

<400> 112

Met Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Glu Gly Arg Phe Leu Asn

225 230 235 240

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

245 250 255

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

260 265 270

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

275 280 285

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

290 295 300

Leu Leu Lys Leu Val Lys Ser Phe His Trp Met Gly Leu Val His Ile

305 310 315 320

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

325 330 335

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

340 345 350

Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser Thr Pro Asn Thr

355 360 365

Tyr Ile Cys Met Gln Arg Thr Val

370 375

<210> 113

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide signal sequence MHCI

<400> 113

Met Gly Leu Gly Pro Val Phe Leu Leu Leu Ala Gly Ile Phe Pro Phe

1 5 10 15

Ala Pro Pro Gly Ala Ala Ala

20

<210> 114

<211> 69

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding

MHCI signal sequence

<400> 114

atgggccttg gcccagtgtt tctgctgttg gcaggcattt tcccttttgc tccgcccggc 60

gccgcagcc 69

<210> 115

<211> 711

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding human IgG1 Fc with

IEGR linker

<400> 115

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg c 711

<210> 116

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding a fusion protein

Fc human IgG1-NKG2D

<400> 116

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcatatcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 117

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding a fusion protein

Fc human IgG1-NKG2D Y152A

<400> 117

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcagctcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 118

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human IgG1 Fc-ectodomain

NKG2D Y199A

<400> 118

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcataccatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgctata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 119

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human IgG1 Fc-ectodomain

NKG2D Y152A/Y199A

<400> 119

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcagctcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgctata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 120

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y199F eNKG2D1

<400> 120

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcataccatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 121

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152S eNKG2D2

<400> 121

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcataccatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 122

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152T eNKG2D3

<400> 122

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcaactcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 123

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152V eNKG2D4

<400> 123

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcagtgcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 124

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152A/Y199F eNKG2D5

<400> 124

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcagctcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 125

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152L/Y199F eNKG2D6

<400> 125

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcactgcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 126

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152S/Y199F eNKG2D7

<400> 126

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcaagtcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 127

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152T/Y199F eNKG2D8

<400> 127

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcaactcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 128

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152V/Y199F eNKG2D9

<400> 128

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcagtgcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 129

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y199D eNKG2D10

<400> 129

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcatatcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgatata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 130

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y199E eNKG2D11

<400> 130

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcataccatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgagata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 131

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152D/Y199D eNKG2D12

<400> 131

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcagatcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgatata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 132

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152E/Y199E eNKG2D13

<400> 132

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcagagcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcgagata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 133

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152L eNKG2D14

<400> 133

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcactgcatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 134

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding IgG1 Fc fusion protein

human-NKG2D Y152F/Y199F eNKG2D15

<400> 134

atggacccga aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaaga tcgaaggacg cttcttaaac 720

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 780

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 840

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 900

gaggaccagg atttacttaa actggtgaag tcattccatt ggatgggact agtacacatt 960

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 1020

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggcttcata 1080

gaaaactgtt caactccaaa tacatacatc tgcatgcaaa ggactgtg 1128

<210> 135

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide human kappa light chain

<400> 135

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 136

<211> 330

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide CH1-CH2-CH3 of human IgG1 heavy chain

D265A/N297A

<400> 136

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 137

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide heavy chain of trastuzumab

<400> 137

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 138

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide light chain of trastuzumab

<400> 138

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

AsnArgGlyGluCys

210

<210> 139

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide heavy chain of rituximab

<400> 139

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 140

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide light chain of rituximab

<400> 140

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 141

<211> 404

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide rituximab LC_ULBP2.wt

<400> 141

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser

210 215 220

Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg

225 230 235 240

Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr

245 250 255

Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro

260 265 270

Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro

275 280 285

Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Arg Asp Ile

290 295 300

Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg

305 310 315 320

Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln

325 330 335

Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg

340 345 350

Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp

355 360 365

Glu Asn Asp Lys Val Val Ala Met Ser Phe His Tyr Phe Ser Met Gly

370 375 380

Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr

385 390 395 400

Leu Glu Pro Ser

<210> 142

<211> 187

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ULPB2 alpha1-alpha2 variant R80W

<400> 142

Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg

1 5 10 15

Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr

20 25 30

Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro

35 40 45

Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro

50 55 60

Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile

65 70 75 80

Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg

85 90 95

Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln

100 105 110

Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg

115 120 125

Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp

130 135 140

Glu Asn Asp Lys Val Val Ala Met Ser Phe His Tyr Phe Ser Met Gly

145 150 155 160

Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr

165 170 175

Leu Glu Pro Ser Ala Gly Ala Pro Pro Met Val

180 185

<210> 143

<211> 180

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP2 alpha1-alpha2 variant ULBP2.C

<400> 143

Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg

1 5 10 15

Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr

20 25 30

Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro

35 40 45

Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro

50 55 60

Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile

65 70 75 80

Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg

85 90 95

Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln

100 105 110

Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg

115 120 125

Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp

130 135 140

Glu Asn Asp Lys Val Val Ala Thr Ile Leu Trp Gln Thr Ser Met Gly

145 150 155 160

Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr

165 170 175

Leu Glu Pro Ser

180

<210> 144

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding ULBP2 alpha1-alpha2

variant ULBP2.C

<400> 144

gagccccata gtctgagcta cgacatcaca gttattccca agttcaggcc cggaccgcgc 60

tggtgtgccg tgcaaggaca agtcgacgaa aaaacctttc ttcattacga ttgcggaaat 120

aagactgtaa cgccagtctc tcctttaggt aagaagttaa acgtcactac ggcgtggaag 180

gcacaaaacc ccgtcctgcg cgaggtcgtc gacatcctga ctgaacaatt gtgggacatc 240

cagctcgaga attacactcc aaaggagcct cttaccctgc aggctagaat gtcttgcgag 300

caaaaggcag agggccactc ctccggcagc tggcagttca gtttcgacgg acaaatcttt 360

ctgttattcg attcagagaa gagaatgtgg actacagttc accccggtgc ccgtaaaatg 420

aaggagaagt gggaaaacga caaagtggtg gcgactattc tgtggcagac ttcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

<210> 145

<211> 180

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP2 alpha1-alpha2 variant ULBP2.R

<400> 145

Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg

1 5 10 15

Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr

20 25 30

Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro

35 40 45

Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro

50 55 60

Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile

65 70 75 80

Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg

85 90 95

Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln

100 105 110

Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg

115 120 125

Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp

130 135 140

Glu Asn Asp Lys Val Val Ala Thr Leu Leu Trp Gly Trp Ser Met Gly

145 150 155 160

Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr

165 170 175

Leu Glu Pro Ser

180

<210> 146

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding ULBP2 alpha1-alpha2

variant ULBP2.R

<400> 146

gagccccata gtctgagcta cgacatcaca gttattccca agttcaggcc cggaccgcgc 60

tggtgtgccg tgcaaggaca agtcgacgaa aaaacctttc ttcattacga ttgcggaaat 120

aagactgtaa cgccagtctc tcctttaggt aagaagttaa acgtcactac ggcgtggaag 180

gcacaaaacc ccgtcctgcg cgaggtcgtc gacatcctga ctgaacaatt gtgggacatc 240

cagctcgaga attacactcc aaaggagcct cttaccctgc aggctagaat gtcttgcgag 300

caaaaggcag agggccactc ctccggcagc tggcagttca gtttcgacgg acaaatcttt 360

ctgttattcg attcagagaa gagaatgtgg actacagttc accccggtgc ccgtaaaatg 420

aaggagaagt gggaaaacga caaagtggtg gcgactttgt tgtgggggtg gtcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

<210> 147

<211> 180

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP2 alpha1-alpha2 variant ULBP2.AA

<400> 147

Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg

1 5 10 15

Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr

20 25 30

Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro

35 40 45

Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro

50 55 60

Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile

65 70 75 80

Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg

85 90 95

Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln

100 105 110

Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg

115 120 125

Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp

130 135 140

Glu Asn Asp Lys Val Val Ala Thr Met Phe Trp Ser Trp Ser Met Gly

145 150 155 160

Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr

165 170 175

Leu Glu Pro Ser

180

<210> 148

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding ULBP2 alpha1-alpha2

variant ULBP2.AA

<400> 148

gagccccata gtctgagcta cgacatcaca gttattccca agttcaggcc cggaccgcgc 60

tggtgtgccg tgcaaggaca agtcgacgaa aaaacctttc ttcattacga ttgcggaaat 120

aagactgtaa cgccagtctc tcctttaggt aagaagttaa acgtcactac ggcgtggaag 180

gcacaaaacc ccgtcctgcg cgaggtcgtc gacatcctga ctgaacaatt gtgggacatc 240

cagctcgaga attacactcc aaaggagcct cttaccctgc aggctagaat gtcttgcgag 300

caaaaggcag agggccactc ctccggcagc tggcagttca gtttcgacgg acaaatcttt 360

ctgttattcg attcagagaa gagaatgtgg actacagttc accccggtgc ccgtaaaatg 420

aaggagaagt gggaaaacga caaagtggtg gcgactatgt tttggagttg gtcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

<210> 149

<211> 180

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP2 alpha1-alpha2 variant ULBP2.AB

<400> 149

Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg

1 5 10 15

Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr

20 25 30

Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro

35 40 45

Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro

50 55 60

Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile

65 70 75 80

Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg

85 90 95

Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln

100 105 110

Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg

115 120 125

Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp

130 135 140

Glu Asn Asp Lys Val Val Ala Thr Leu Met Trp Gln Trp Ser Met Gly

145 150 155 160

Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr

165 170 175

Leu Glu Pro Ser

180

<210> 150

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding ULBP2 alpha1-alpha2

variant ULBP2.AB

<400> 150

gagccccata gtctgagcta cgacatcaca gttattccca agttcaggcc cggaccgcgc 60

tggtgtgccg tgcaaggaca agtcgacgaa aaaacctttc ttcattacga ttgcggaaat 120

aagactgtaa cgccagtctc tcctttaggt aagaagttaa acgtcactac ggcgtggaag 180

gcacaaaacc ccgtcctgcg cgaggtcgtc gacatcctga ctgaacaatt gtgggacatc 240

cagctcgaga attacactcc aaaggagcct cttaccctgc aggctagaat gtcttgcgag 300

caaaaggcag agggccactc ctccggcagc tggcagttca gtttcgacgg acaaatcttt 360

ctgttattcg attcagagaa gagaatgtgg actacagttc accccggtgc ccgtaaaatg 420

aaggagaagt gggaaaacga caaagtggtg gcgactctta tgtggcagtg gtcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

<210> 151

<211> 180

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide, ULBP2 alpha1-alpha2 variant ULBP2.S3

<400> 151

Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg

1 5 10 15

Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr

20 25 30

Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro

35 40 45

Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro

50 55 60

Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile

65 70 75 80

Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg

85 90 95

Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln

100 105 110

Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg

115 120 125

Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp

130 135 140

Glu Asn Asp Lys Val Val Ala Thr Lys Leu Tyr Leu Trp Ser Met Gly

145 150 155 160

Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr

165 170 175

Leu Glu Pro Ser

180

<210> 152

<211> 404

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide rituximab LC_ULBP2.S3

<400> 152

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser

210 215 220

Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg

225 230 235 240

Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr

245 250 255

Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro

260 265 270

Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro

275 280 285

Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile

290 295 300

Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg

305 310 315 320

Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln

325 330 335

Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg

340 345 350

Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp

355 360 365

Glu Asn Asp Lys Val Val Ala Thr Lys Leu Tyr Leu Trp Ser Met Gly

370 375 380

Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr

385 390 395 400

Leu Glu Pro Ser

<210> 153

<211> 634

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide rituximab HC_ULBP2.R80W

<400> 153

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Ser Glu Pro His Ser Leu Ser Tyr Asp Ile Thr

450 455 460

Val Ile Pro Lys Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly

465 470 475 480

Gln Val Asp Glu Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr

485 490 495

Val Thr Pro Val Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala

500 505 510

Trp Lys Ala Gln Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr

515 520 525

Glu Gln Leu Trp Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro

530 535 540

Leu Thr Leu Gln Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His

545 550 555 560

Ser Ser Gly Ser Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu

565 570 575

Phe Asp Ser Glu Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg

580 585 590

Lys Met Lys Glu Lys Trp Glu Asn Asp Lys Val Val Ala Met Ser Phe

595 600 605

His Tyr Phe Ser Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu

610 615 620

Met Gly Met Asp Ser Thr Leu Glu Pro Ser

625 630

<210> 154

<211> 156

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide ectodomain NKG2D.AF

<400> 154

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr

20 25 30

Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn

35 40 45

Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln

50 55 60

Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys

65 70 75 80

Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly

85 90 95

Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser

100 105 110

Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp

115 120 125

Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser

130 135 140

Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

145 150 155

<210> 155

<211> 629

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide complete chimeric receptor

antigen NKG2D.wt_CD8hingeTM_4-1BB_CD3-zeta_EGFP

<400> 155

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr

20 25 30

Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn

35 40 45

Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln

50 55 60

Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys

65 70 75 80

Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly

85 90 95

Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser

100 105 110

Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp

115 120 125

Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser

130 135 140

Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro

145 150 155 160

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

165 170 175

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

180 185 190

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

195 200 205

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

210 215 220

Cys Ser Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

225 230 235 240

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

245 250 255

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

260 265 270

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln

275 280 285

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

290 295 300

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

305 310 315 320

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

325 330 335

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

340 345 350

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

355 360 365

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser

370 375 380

Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr

385 390 395 400

Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His

405 410 415

Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys

420 425 430

Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp

435 440 445

Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg

450 455 460

Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro

465 470 475 480

Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn

485 490 495

Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn

500 505 510

Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu

515 520 525

Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met

530 535 540

Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His

545 550 555 560

Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn

565 570 575

Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu

580 585 590

Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His

595 600 605

Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met

610 615 620

Asp Glu Leu Tyr Lys

625

<210> 156

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a complete chimeric receptor

antigen NKG2D.wt_CD8hingeTM_4-1BB_CD3-zeta_EGFP

<400> 156

atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60

cctctgttca atcaagaggt gcagatccct ctgaccgaga gctactgtgg cccctgtcct 120

aagaactgga tctgctacaa gaacaactgc taccagttct tcgacgagag caagaattgg 180

tacgagagcc aggccagctg catgagccag aatgccagcc tgctgaaggt gtacagcaaa 240

gaggaccagg atctgctgaa gctggtcaag agctaccact ggatgggact cgtgcacatc 300

cctacaaacg gcagctggca gtgggaggac ggctctatcc tgtctcctaa cctgctgacc 360

atcatcgaga tgcagaaggg cgactgcgcc ctgtacgcca gcagctttaa gggctacatc 420

gagaactgca gcacccctaa cacctacatc tgtatgcagc ggaccgtgac caccacacca 480

gctcctagac ctccaactcc tgctcctaca atcgccagcc agcctctgtc tctgaggcca 540

gaagcttgta gacctgctgc aggcggagcc gtgcatacaa gaggactgga tttcgcctgc 600

gacatctaca tctgggcccc tctggctgga acatgtggcg tgctgctgct gagcctggtc 660

atcaccctgt actgcagcct gaagcggggc agaaagaagc tgctgtacat ctttaagcag 720

cccttcatgc ggcccgtgca gaccacacaa gaggaagatg gctgctcctg cagattcccc 780

gaggaagaag aaggcggctg cgagctgaga gtgaagttca gccgttctgc cgacgctccc 840

gcctataagc agggacagaa ccagctgtac aacgagctga acctggggag aagagaagag 900

tacgacgtgc tggacaagcg gagaggcaga gatcctgaga tgggcggcaa gccgacgg 960

aagaatcctc aagaggggcct gtataatgag ctgcagaaag acaagatggc cgaggcctac 1020

agcgagatcg gaatgaaggg cgagcgcaga agaggcaagg gacacgatgg actgtaccag 1080

ggcctgagca ccgccaccaa ggatacctat gatgccctgc acatgcaggc cctgcctcca 1140

agatcaggct ctggttctgg cagcggcagc atggtgtcta aaggcgagga actgttcacc 1200

ggcgtggtgc ccattctggt ggaactggac ggggatgtga acggccacaa gtttagcgtt 1260

agcggcgaag gcgaagggga tgccacatac ggaaagctga ccctgaagtt catctgcacc 1320

accggcaagc tgcctgtgcc ttggcctaca ctggtcacca cactgacata cggcgtgcag 1380

tgctttagca gataccccga ccatatgaag cagcacgact tcttcaagtc cgccatgcct 1440

gagggctacg tgcaagagcg gaccatcttc tttaaggacg acggcaacta caagaccagg 1500

gccgaagtga agtttgaggg cgacaccctg gtcaaccgga tcgagctgaa gggcatcgac 1560

ttcaaagagg atggcaacat cctgggccac aagctcgagt acaactacaa cagccacaac 1620

gtgtacatca tggccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccggcac 1680

aacatcgagg acggcagcgt tcagctggcc gatcactacc agcagaacac ccctatcgga 1740

gatggccctg tgctgctccc cgacaatcac tacctgagca cacagagcgc cctgagcaag 1800

gaccccaacg agaagaggga tcacatggtg ctgctggaat ttgtgaccgc cgcaggcatc 1860

accctcggca tggacgaact gtacaaa 1887

<210> 157

<211> 629

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide complete chimeric antigen receptor

NKG2D.YA_CD8hingeTM_4-1BB_CD3-zeta_EGFP

<400> 157

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr

20 25 30

Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn

35 40 45

Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln

50 55 60

Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys

65 70 75 80

Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly

85 90 95

Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser

100 105 110

Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp

115 120 125

Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser

130 135 140

Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro

145 150 155 160

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

165 170 175

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

180 185 190

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

195 200 205

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

210 215 220

Cys Ser Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

225 230 235 240

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

245 250 255

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

260 265 270

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln

275 280 285

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

290 295 300

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

305 310 315 320

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

325 330 335

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

340 345 350

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

355 360 365

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser

370 375 380

Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr

385 390 395 400

Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His

405 410 415

Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys

420 425 430

Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp

435 440 445

Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg

450 455 460

Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro

465 470 475 480

Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn

485 490 495

Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn

500 505 510

Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu

515 520 525

Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met

530 535 540

Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His

545 550 555 560

Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn

565 570 575

Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu

580 585 590

Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His

595 600 605

Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met

610 615 620

Asp Glu Leu Tyr Lys

625

<210> 158

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a complete chimeric

antigen receptor NKG2D.YA_CD8hingeTM_4-1BB_CD3-zeta_EGFP

<400> 158

atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60

cctctgttca atcaagaggt gcagatccct ctgaccgaga gctactgtgg cccctgtcct 120

aagaactgga tctgctacaa gaacaactgc taccagttct tcgacgagag caagaattgg 180

tacgagagcc aggccagctg catgagccag aatgccagcc tgctgaaggt gtacagcaaa 240

gaggaccagg atctgctgaa gctggtcaag agcgcccact ggatgggact cgtgcacatc 300

cctacaaacg gcagctggca gtgggaggac ggctctatcc tgtctcctaa cctgctgacc 360

atcatcgaga tgcagaaggg cgactgcgcc ctgtacgcca gcagctttaa gggctacatc 420

gagaactgca gcacccctaa cacctacatc tgtatgcagc ggaccgtgac caccacacca 480

gctcctagac ctccaactcc tgctcctaca atcgccagcc agcctctgtc tctgaggcca 540

gaagcttgta gacctgctgc aggcggagcc gtgcatacaa gaggactgga tttcgcctgc 600

gacatctaca tctgggcccc tctggctgga acatgtggcg tgctgctgct gagcctggtc 660

atcaccctgt actgcagcct gaagcggggc agaaagaagc tgctgtacat ctttaagcag 720

cccttcatgc ggcccgtgca gaccacacaa gaggaagatg gctgctcctg cagattcccc 780

gaggaagaag aaggcggctg cgagctgaga gtgaagttca gccgttctgc cgacgctccc 840

gcctataagc agggacagaa ccagctgtac aacgagctga acctggggag aagagaagag 900

tacgacgtgc tggacaagcg gagaggcaga gatcctgaga tgggcggcaa gccgacgg 960

aagaatcctc aagaggggcct gtataatgag ctgcagaaag acaagatggc cgaggcctac 1020

agcgagatcg gaatgaaggg cgagcgcaga agaggcaagg gacacgatgg actgtaccag 1080

ggcctgagca ccgccaccaa ggatacctat gatgccctgc acatgcaggc cctgcctcca 1140

agatcaggct ctggttctgg cagcggcagc atggtgtcta aaggcgagga actgttcacc 1200

ggcgtggtgc ccattctggt ggaactggac ggggatgtga acggccacaa gtttagcgtt 1260

agcggcgaag gcgaagggga tgccacatac ggaaagctga ccctgaagtt catctgcacc 1320

accggcaagc tgcctgtgcc ttggcctaca ctggtcacca cactgacata cggcgtgcag 1380

tgctttagca gataccccga ccatatgaag cagcacgact tcttcaagtc cgccatgcct 1440

gagggctacg tgcaagagcg gaccatcttc tttaaggacg acggcaacta caagaccagg 1500

gccgaagtga agtttgaggg cgacaccctg gtcaaccgga tcgagctgaa gggcatcgac 1560

ttcaaagagg atggcaacat cctgggccac aagctcgagt acaactacaa cagccacaac 1620

gtgtacatca tggccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccggcac 1680

aacatcgagg acggcagcgt tcagctggcc gatcactacc agcagaacac ccctatcgga 1740

gatggccctg tgctgctccc cgacaatcac tacctgagca cacagagcgc cctgagcaag 1800

gaccccaacg agaagaggga tcacatggtg ctgctggaat ttgtgaccgc cgcaggcatc 1860

accctcggca tggacgaact gtacaaa 1887

<210> 159

<211> 629

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide complete chimeric antigen receptor

NKG2D.AF_CD8hingeTM_4-1BB_CD3-zeta_EGFP

<400> 159

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr

20 25 30

Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn

35 40 45

Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln

50 55 60

Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys

65 70 75 80

Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Ala His Trp Met Gly

85 90 95

Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser

100 105 110

Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp

115 120 125

Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Phe Ile Glu Asn Cys Ser

130 135 140

Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro

145 150 155 160

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

165 170 175

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

180 185 190

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

195 200 205

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

210 215 220

Cys Ser Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

225 230 235 240

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

245 250 255

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

260 265 270

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln

275 280 285

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

290 295 300

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

305 310 315 320

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

325 330 335

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

340 345 350

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

355 360 365

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Ser Gly Ser

370 375 380

Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr

385 390 395 400

Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His

405 410 415

Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys

420 425 430

Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp

435 440 445

Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg

450 455 460

Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro

465 470 475 480

Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn

485 490 495

Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn

500 505 510

Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu

515 520 525

Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met

530 535 540

Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His

545 550 555 560

Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn

565 570 575

Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu

580 585 590

Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His

595 600 605

Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met

610 615 620

Asp Glu Leu Tyr Lys

625

<210> 160

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a complete chimeric receptor

antigen NKG2D.AF_CD8hingeTM_4-1BB_CD3-zeta_EGFP

<400> 160

atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60

cctctgttca atcaagaggt gcagatccct ctgaccgaga gctactgtgg cccctgtcct 120

aagaactgga tctgctacaa gaacaactgc taccagttct tcgacgagag caagaattgg 180

tacgagagcc aggccagctg catgagccag aatgccagcc tgctgaaggt gtacagcaaa 240

gaggaccagg atctgctgaa gctggtcaag agcgcccact ggatgggact cgtgcacatc 300

cctacaaacg gcagctggca gtgggaggac ggctctatcc tgtctcctaa cctgctgacc 360

atcatcgaga tgcagaaggg cgactgcgcc ctgtacgcca gcagctttaa gggcttcatc 420

gagaactgca gcacccctaa cacctacatc tgtatgcagc ggaccgtgac caccacacca 480

gctcctagac ctccaactcc tgctcctaca atcgccagcc agcctctgtc tctgaggcca 540

gaagcttgta gacctgctgc aggcggagcc gtgcatacaa gaggactgga tttcgcctgc 600

gacatctaca tctgggcccc tctggctgga acatgtggcg tgctgctgct gagcctggtc 660

atcaccctgt actgcagcct gaagcggggc agaaagaagc tgctgtacat ctttaagcag 720

cccttcatgc ggcccgtgca gaccacacaa gaggaagatg gctgctcctg cagattcccc 780

gaggaagaag aaggcggctg cgagctgaga gtgaagttca gccgttctgc cgacgctccc 840

gcctataagc agggacagaa ccagctgtac aacgagctga acctggggag aagagaagag 900

tacgacgtgc tggacaagcg gagaggcaga gatcctgaga tgggcggcaa gccgacgg 960

aagaatcctc aagaggggcct gtataatgag ctgcagaaag acaagatggc cgaggcctac 1020

agcgagatcg gaatgaaggg cgagcgcaga agaggcaagg gacacgatgg actgtaccag 1080

ggcctgagca ccgccaccaa ggatacctat gatgccctgc acatgcaggc cctgcctcca 1140

agatcaggct ctggttctgg cagcggcagc atggtgtcta aaggcgagga actgttcacc 1200

ggcgtggtgc ccattctggt ggaactggac ggggatgtga acggccacaa gtttagcgtt 1260

agcggcgaag gcgaagggga tgccacatac ggaaagctga ccctgaagtt catctgcacc 1320

accggcaagc tgcctgtgcc ttggcctaca ctggtcacca cactgacata cggcgtgcag 1380

tgctttagca gataccccga ccatatgaag cagcacgact tcttcaagtc cgccatgcct 1440

gagggctacg tgcaagagcg gaccatcttc tttaaggacg acggcaacta caagaccagg 1500

gccgaagtga agtttgaggg cgacaccctg gtcaaccgga tcgagctgaa gggcatcgac 1560

ttcaaagagg atggcaacat cctgggccac aagctcgagt acaactacaa cagccacaac 1620

gtgtacatca tggccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccggcac 1680

aacatcgagg acggcagcgt tcagctggcc gatcactacc agcagaacac ccctatcgga 1740

gatggccctg tgctgctccc cgacaatcac tacctgagca cacagagcgc cctgagcaag 1800

gaccccaacg agaagaggga tcacatggtg ctgctggaat ttgtgaccgc cgcaggcatc 1860

accctcggca tggacgaact gtacaaa 1887

<210> 161

<211> 470

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic sequence, H-chain 3BNC60 (Fc IgG1 with

mutations D265A/N297A (Kabat numbering))

<400> 161

Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu

1 5 10 15

Ala Gln Val His Leu Ser Gln Ser Gly Ala Ala Val Thr Lys Pro Gly

20 25 30

Ala Ser Val Arg Val Ser Cys Glu Ala Ser Gly Tyr Lys Ile Ser Asp

35 40 45

His Phe Ile His Trp Trp Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp

50 55 60

Val Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro Asn Asn Pro Arg Gln

65 70 75 80

Phe Gln Gly Arg Val Ser Leu Thr Arg Gln Ala Ser Trp Asp Phe Asp

85 90 95

Thr Tyr Ser Phe Tyr Met Asp Leu Lys Ala Val Arg Ser Asp Asp Thr

100 105 110

Ala Ile Tyr Phe Cys Ala Arg Gln Arg Ser Asp Phe Trp Asp Phe Asp

115 120 125

Val Trp Gly Ser Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

225 230 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210> 162

<211> 414

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic sequence, L-chain 3BNC60 (with

added ULBP2.S3)

<400> 162

Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu

1 5 10 15

Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Arg Val

20 25 30

Gly Asp Thr Val Thr Ile Thr Cys Gln Ala Asn Gly Tyr Leu Asn Trp

35 40 45

Tyr Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Gly

50 55 60

Ser Lys Leu Glu Arg Gly Val Pro Ala Arg Phe Ser Gly Arg Arg Trp

65 70 75 80

Gly Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu Gln Pro Glu Asp Val

85 90 95

Ala Thr Tyr Phe Cys Gln Val Tyr Glu Phe Ile Val Pro Gly Thr Arg

100 105 110

Leu Asp Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

115 120 125

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

130 135 140

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

145 150 155 160

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

165 170 175

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

180 185 190

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

195 200 205

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Ala

210 215 220

Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser Glu Pro His Ser Leu Ser

225 230 235 240

Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg Pro Gly Pro Arg Trp Cys

245 250 255

Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu His Tyr Asp Cys

260 265 270

Gly Asn Lys Thr Val Thr Pro Val Ser Pro Leu Gly Lys Lys Leu Asn

275 280 285

Val Thr Thr Ala Trp Lys Ala Gln Asn Pro Val Leu Arg Glu Val Val

290 295 300

Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile Gln Leu Glu Asn Tyr Thr

305 310 315 320

Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu Gln Lys

325 330 335

Ala Glu Gly His Ser Ser Gly Ser Trp Gln Phe Ser Phe Asp Gly Gln

340 345 350

Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg Met Trp Thr Thr Val His

355 360 365

Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn Asp Lys Val Val

370 375 380

Ala Thr Lys Leu Tyr Leu Trp Ser Met Gly Asp Cys Ile Gly Trp Leu

385 390 395 400

Glu Asp Phe Leu Met Gly Met Asp Ser Thr Leu Glu Pro Ser

405 410

<210> 163

<211> 470

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic sequence, H-chain 3BNC117 (Fc IgG1

with mutations D265A/N297A (Kabat numbering))

<400> 163

Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu

1 5 10 15

Ala Gln Val Gln Leu Leu Gln Ser Gly Ala Ala Val Thr Lys Pro Gly

20 25 30

Ala Ser Val Arg Val Ser Cys Glu Ala Ser Gly Tyr Asn Ile Arg Asp

35 40 45

Tyr Phe Ile His Trp Trp Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp

50 55 60

Val Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro Asn Asn Pro Arg Gln

65 70 75 80

Phe Gln Gly Arg Val Ser Leu Thr Arg His Ala Ser Trp Asp Phe Asp

85 90 95

Thr Tyr Ser Phe Tyr Met Asp Leu Lys Ala Leu Arg Ser Asp Asp Thr

100 105 110

Ala Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp Tyr Trp Asp Phe Asp

115 120 125

Val Trp Gly Ser Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

225 230 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210> 164

<211> 414

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic sequence, L-chain 3BNC117 (with

added ULBP2.S3)

<400> 164

Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu

1 5 10 15

Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

20 25 30

Gly Asp Thr Val Thr Ile Thr Cys Gln Ala Asn Gly Tyr Leu Asn Trp

35 40 45

Tyr Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Gly

50 55 60

Ser Lys Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly Arg Arg Trp

65 70 75 80

Gly Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu Gln Pro Glu Asp Ile

85 90 95

Ala Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val Val Pro Gly Thr Arg

100 105 110

Leu Asp Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

115 120 125

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

130 135 140

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

145 150 155 160

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

165 170 175

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

180 185 190

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

195 200 205

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Ala

210 215 220

Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser Glu Pro His Ser Leu Ser

225 230 235 240

Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg Pro Gly Pro Arg Trp Cys

245 250 255

Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu His Tyr Asp Cys

260 265 270

Gly Asn Lys Thr Val Thr Pro Val Ser Pro Leu Gly Lys Lys Leu Asn

275 280 285

Val Thr Thr Ala Trp Lys Ala Gln Asn Pro Val Leu Arg Glu Val Val

290 295 300

Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile Gln Leu Glu Asn Tyr Thr

305 310 315 320

Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu Gln Lys

325 330 335

Ala Glu Gly His Ser Ser Gly Ser Trp Gln Phe Ser Phe Asp Gly Gln

340 345 350

Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg Met Trp Thr Thr Val His

355 360 365

Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn Asp Lys Val Val

370 375 380

Ala Thr Lys Leu Tyr Leu Trp Ser Met Gly Asp Cys Ile Gly Trp Leu

385 390 395 400

Glu Asp Phe Leu Met Gly Met Asp Ser Thr Leu Glu Pro Ser

405 410

<210> 165

<211> 479

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic Sequence

<400> 165

Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu

1 5 10 15

Ala Gln Met Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser

20 25 30

Glu Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Ala Ser Ile Ser Asp

35 40 45

Ser Tyr Trp Ser Trp Ile Arg Arg Ser Pro Gly Lys Gly Leu Glu Trp

50 55 60

Ile Gly Tyr Val His Lys Ser Gly Asp Thr Asn Tyr Ser Pro Ser Leu

65 70 75 80

Lys Ser Arg Val Asn Leu Ser Leu Asp Thr Ser Lys Asn Gln Val Ser

85 90 95

Leu Ser Leu Val Ala Ala Thr Ala Ala Asp Ser Gly Lys Tyr Tyr Cys

100 105 110

Ala Arg Thr Leu His Gly Arg Arg Ile Tyr Gly Ile Val Ala Phe Asn

115 120 125

Glu Trp Phe Thr Tyr Phe Tyr Met Asp Val Trp Gly Asn Gly Thr Gln

130 135 140

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

145 150 155 160

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

165 170 175

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

180 185 190

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

195 200 205

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

210 215 220

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

225 230 235 240

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

245 250 255

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

275 280 285

Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

385 390 395 400

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 166

<211> 420

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic sequence, L-chain of PGT121 (with

added ULBP2.S3)

<400> 166

Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu

1 5 10 15

Ala Ser Asp Ile Ser Val Ala Pro Gly Glu Thr Ala Arg Ile Ser Cys

20 25 30

Gly Glu Lys Ser Leu Gly Ser Arg Ala Val Gln Trp Tyr Gln His Arg

35 40 45

Ala Gly Gln Ala Pro Ser Leu Ile Ile Tyr Asn Asn Gln Asp Arg Pro

50 55 60

Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Pro Asp Ser Pro Phe Gly

65 70 75 80

Thr Thr Ala Thr Leu Thr Ile Thr Ser Val Glu Ala Gly Asp Glu Ala

85 90 95

Asp Tyr Tyr Cys His Ile Trp Asp Ser Arg Val Pro Thr Lys Trp Val

100 105 110

Phe Gly Gly Gly Thr Thr Leu Thr Val Leu Arg Thr Val Ala Ala Pro

115 120 125

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

130 135 140

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

145 150 155 160

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

165 170 175

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

180 185 190

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

195 200 205

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

210 215 220

Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly Ser

225 230 235 240

Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg

245 250 255

Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr

260 265 270

Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro

275 280 285

Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro

290 295 300

Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp Ile

305 310 315 320

Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg

325 330 335

Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln

340 345 350

Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg

355 360 365

Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp

370 375 380

Glu Asn Asp Lys Val Val Ala Thr Lys Leu Tyr Leu Trp Ser Met Gly

385 390 395 400

Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr

405 410 415

Leu Glu Pro Ser

420

<210> 167

<211> 479

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic sequence, H chain 10-1074 (Fc IgG1

with mutations D265A/N297A (Kabat numbering))

<400> 167

Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu

1 5 10 15

Ala Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser

20 25 30

Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met Asn Asn

35 40 45

Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp

50 55 60

Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu

65 70 75 80

Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser

85 90 95

Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr Tyr Cys

100 105 110

Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly

115 120 125

Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr

130 135 140

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

145 150 155 160

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

165 170 175

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

180 185 190

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

195 200 205

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

210 215 220

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

225 230 235 240

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

245 250 255

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

275 280 285

Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu

290 295 300

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

355 360 365

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

385 390 395 400

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

435 440 445

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

450 455 460

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 168

<211> 428

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic sequence, L-chain 10-1074 (c

added ULBP2.S3)

<400> 168

Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu

1 5 10 15

Ala Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr Ala Arg

20 25 30

Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln Trp Tyr

35 40 45

Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn Asn Gln

50 55 60

Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro Asp Ile

65 70 75 80

Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly

85 90 95

Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser Gly Phe

100 105 110

Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu Arg Thr Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly

225 230 235 240

Gly Gly Ser Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro

245 250 255

Lys Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp

260 265 270

Glu Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro

275 280 285

Val Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala

290 295 300

Gln Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu

305 310 315 320

Trp Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu

325 330 335

Gln Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly

340 345 350

Ser Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser

355 360 365

Glu Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys

370 375 380

Glu Lys Trp Glu Asn Asp Lys Val Val Ala Thr Lys Leu Tyr Leu Trp

385 390 395 400

Ser Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met

405 410 415

Asp Ser Thr Leu Glu Pro Ser Leu Ile Ser Gly Arg

420 425

<210> 169

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic sequence binding the 3BNC60 epitope

and 3BNC117

<400> 169

Ser Ser Gly Gly Asp Pro Glu Ile Val Thr His

1 5 10

<210> 170

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic sequence binding PGF12 epitope and

10-1074

<400> 170

Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Arg Ile Arg Ile Gln Arg

1 5 10 15

Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys Ile Gly Asn Met Arg

20 25 30

Gln Ala His Cys

35

<---

Claims (16)

1. A modified non-natural ligand for a modified non-natural NKG2D receptor for delivering a cell displaying the modified non-natural NKG2D receptor to an HIV-infected cell, wherein the ligand (a) contains (i) SEQ ID NO: 63 or a mutant thereof having T at position 154; M, K, W, L or T at position 155; L or M at position 156; E, T, S, Q, Y or R at position 157; L, V, I or T at position 158; and/or W or I at position 159; or (ii) SEQ ID NO: 65 or a mutant thereof having D, W, R, Y or L at position 155; L or M at position 156; I, Y, V or L at position 157; R, L or T at position 158; and/or R, I, W or K at position 159; and (b) has an attached heterologous molecule that selectively binds an HIV protein displayed on the surface of a cell infected with HIV, wherein said heterologous molecule selectively binds to an epitope sequence that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 169 and SEQ ID NO: 170, and wherein the modified ligand with its attached heterologous molecule can selectively bind to the modified non-natural NKG2D CAR cell receptor and cause destruction of the HIV-infected cell. 2. A modified non-natural ligand according to claim 1, characterized in that said modified non-natural ligand of the modified non-natural NKG2D receptor contains a non-natural α1-α2 domain of the NKG2D ligand. 3. The modified non-natural ligand according to claim 2, characterized in that the NKG2D ligand is selected from the group consisting of MICA, MICB and ULBP 1-6. 4. A modified non-natural ligand according to claim 2 or 3, characterized in that the α1-α2 domain of the non-natural NKG2D ligand is at least 80% identical to a native or natural α1-α2 domain of an NKG2D ligand selected from the group consisting of SEQ ID NO: 1-19. 5. A modified non-natural ligand according to claim 1, characterized in that said modified non-natural ligand comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 68-72. 6. A modified non-natural ligand according to claim 1, characterized in that said modified non-natural ligand binds to a non-natural NKG2D receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 54 and SEQ ID NO: 154. 7. A CAR cell for killing HIV-infected cells, wherein the CAR cell has a modified non-natural ligand according to claim 1 associated therewith, wherein said CAR cell is associated with a plurality of modified non-natural ligands with different, separate heterologous molecules that bind different epitopes, proteins or other molecules on the surface of an HIV-infected cell, and wherein the CAR cell is a CAR T cell or a CAR NK cell. 8. The CAR cell of claim 7, wherein said CAR cell comprises a modified non-natural NKG2D receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 54 and SEQ ID NO: 154, and, wherein a modified non-natural ligand that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 68-72, to which is attached a heterologous molecule or atom that does not bind an HIV protein, is further linked to said CAR cell. 9. The CAR cell of claim 7 or 8, wherein said heterologous molecule or atom that does not bind an HIV protein modulates the function of the CAR cell. 10. The CAR cell of claim 9, wherein said function is a function selected from the group consisting of proliferation, differentiation, ablation, imaging, immunosuppression antagonism, homing, and cytolysis of a cell not infected with HIV.

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