RU2832538C2 - Non-natural nkg2d receptors which do not directly transmit signal to cells with which they are bound - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to non-natural NKG2D receptors associated with mammalian cell surfaces and can be used to deliver a payload to a T-cell, macrophage or NK-cell of a mammal. Disclosed is a non-natural modified NKG2D receptor, which contains an amino acid sequence selected from SEQ ID NO: 18–35.

EFFECT: invention provides obtaining non-natural NKG2D receptors binding to non-natural, modified domains α1-α2 ligands NKG2D, but not with natural ligands NKG2D, containing SEQ ID NO: 4.

6 cl, 24 dwg, 8 ex

Description

Prerequisites for the creation of an invention

Field to which the invention relates

[0001] The present invention generally relates to a non-natural ectodomain of a non-natural NKG2D receptor associated with a mammalian cell, wherein the receptor does not directly activate or signal the mammalian cell upon binding to a non-natural NKG2D ligand modified to specifically bind to the non-natural NKG2D receptor, and to heterologous molecules attached to the modified α1-α2 domains of the NKG2D ligand.

Background information

NKG2D is an activating receptor expressed as a homodimeric type II integral protein on the surface of natural killer (NK) cells and some T cells and macrophages. When bound to one of its eight natural ligands, expressed primarily on the surface of injured cells, NKG2D activates the NK cell to kill the injured cell, or if expressed on T cells, ligand-occupied NKG2D stimulates the activated T cell to exert its effector function. Three-dimensional structures have been determined for the ectodomain of native human NKG2D, several of its soluble natural ligands, and, in some cases, for the associated complex of soluble ligand and receptor ectodomain. The monomeric α1-α2 domains of NKG2D ligands bind specifically to the two ectodomains of the native NKG2D homodimer.

The essence of the invention

[0002] The present invention relates to non-natural NKG2D receptors associated with mammalian cell surfaces, wherein the non-natural receptors do not directly signal or directly activate the cell if the receptor is associated with cognate non-natural α1-α2 NKG2D ligand domains modified to specifically bind to the non-natural NKG2D receptors. The non-natural α1-α2 NKG2D ligand domains may be attached to heterologous atoms or molecules, including polypeptides, and in some embodiments, cytokines or modified cytokines, antibodies or antibody fragments. Direct activation or direct signaling to the cell is not mediated by the associated non-natural NKG2D receptor and does not occur even upon activation of immunological synapses.

Brief description of the drawings

[0003] Fig. 1A-1B: (Fig. 1A) Alignment of the native ectodomain of NKG2D.wt (SEQ ID NO: 17) with the non-native variants NKG2D.YA (SEQ ID NO: 18) and NKG2D.AF (SEQ ID NO: 25). Positions Y152 and Y199 are indicated, with mutated residues present in the non-native variants highlighted in gray. (Fig. 1B) Alignment of the α1-α2 domain of native/wild-type ULBP2 (SEQ ID NO: 4) and non-native ULBP2 variants, including ULBP2.R80W (SEQ ID NO: 108). Residues that play an important role in binding of the non-native ULBP2 variants to the non-native receptors NKG2D.YA or NKG2D.AF are highlighted in gray. The positions of residue R80 are indicated, as well as the region M154-F159, which was analyzed for binding of orthogonal variants to NKG2D.YA (ULBP2.S3, SEQ ID NO: 127) or NKG2D.AF (ULBP2.C, SEQ ID NO: 111; ULBP2.R, SEQ ID NO: 113; ULPB2.AA, SEQ ID NO: 115; and ULBP2.AB, SEQ ID NO: 117).

[0004] Figure 2: Comparison of non-natural Fc-NKG2D fusion proteins analyzed by size exclusion chromatography on an Akta HiLoad 16/600 Superdex 200 column. Migration of correctly assembled material was illustrated as a discrete symmetrical peak that elutes at higher volumes, while aggregated material elutes earlier at lower volumes. The site and nature of the modifications are represented by amino acid residues Y152 or Y199, or both (SEQ ID NOs: 48, 43, 42, 58, 41, and 40, starting from the top of the figure).

[0005] Figure 3: Profiles of non-naturally occurring Fc-eNKG2D variants with one or two amino acid substitutions were analyzed by size exclusion chromatography on an Akta Superdex 200 Increase 10/300 GL column. The migration of correctly assembled material was illustrated as a discrete symmetrical peak that elutes at higher volumes, while the aggregated material, which is characterized by a broad peak or a series of low-amplitude peaks, elutes at lower volumes. The letters in parentheses represent amino acids at positions 152 and 199 (SEQ ID NOs: 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, and 40 in order from the top), respectively.

[0006] Figure 4: ELISA assay for binding of wild-type ULBP2, MICwed- and MIC25-rituximab antibodies to Fc-eNKG2D candidates. Key residues are indicated at the top of the figure, but since many of the curves overlap, individual curves have also been labeled in each plot.

[0007] Figure 5: Binding of eNKG2D variants to wild-type ligands. Wild-type ligands (all presented as Fc-fusion) were captured on Octet AHC biosensors, and each native NKG2D, NKG2D.Y152A, or eNKG2D5 (Y152A/Y199F) Fc-fusion was titrated from 300 nM to 0.41 nM. Maximal binding responses were quantified using Octet (note the different ordinates for each plot).

[0008] Fig. 6: ELISA titration assay of individual phage variants to confirm selective binding to Fc-NKG2D.AF and reduction or elimination of binding to Fc-NKG2D.wt. The mutations are described in detail in Fig. 22.

[0009] Fig. 7: ELISA data for four MicAbody antibodies against non-natural α1-α2 ULBP2 variants binding to NKG2D.wt, NKG2D.YA, and NKG2D.AF. Fc-NKG2D variants were used as capture agents. MicAbodies were titrated and detected using HRP-conjugated anti-human kappa chains.

[0010] Fig. 8: ULBP2.C (SEQ ID NO: 111), ULBP2.R (SEQ ID NO: 113), ULBP2.AA (SEQ ID NO: 115), and ULBP2.AB (SEQ ID NO: 117) were assessed for changes in peptide-MHCI immunogenicity compared to wild-type ULBP2 (SEQ ID NO: 4) using the NetMHC4.0 server queried for representative HLA supertype variants. For the parent sequence, the variable region (residues 154-159 according to the alignment in Fig. 1B) for each variant was entered along with nine residues located upstream and downstream (24 residues in total), and the 9-mer peptide windows were assessed for predicted immunogenicity. Dark gray boxes correspond to peptides that were predicted to have a high probability of binding to the MHCI pocket (defined as a percentage rank < 0.5) and therefore a high probability of being present. Light gray boxes correspond to weakly predicted binding (percentage rank < 2). See Example 5 for a detailed description.

[0011] Fig. 9: ULBP2.C (SEQ ID NO: 111), ULBP2.R (SEQ ID NO: 113), ULBP2.AA (SEQ ID NO: 115), and ULBP2.AB (SEQ ID NO: 117) were assessed for altered class II peptide-MHC immunogenicity compared to wild-type ULBP2 (SEQ ID NO: 4) using the NetMHCII 2.3 server queried for HLA-DR, HLA-DQ, HLA-DP. For the parent sequence, the variable region (residues 154-159 according to the alignment as described in Fig. 1B) for each variant was entered along with 15 residues located left and right (36 residues in total), and 15-mer windows of peptides were assessed for predicted immunogenicity. Dark grey boxes represent peptides that are highly likely to bind to the MHCII pocket and are therefore likely to be present and immunogenic. Light grey boxes represent weak predicted binding.

[0012] Fig. 10A-10B: ELISA assay for binding of rituximab-MicAbodies consisting of ULBP2.wt (wild type), ULBP2.R80W (which has increased affinity for wild type NKG2D), ULPB2.S3 (a selected orthogonal variant of NKG2D.YA) or ULBP2.R (a selected orthogonal variant of NKG2D.AF) to native NKG2D.wt, NKG2D.YA and NKG2D.AF. (Fig. 10A) ELISA curves. The decrease in absorbance at 458 nm for some assays at higher concentrations is an artifact that is often observed in high affinity interactions at higher concentrations due to precipitation of the TMB-Ultra reagent used for development in the ELISA. (Fig. 10B) EC ₅₀ values (expressed in nM) determined using GraphPad Prism software based on the curves in (A). nd=not determined due to lack of relationship between elevated concentration and binding.

[0013] Fig. 11A-11C: In vitro cytolytic assays using CD8 effector cells that were either untransduced or transduced with NKG2D.wt, NKG2D.YA, or NKG2D.AF CAR constructs consisting of the CD8a hinge/transmembrane domain and the 4-1BB and CD3zeta intracellular signaling domains. Target cells were preloaded with calcein and treated with effector cells at increasing effector-to-target (E:T) ratios. The amount of calcein released was determined after five hours. (Fig. 11A) - lysis of HeLa cells, (Fig. 11B) - lysis of HeLa cells transfected to overexpress surface ULBP1, and (Fig. 11C) - cytolysis of HeLa cells expressing non-native ULBP2.R on their surface. Error bars represent the standard deviation of technical replicates in the experiment.

[0014] Fig. 12A-12B: MicAbody-mediated cytolysis of tumor lines by NKG2D CAR CD8 T cells. (Fig. 12A): Ramos cells, which express CD20, the target of rituximab, were preloaded with calcein and treated with NKG2D.AF or NKG2D.YA CAR cells at an E:T ratio of 20:1 along with increasing concentrations of rituximab-Micabody against ULBP2.S3 or ULBP2.R. The level of cytolysis was quantified after coincubation for two hours. (Fig. 12B): The murine tumor line CT26, transfected to express human Her2, was used as a target for cytolysis in parallel with Ramos cells. NKG2D.AF-CAR-CD8 T cells were pretreated with a saturating concentration (5 nM) of rituximab-ULBP2.R, trastuzumab-ULBP2.R, or an equimolar mixture of the two MicAbodies. Unbound MicAbody was removed by washing and CD8 cells were added to target cells at two different E:T ratios. Cytolysis was assessed after two hours.

[0015] Fig. 13A-13D: Potential MicAbody and MicAdaptor formats are illustrated. (Fig. 13A): Various Fc variants of antibodies used in the development of MicAbody and MicAdaptor reagents, including: (a) wild-type human IgG1 Fc, (b) two mutations that confer Fc-ADCC deficiency, and (c) mutations conferring an electrostatic interaction in each Fc-Fc1 or Fc2, allowing the production of heterodimeric Fc molecules that also contain mutations associated with ADCC deficiency. (Fig. 13B): An example of how orthogonal ligands can be linked to the C-terminus of (a) the heavy chain or (b) the light chain to produce MicAbody reagents. (Fig. 13C): Examples of MicAdaptors with direct attachment of orthogonal ligands without antibody components. (Fig. 13D): Illustration of a variety of MicAdaptor molecules that can be generated in the presence of human IgG1 Fc to enhance serum stability and may or may not include (a, b, c; designated as "Fc1/Fc2" in text and legend) or not (d, e) heterodimeric Fc mutations depending on the valence of the desired molecule for either heterologous loading or orthogonal ligand. Use of a full-length antibody structure may also be included depending on the desired loading, valence, and functionality (f, g), and this structure may be a hybrid of heavy or light chains.

[0016] Fig. 14: Schematic representation of CAR constructs, silent CARs, and other CAR variants. SEQ ID NOs are provided for each construct.

[0017] Fig. 15A-15C: Selective delivery of cytokine fusions to CD8 T cells expressing NKG2D.YA-CAR. (Fig. 15A) CD8 cells expressing either NKG2D.wt-CAR (SEQ ID NO: 151) or NKG2D.YA-CAR, SEQ ID NO: 153) were treated with 30 or 300 IU/mL recombinant human IL2 (rhIL2) or IL15 (rhIL15), or mutant IL2/orthogonal ligand fusion variants of IL15 or heterodimeric Fc fusions (Fc1/Fc2). Three days later, proliferation was quantified using the WST cell proliferation assay. The data presented here were normalized to a no-cytokine control. (Fig. 15B) Human T cells transduced with a vector encoding the NKG2D.YA-CAR were exposed to various ligand-cytokine fusion molecules for seven days, and the percentage of GFP ^{+ CAR−} cells in culture was monitored over time. (Fig. 15C) The orthogonal ligand enhances the delivery of IL21 and mutant IL21 reagents to NKG2D.YA-CAR cells and promotes their distribution to untransduced cells over three days of culture, as determined by WST analysis. IU/mL cytokine or cytokine-MicAdaptor are indicated in parentheses on the x-axis. See Fig. 24 for MicAdaptor SEQ ID NO.

[0018] Fig. 16A-16C: Data from a study on the sufficiency of the NKG2D.YA ectodomain alone to stimulate delivery of the orthogonal ligand-cytokine fusion into cells. (Fig. 16A) The NKG2D.YA ectodomain alone (SEQ ID NO: 157) was unable to directly kill Ramos cells induced by rituximab-ULBP2.S3 MicAbody (SEQ ID NOS: 98 and 129). (Fig. 16B) WST proliferation assay following three days of treatment with various cytokine reagents. (Fig. 16C) The WST proliferation assay demonstrated that NKG2D.YA-CAR interactions with the orthogonal ligand, but in the absence of the fusion cytokine or cytokine mutant, were not sufficient to stimulate cell proliferation. See Fig. 24 for MicAdaptor SEQ ID NO. The amount of IU/ml of cytokines and cytokine-MicAdaptors is given in brackets in the legends to the abscissa axis.

[0019] Fig. 17A-17C: (Fig. 17A) WST proliferation assay using different costimulatory domain mutants (SEQ ID NOs: 161, 163, and 165) following incubation with the indicated cytokine or cytokine-MicAdaptor for three days. (Fig. 17B): The same CAR costimulatory domain mutants were assessed for their ability to efficiently lyse calcein-loaded Ramos target cells in the presence of rituximab-ULBP2.S3 MicAbody (SEQ ID NOs: 98 and 129). (Fig. 17C): Co-expression of the NKG2D.YA ectodomain (NKG2D.YA-ecd) with the complete CD19scFv-CAR is sufficient to stimulate proliferation as assessed by WST assay after three days of incubation with cytokine reagents. See Fig. 24 for MicAdaptor SEQ ID NO.

[0020] Fig. 18: Brief overview of candidate, non-natural variant Fc-eNKG2D mutations, and aggregated protein properties determined by size exclusion chromatography (SEC).

[0021] Figure 19: Percent saturation (Rmax) of eNKG2D variants normalized to wild-type NKG2D binding to MICwed-MicAbody or MIC25-MicAbody. Wild-type Fc-NKG2D and each Fc-eNKG2D receptor were captured on AHC biosensors and then exposed to trastuzumab-specific MicAbody at 20 nM. Dissociation kinetics were then monitored and the Rmax values of the Fc-eNKG2D hybrids were ranked. These samples (nt) were not tested due to significant aggregation or inadequate amounts of material expressed or recovered after SEC fractionation.

[0022] Fig. 20: EC50 values (nM) for the Fc-eNKG2D ELISA as shown in Fig. 3. nt=not tested; nb=no binding or very weak binding even at 300 nM, and therefore EC50 value was not calculated.

[0023] Fig. 21: Subpopulation of combinatorial mutations in ULBP2 that resulted in phage clones with selective binding to NKG2D.AF compared to native NKG2D.wt, as confirmed by spot ELISA. Mutants were ranked by the frequency of occurrence of the selected phages.

[0024] Fig. 22: Specificity of NKG2D.AF-selected ULBP2 variants for rituximab-MicAbody that retained binding to NKG2D.AF as assessed by quantitative ELISA. The specific amino acid modifications of each ULBP2 variant are shown as their binding ratios to the Fc-NKG2D.wt fusion compared to the Fc-NKG2D.AF fusion. The amino acid residue positions of ULBP2 are shown in Fig. 1B.

[0025] Fig. 23: Selected mutations at the indicated amino acid positions of ULBP2.R80W (Fig. 1B; SEQ ID NO: 108) that resulted in Y152A-specific phage clones.

[0026] Fig. 24: SEQ ID NO: MicAdaptor and method for purification via transient transfections.

Detailed description of the invention

[0027] Natural killer (NK) cells and certain (CD8+ αβ and γδ) T cells of the immune system play an important role in humans and other mammals as first-line innate defenses against tumor and infected cells (Cerwenka, A., and LL Lanier. 2001. NK cells, viruses and cancer. Nat. Rev. Immunol. 1:41-49). NK cells and some T cells have on their surface NKG2D, a prominent, homodimeric, surface immunoreceptor responsible for recognizing target cells and activating natural defenses against the pathological 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 lectin-like C-type extracellular (ecto-)domain that binds eight different cognate ligands, with the best-studied ligands being those with 84% sequence identity or homology to that of monomeric MICA and MICB, polymorphic analogues of the major histocompatibility complex (MHC) class I (MIC) chain-related glycoproteins (Weis et al. 1998. The C-type lectin superfamily of the immune system. Immunol. Rev. 163: 19–34; Bahram et al. 1994. A second lineage of mammalian MHC class I genes. PNAS 91:6259–6263; Bahram et al. 1996a. Nucleotide sequence of the human MHC class I MICA gene. Immunogenetics 44: 80-81; Bahram and Spies TA. 1996. Nucleotide sequence of human MHC class I MICB cDNA. Immunogenetics 43: 230-233). Non-pathological expression of MICA and MICB is to some extent restricted to intestinal epithelium, keratinocytes, endothelial cells, and monocytes, but aberrant expression of these MIC surface proteins occurs in response to many types of cellular stress, such as proliferation, oxidation, and heat shock, and is a marker of cellular pathology (Groh et al. 1996. Cell stress-regulated human MHC class I gene expressed in GI epithelium. PNAS 93: 12445–12450; Groh et al. 1998. Recognition of stress-induced MHC molecules by intestinal γδ T cells. Science 279: 1737–1740; Zwirner et al. 1999. Differential expression of MICA by endothelial cells, fibroblasts, keratinocytes and monocytes. Human Immunol. 60: 323-330). Abnormal expression of MIC proteins also appears to be involved in the development of 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 in enabling the immune system to identify and respond to a wide range of dangerous signals, thereby 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).

[0028] Viral infection is the most common inducer of MIC protein expression and identifies virus-infected cells for attack by NK or T cells (Groh et al. 1998; Groh et al. 2001. Co-stimulation of CD8+ 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 their host cell, CMV and other viruses have evolved mechanisms that prevent the expression of MIC proteins on the surface of the cell they infect, allowing these viruses to “escape” targeting by 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).

[0029] Despite stress, many malignant cells, such as lung cancer cells and brain glioblastomas, also escape expression of MIC proteins and, as a result, may be particularly aggressive because they also 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).

[0030] A high resolution structure of human MICA bound to NKG2D has been elucidated and it has been demonstrated 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 No. 1HYR). The α3 domain of MICA, like MICB, is linked to the α1-α2 scaffold domain via a short flexible peptide linker and itself is intrinsically a "spacer" located between the scaffold and the surface of the MIC-expressing cell. The three-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).

[0031] T cells, NK cells, and macrophages can be modified using gene transfer technologies to directly and stably express on their surface antibody binding domains that confer specificity to a new antigen (Saar Gill & Carl H. June. Going viral: Chimeric Antigen Receptor (CAR) T cell therapy for hematological malignancies. Immunological Reviews 2015. Vol. 263: 68–89; Wolfgang Glienke, Ruth Esser, Christoph Priesner, Julia D. Suerth, Axel Schambach, Winfried S. Wels, Manuel Grez, Stephan Kloess, Lubomir Arseniev and Ulrike Koehl. 2015. Advantages and applications of CAR-expressing natural killer cells. Front. Pharmacol. doi: 10.3389/fphar.2015.00021). This approach uses CAR T cells, which fuse the antigen recognition domain of a specific antigen with a hybrid intracellular domain of the CD3-zeta chain. The CD3-zeta chain is the primary transducer of signals from the ectodomain of endogenous T cell receptors (TCRs) to the intracellular space. CARs engineered with the CD3-zeta chain and costimulatory molecules such as CD27, CD28, ICOS, 4-1BB, or OX40 trigger CAR T cell activation upon binding to the target antigen in a manner similar to that seen with the endogenous T cell receptor but independent of the major histocompatibility complex (MHC).

[0032] Several non-natural α1-α2 domains of NKG2D ligands have been described that are modified to bind the natural human NKG2D receptor 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, we describe the attachment of non-natural NKG2D receptors to the surface of mammalian cells in a form that retains specific binding of modified non-natural NKG2D ligands to attached heterologous molecules, but the non-natural receptors avoid direct or cis-activation or intracellular signaling in the mammalian cell, even when the cell forms an immunological synapse with the cell or other surface targeted by the heterologous molecule. The unnatural NKG2D receptors themselves have been mutated at one or two specific sites, and each of these mutations results in disruption or loss of binding to all natural α1-α2 domains of NKG2D ligands (David J. Culpepper, Michael K. Maddox, Andrew B. Caldwell, and Benjamin J. McFarland. Systematic mutation and thermodynamic analysis of central tyrosine pairs in polyspecific NKG2D receptor interactions. Mol. Immunol. 2011 January; 48(4): 516–523; USPTO application 14/562534; USPTO provisional application 62/088456)). The present invention provides CARs that, when bound to a mammalian cell surface, provide a silenced receptor that can serve as a surrogate receptor with high binding affinity for heterologous atoms or molecules on the cell surface. Accordingly, by means of linked non-natural ligands specific for a non-natural modified NKG2D receptor, heterologous molecules containing, for example, a defective cytokine, can be specifically delivered to the silenced receptor on the surface of a mammalian cell, but not to cells lacking the cognate silenced receptor. Once bound to a cell bearing the silenced receptor, the defective heterologous molecule can bind to the corresponding receptor subunits on the cell surface where such binding is maintained, and thereby directly signal the cell as if it were stimulated by the wild-type ligand.

[0033] It is apparent that a CAR consisting of an inert non-natural NKG2D, CD3-zeta, and a costimulatory domain such as CD28, 4-1BB, ICOS, or OX40, and present on a mammalian cell, is capable of directly stimulating and activating the CAR cell after formation of an immunological synapse. Activation of such a second or third generation CAR T cell is dependent on the function of the CD3-zeta domain and the function of at least one costimulatory domain, such as 4-1BB or CD28. However, such a CAR, such as a silent CAR, can serve as a surrogate receptor with high binding affinity for cognate non-natural ligands bound to heterologous molecules that have defective binding to their corresponding natural receptor or receptor subunit(s). This high affinity binding allows the heterologous molecule attached to the non-natural ligand to transmit signals into the cell via their respective other receptor subunits, for which such binding is conserved.

[0034] It is important to note that if the CD3-zeta domain of such a direct activation competent CAR is selectively inactivated, it can still act as a silent CAR and activate heterologous molecules that are bound to a cognate non-natural ligand and that have defective binding to their respective natural receptor or to a receptor subunit(s) to transmit signals to the cell via their respective other receptor subunits. If the costimulatory domain of a CAR, such as 4-1BB, is inactivated but the active CD3-zeta domain is retained, the CAR cannot serve as a silent receptor. That is, although CD3-zeta is not required in this case, a functional costimulatory domain is required for a heterologous molecule, such as a defective cytokine, bound to a cognate non-natural ligand bound to the receptor to mediate the appropriate signaling to the CAR cell.

[0035] The present inventors have unexpectedly discovered that there is a need for a costimulatory domain, but not a CD3-zeta domain, in order for a heterologous defective cytokine linked to a cognate non-natural ligand to mediate the appropriate signaling in a CAR cell. Furthermore, the present invention discloses that the costimulatory domain can act in a cis- or trans-orientation relative to the silent receptor to which the cognate ligand linked to the defective heterologous molecule is linked.

[0036] If a heterologous molecule, such as an antibody or antibody fragment, targeting a particular molecule binds to a cognate non-natural NKG2D ligand, which in turn binds to a silent receptor, the mammalian cell bearing the silent receptor will return to the surface to which it is directed by the targeting heterologous molecule. Even if a "synapse" occurs between the cell bearing the silent receptor and the surface of the target cell, the cell will not be activated by the silent receptor.

[0037] Because there are multiple copies of the non-naturally occurring NKG2D-based silent receptors of the invention on the cell surface, homing and/or selective activation by heterologous molecules can be multiplexed or sequentially altered during processing or treatment regimens.

[0038] A cell bearing a silenced CAR receptor may also express other receptor(s) or CARs orthogonal to the silenced CAR and act independently of the silenced CAR, thereby providing specific and direct activation or signaling in some other manner to the same cell upon appropriate stimulation. The other or "second" orthogonal CAR may be a conventional single-chain Fv (scFv) CAR or a second orthogonal non-naturally occurring NKG2D-based modified CAR with its own cognate non-naturally occurring α1-α2 ligand(s) (reference to provisional application AF). The ability to engineer immune effector cells with more than one orthogonal non-natural CAR, silent or active, and multiple related non-natural ligands with attached heterologous molecules or atoms will greatly expand the capabilities, flexibility, and quality control of adoptive cell therapy (ACT).

[0039] In the course of characterizing a silenced CAR on a cell and its dependence on a cis- or trans-acting costimulatory domain such as 4-1BB, it was found that, compared to an unmodified human T cell, a human T cell expressing a silenced CAR with a costimulatory domain exhibits a significantly enhanced response to native IL-2 or a cognate non-natural ligand bound to either native or mutant IL-2 that has low binding affinity for the receptor α subunit. This observation has important implications for the preferential expansion ex vivo or in vivo of cells expressing a CAR consisting of a costimulatory domain with or without a CD3 zeta domain.

[0040] As used herein, the terms "soluble MIC protein,""solubleMICA," and "soluble MICB" refer to a MIC protein that contains α1-α2 domains with or without the α3 domain of the MIC protein and lacks transmembrane or intracellular domains. The NKG2D ligands, ULBP1-6, do not naturally have an α3 domain (Cerwenka A, Lanier LL. 2004. NKG2D ligands: unconventional MHC class I-like molecules exploited by viruses and cancer. Tissue Antigens 61 (5): 335-43. doi:10.1034/j.1399-0039.2003.00070.x. PMID: 12753652). "NKG2D ligand α1-α2 domain" refers to the domain of the ligand protein that binds to the NKG2D receptor.

[0041] 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 naturally occurring α1-α2 domain of an NKG2D ligand (SEQ ID NOS: 1-9 for MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and OMCP, respectively). In other embodiments, the modified α1-α2 domain is 85% identical to a native or naturally occurring α1-α2 domain of an NKG2D ligand. In other embodiments, the modified α1-α2 domain is 90% identical to the native or natural α1-α2 domain of a natural NKG2D ligand protein and binds to non-natural NKG2D.

[0042] Preferably, the modified or non-natural α1-α2 domains of the non-natural MIC proteins of the invention are at least 80% identical or homologous to a native or natural α1-α2 domain of one of the 8 human NKG2D ligand proteins (SEQ ID NOs: 1-8) and bind to a non-natural NKG2D ectodomain. In some embodiments, the non-natural α1-α2 domain is 85% identical to a native or natural α1-α2 domain of an NKG2D ligand protein and binds to a non-natural NKG2D. In other embodiments, the non-natural α1-α2 scaffold domain is 90%, 95%, 96%, 97%, 98%, or 99% identical to a native or natural α1-α2 scaffold of a natural α1-α2 domain of a human protein and binds to non-natural NKG2D.

[0043] In some embodiments, a heterologous molecular label can be attached to the N-terminus or C-terminus of the non-natural α1-α2 domain of the soluble MIC protein or the domain of the attached heterologous peptide or protein to facilitate purification of the soluble ligand. Label sequences include peptides such as poly-histidine, myc peptide, FLAG tag, streptavidin-like tag, or a small molecule such as biotin. Such labels can be removed after isolation of the MIC molecule by methods known to those skilled in the art.

[0044] Specific mutations in the α1-α2 domains of NKG2D ligands can be introduced to create non-natural α1-α2 domains that bind to non-natural NKG2D receptors that have themselves been engineered to have reduced affinity for natural NKG2D ligands. This can be accomplished, for example, by genetic engineering. The non-natural NKG2D receptor modified in this way can be used to create an NKG2D-based CAR on the surface of NK cells, T cells, macrophages, or other cells of the immune system that can bind to molecules consisting of non-natural α1-α2 domains. These non-natural NKG2D receptors and their cognate non-natural NKG2D ligands will provide important advantages in safety, efficacy, and manufacturing properties for the treatment of cancer and viral infections compared to currently available CAR-T cells and CAR-NK cells, as described below. If the intracellular signaling of non-natural NKG2D receptors on the surface of mammalian cells was blocked as described in the present invention, then these CARs of the invention can act as surrogate receptors with high binding affinity for other defective heterologous molecules such as cytokines, chemokines, lymphokines, cytotoxins, and atoms linked or conjugated to orthogonal NKG2D ligands. This allows for the delivery of heterologous molecules directly and specifically to the cell bearing the silenced receptor, without direct activation of the host cell by the silenced receptor per se . Furthermore, heterologous molecules that bind to specific targets, and thereby to cells or other surfaces bearing such targets, may provide specific homing functions to a cell bearing a silent receptor without accidentally activating or stimulating it.

[0045] CAR-T or CAR-NK cells composed of ectodomains of non-natural NKG2D receptors that do not bind or bind very poorly to natural NKG2D ligands will not be activated by any natural ligands and, therefore, will not be toxigenic like cells expressing a CAR based on the natural NKG2D receptor. Furthermore, the ectodomains of non-natural NKG2D receptors on cells will not be inhibited by natural NKG2D ligands in soluble form 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, if such CAR cells bearing ectodomains of non-natural NKG2D receptors are bound by bispecific molecules to the cognate non-natural α1-α2 domains of the invention and to its heterologous targeting motif that has been found to bind to its target, the CAR will be activated and express CAR cell effector functions.

[0046] Since CAR-T or CAR-NK cells composed of ectodomains of the non-natural NKG2D receptor are not activated unless a bound bispecific molecule composed of the related non-natural α1-α2 domain is present, their activation can be controlled by administering bispecific molecules that, as biopharmaceuticals, will have pharmacokinetics and pharmacodynamics well known to those skilled in the art. In the event of adverse effects, the physician, instead of using an induced "suicide" mechanism to destroy the infused CAR cells, as is currently done, can simply change the dosing schedule of the bispecific molecule (Monica Casucci and Attilio Bondanza. Suicide Gene Therapy to Increase the Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes. J Cancer. 2011; 2: 378-382). Furthermore, such bispecific molecules with different specific targeting motifs can be administered simultaneously or sequentially to prevent and reverse tumor resistance resulting from loss of the target antigen without the need to generate, expand and infuse several 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 administered bispecific molecule according to the invention, the manufacturing processes will be simplified and less expensive.

[0047] Examples of parent or recipient proteins or polypeptides that are candidates for attachment to the non-natural α1-α2 domains of NKG2D ligands include, but are not limited to, antibodies, proteins consisting of Ig folds or Ig domains, including modified Fc domains that recruit natural molecules or are incapable of recruiting or binding natural molecules, globulins, albumins, fibronectins and fibronectin domains, integrins, fluorescent proteins, enzymes, outer membrane proteins, receptor proteins, T cell receptors, chimeric antigen receptors, viral antigens, viral capsids, viral ligands for cellular receptors, hormones, cytokines and modified cytokines such as interleukins, knottins, cyclic peptides or polypeptides, proteins, belonging to the family of major histocompatibility complex (MHC) molecules, MIC proteins, lectins and ligands for lectins. Non-protein molecules such as polysaccharides, dendrimers, polyglycols, peptidoglycans, antibiotics and polyketides can also be attached to the modified α1-α2 domains of NKG2D ligands.

[0048] Thus, the present invention allows to expand the diversity and practicality of this remarkable, very promising immunological approach to the treatment of cancer using CAR-T cells, CAR-NK cells and CAR-macrophage-like cells, and thereby solve many of the currently existing problems.

[0049] As used herein, the terms "peptide,""polypeptide," and "protein" are synonymous; and the terms "heterologous molecule,""heterologouspeptide,""heterologoussequence," or "heterologous atom" mean a molecule, peptide, nucleic acid, or amino acid sequence or atom, respectively, that is not naturally occurring or is not normally in physical association with the molecule in question. As used herein, the terms "non-natural" and "modified" are synonymous. As used herein, the terms "natural,""native," and "wild type" are synonymous, and the terms "NKG2D" and "NKG2D receptor" are also synonymous. As used herein, the term "antibody" is considered in the broadest sense and particularly encompasses monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they possess the desired biological activity. "Antibody fragments" include a portion of an antibody, preferably comprising its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , Fv fragments, and inserted Fv; diabodies; straight chain antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment(s).

[0050] The term "comprising," as used herein as a synonym for "including," "constituting," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The term "consisting of" excludes any element, step, or ingredient not recited in the claims. The term "consisting essentially of" limits the scope of the claims to the recited materials or steps and to elements that do not materially affect the basic and novel characteristics of the claimed invention. The present invention discloses embodiments of the compositions and methods of the invention that fall within the scope of each of these terms. Thus, when describing a composition or method that contains the recited elements or steps, particular embodiments of the invention are contemplated in which the composition or method essentially consists of or consists of these elements or steps.

[0051] All documents cited herein are hereby incorporated by reference in their entirety, whether or not specifically incorporated previously. As used herein, the articles "a," "an," and the word "any" may refer to both singular and plural nouns.

[0052] From the complete description of the invention, it will be apparent to those skilled in the art that the present invention can be practiced using a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without undue experimentation. Although the present invention has been described with respect to its specific embodiments, it should be noted that additional modifications can be made thereto. The present application covers any changes, applications or adaptations according to the invention that are generally consistent with the principles of the invention and includes such departures from the disclosure of the present invention that are within the known or customary practice in the art to which the present invention pertains and can be applied to the above-mentioned essential features.

EXAMPLES

Modified ectodomain of NKG2D receptor and modified α1-α2 domains of NKG2D ligands

[0053] Example 1. Substitution of tyrosine 152 with alanine (Y152A) and tyrosine 199 with phenylalanine (Y199F) of the human NKG2D receptor to create an inert NKG2D ectodomain

[0054] It has been previously demonstrated that mutations at tyrosine 152 or tyrosine 199 in human NKG2D, equivalent to positions 73 and 120 of the NKG2D ectodomain (Fig. 1A, SEQ ID NO:17), can significantly reduce the level of 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 although mutation of any tyrosine residue strongly affects the ability of NKG2D to bind to its natural ligands, simultaneous mutation of tyrosine 152 (Y152) and tyrosine 199 (Y199) virtually eliminates the ability of the receptor to interact with all native ligands. Therefore, we sought to investigate individual and combinatorial substitutions of Y152 and Y199 and characterize them with respect to their biochemical behavior in order to identify variants with both single and double mutations that are unable to interact with any natural ligands. Variants that were also well expressed and assembled were of particular interest because they were inert ligands that could be readily recovered for analysis.

[0055] The native NKG2D ectodomain (wild type) (NKG2D.wt, SEQ ID NO: 17) and candidate ectodomains of a non-naturally occurring NKG2D variant (SEQ ID NOs: 18-35), also referred to as "engineered NKG2D" or "eNKG2D", were cloned as fusions with the C-terminus of human IgG1 Fc (lacking the Fab domains) via a short Ile-Glu-Gly-Arg linker recognized by factor Xa (SEQ ID NO: 38), and these domains are interchangeably referred to as Fc-NKG2D.wt or NKG2D.wt and Fc-eNKG2D or eNKG2D (SEQ ID NOs: 40-58). gBlocks® DNA fragments (Integrated DNA Technologies, San Diego, CA) corresponding to the MHCI signal sequence (SEQ ID NOs: 36 and 37), human IgG1 Fc with linker (SEQ ID NO: 39), and NKG2D ectodomain variants (SEQ ID NOs: 59-77) were synthesized and inserted into pD2610-V12 (ATUM, Newark, CA). DNA constructs in which substitutions were experimentally introduced at positions Y152, Y199, or a combination of Y152/Y199 substitutions (Fig. 18) were transiently expressed in Expi293 ^™ cells (ThermoFisher Scientific, Waltham, MA), and the secreted protein was purified by protein A affinity chromatography (catalog #20334, Pierce Biotechnology, Rockford, IL). Eluted material was characterized by size exclusion chromatography (SEC) on Akta Pur Superdex columns, and correctly collected material of the appropriate size was fractionated and isolated from aggregated peaks before inclusion in analyses.

[0056] SEC characterization of the purified NKG2D.Y199A-Fc hybrid revealed that the hybrid consists predominantly of aggregated material (Figure 2). In comparison, both the natural Fc-NKG2D hybrid and the Fc-NKG2D.Y152A hybrid exhibit a discrete, non-aggregated peak that can be easily distinguished from the more rapidly migrating aggregate. The effect of the Y199A substitution on aggregation was also observed in the Fc-NKG2D hybrid variant with the double Y152A/Y199A substitution, indicating that this substitution does not affect protein misfolding (Figure 2). Therefore, this aspect of incorporating Y199A with any combination of Y152 mutations into NKG2D variants presents a particular challenge in obtaining the material needed for subsequent engineering efforts and hinders assembly and cell surface presentation. As a consequence, an attempt was made to explore other substitutions at positions Y152 and Y199 that could be combined to yield a more stable molecule. The Y152 and Y199 eNKG2D combinatorial mutant candidates were evaluated as Fc-fusions and are detailed in Fig. 18. Furthermore, all purified and expressed Fc-eNKG2D fusion candidates were profiled by SEC and their chromatograms revealed varying levels of aggregate formation (Figs. 2 and 3, Fig. 18). Among the individual amino acid substitutions explored at residue 152, alanine, serine, threonine, and valine did not affect the assembly of the Fc-NKG2D molecule, whereas Y152-leucine (Y152L) yielded a highly aggregated material. Like alanine, neither glutamate nor aspartate were retained at position 199, although phenylalanine only slightly increased aggregate formation. Of the mutation combinations studied, Y152A/Y199F, Y152S/Y199F, Y152T/Y199F, and Y152F/Y199F did not adversely affect formation of the desired dimer, whereas the other combinations resulted in increased levels of aggregation (Fig. 18, Figs. 2 and 3).

[0057] Example 2: Generation of bispecific antibody-based molecules, “MicAbodies,” with variants of a non-natural NKG2D ligand

[0058] To generate non-naturally occurring MicA variants associated with human IgG1, DNA polynucleotides encoding the α1-α2 domains, such as MICwed (SEQ ID NO: 79) and MIC25 (SEQ ID NO: 81), were amplified by PCR using primers that also introduced a polynucleotide encoding either an APTSSSGGGGS linker for binding to the C-terminal kappa light chain (SEQ ID NO: 84) or a GGGS linker for binding to the C-terminal heavy chain of human IgG1 (SEQ ID NO: 82). In addition, two mutations were introduced into the CH2 domain of the heavy chain: D265A/N297A (Kabat numbering; Fig. 13A and 13B), which reduced the level of binding to all FcγR receptors, resulting in the elimination of 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: 12) was similarly cloned and fused to DNA polynucleotides encoding linkers and the heavy chain or light chain of IgG1. These bispecific antibodies, referred to as “MicAbody ^TM ” in the singular and “MicAbodies” in the plural, are bivalent for the α1-α2 fusion domain. Examples of antibodies used to generate MicAbodies for the purpose of studying the eNKG2D construct include, but are not limited to, trastuzumab (SEQ ID NOS: 94 and 96) and rituxumab (SEQ ID NOS: 98 and 100), and will hereinafter be referred to as “trastuzumab-MicAbody” (e.g., SEQ ID NOS: 102 and 104) and “rituximab-MicAbody” (e.g., SEQ ID NO: 106), respectively. The fusion constructs were individually inserted into pD2610-V12 (ATUM, Newark, 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 linked to a natural or non-natural NKG2D ligand were cotransfected for transient expression in Expi293 ^™ cells (ThermoFisher Scientific, Waltham, MA). Alternatively, a plasmid encoding the heavy chain linked to either a natural or non-natural NKG2D ligand and a plasmid encoding the light chain were cotransfected. Secreted bispecific antibodies were purified by protein A affinity chromatography (catalog #20334, Pierce Biotechnology, Rockford, IL), and the eluted material was characterized by size exclusion chromatography (SEC) on Akta Pur Superdex columns and fractionated when necessary. In addition, SDS-PAGE analysis was performed on purified samples to confirm the expected molecular masses of the hybrid heavy chain molecules and hybrid light chain molecules.

[0059] Example 3: Identification of modified NK2GD variants that are unable to bind either natural NKG2D-binding ligands or non-natural ligands that exhibit increased binding to wild-type NKG2D

[0060] The binding affinity of the α1-α2 variants to the extracellular domains of wild-type NKG2D and non-natural eNKG2D proteins was analyzed by plate ELISA. Each of the SEC-fractionated natural Fc-NKG2D fusions and non-natural Fc-eNKG2D fusions was coated overnight at 4°C onto individual wells of 96-well Nunc Maxisorp plates (Thermo Fisher Scientific, Waltham, MA) using a coating concentration of 1 μg/mL in phosphate-buffered saline (PBS). Plates were washed three times with 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 fusions 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 substrate solution in the 1-Step ^TM Ultra TMB ELISA assay (Thermo Fisher Scientific, Waltham, MA). Binding of rituximab-MicAbody ULBP2.wt (SEQ ID NOS: 98 and 106) differed between wild-type NKG2D variants and eNKG2D variants with reduced binding to the latter ligand, and MICwed (SEQ ID NOS: 96 and 102) and MIC25 (SEQ ID NOS: 96 and 104) ligand variants were found to be more effective in identifying eNKG2D variants with ablated ligand binding. The binding pattern of each eNKG2D variant to all three bispecific ligands revealed that combinations of NKG2D modifications resulted in the greatest reduction in binding of wild-type and modified ligands, allowing for the selection of top inert NKG2D variants.

[0061] Additional biophysical analysis of eNKG2D variant binding to ligands was also performed using biolayer interferometry (BLI) using the FortéBio Octet system (all from 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 captured on anti-human IgG Fc capture biosensor (AHC) tips. After establishing baselines, the tips were subjected to a series of titrations of Fc-eNKG2D fusion proteins ranging from 300 nM to 0.41 nM, and association/dissociation kinetics were monitored by performing all steps in PBS-BT. Fc-eNKG2D fusion proteins were then captured on AHC tips, and MicAbodies were titrated to characterize binding kinetics.

[0062] To determine the maximal response assessed by native NKG2D binding to MICwed or MIC25, native Fc-NKG2D fusions were captured on AHC biosensors and 20 nM trastuzumab-MICwed or 20 nM trastuzumab-MIC25 MicAbodies were incubated for two minutes, followed by dissociation kinetics assessed over 30 seconds. Binding assays were then performed under the same conditions with the Fc-eNKG2D fusion receptors as the capture agent, and the binding level for each eNKG2D was assessed as a percentage of the maximal binding response achieved with Fc-NKG2D.wt (Figure 19). For MICwed, the responses of all Fc-eNKG2D single mutant variants, except Y199F, were reduced to 50%. The Y199F substitution maintained 100% binding responses. However, all double mutant Fc-eNKG2D variants completely lost their ability to bind MICwed. For MIC25, all single mutant Fc-eNKG2D variants and Y152V/Y199F retained 100% binding responses compared to wild-type Fc-NKG2D binding. However, binding was reduced to 50% for several double mutant Fc-eNKG2D variants, including Y152A/Y199F, Y152S/Y199F, and Y152T/Y199F.

[0063] ELISA assays using Fc-eNKG2D fusions as capture agents were performed using ULBP2.wt, MICwed, MIC25 MicAbodies titrated starting at 300 nM (Figure 4). When available, EC50 values were calculated using GraphPad Prism (Figure 20). Native NKG2D bound ULBP2, MICwed, and MicAbody based on MIC25 with affinities calculated as Kd values of 1.4, 0.007, and 0.005 nM, respectively. Although the binding affinity of ULBP2 and MICwed MicAbodies to all eNKG2D candidates with one mutation was reduced, MIC25 binding to the eNKG2D candidates was maintained. However, all eNKG2D double mutant candidates had significantly reduced or no binding to all three ligands ULBP2, MICwed and MIC25 in the Micabody format.

[0064] eNKG2D variants eNKG2D5 (Y152A/Y199F), eNKG2D7 (Y152S/Y199F), eNKG2D8 (Y152T/Y199F), and eNKG2D9 (Y152V/Y199F) had reduced or no binding to ULBP2, MICwed, and MicAbody based on MIC25 as determined by Octet and ELISA (Figures 19 and 20). Additionally, eNKG2D 5, 7, and 8 had the lowest levels of aggregation, indicating more robust protein assembly following 293T expression (Figure 18). eNKG2D5 (SEQ ID NO: 48) was assessed more closely for binding to wild-type ligands, as MicAbodies were captured on Octet AHC tips. Fc-NKG2D.Y152A with a single mutation (SEQ ID NO: 41) had reduced binding to all natural ligands compared to natural (SEQ ID NO: 40) NKG2D (Fig. 5). The binding response curve of eNKG2D5 (Y152A/Y199F) was further flattened compared to Y152A eNKG2D. eNKG2D5 (Y152A/Y199F, hereafter referred to as “AF” or “NKG2D.AF”) was selected as the primary NKG2D variant for which related selective orthogonal non-natural ligands were designed.

[0065] Example 4: Construction of orthogonal unnatural α1-α2 domains that selectively bound to the unnatural ectodomain of NKG2D.AF

[0066] We used phage display to construct orthogonal unnatural α1-α2 domains that selectively bind to the NKG2D.AF receptor (SEQ ID NO: 48). As a starting point, the unnatural α1-α2 domain of ULBP2.R80W (Figure 1B; SEQ ID NO: 108) with high affinity for the wild-type natural NKG2D ectodomain (NKG2D.wt) was selected as the parent domain for further mutagenesis and phage display screening. Synthetic DNA libraries were generated for the α1-α2 domain of ULBP2.R80W (SEQ ID NO: 108), which has an additional C8S mutation to remove potential disulfide bonds. The codons of the ligand amino acid residues that, when bound, are located in close proximity to positions Y152 and Y199 on the natural NKG2D receptor were replaced with NNK codons, where the libraries consisted of NNK codons at positions 154-159 (Fig. 1B; SEQ ID NO: 110). Libraries were cloned as fusions with the small 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 procedures (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, CF, 3rd. (2011). These α1-α2 phage display libraries were selected for their high binding affinity to the unnatural receptor NKG2D.AF by selectively capturing phage clones bound to biotinylated Fc-NKG2D.AF protein in the presence of the non-biotinylated natural competitor protein Fc-NKG2D.wt. Selective clones were enriched by performing multiple rounds of competitive binding selection with increasing concentrations of non-biotinylated natural Fc-NKG2D.

[0067] Following four rounds of selection, phage clones were individually distributed in a 96-well format and then spot ELISA was performed to confirm preferential binding to non-native plate-bound NKG2D.AF compared to NKG2D.wt. Bound phage were detected using monoclonal antibody E1 against biotinylated phage M13 coat protein (ThermoFisher Scientific, Waltham, MA), using streptavidin-HRP detection reagent (R&D Systems, Minneapolis, MN), and Ultra TMB one-step ELISA development reagent (ThermoFisher Scientific, Waltham, MA). The spot ELISA signal for each clone was expressed as the ratio of phage bound to NKG2D.AF to phage bound to NKG2D.wt. Phages that had ratios greater than or equal to 14 were sequenced to identify specific mutations in the mutagenized regions of NNK. Figure 21 shows the selected amino acid residues for each phage α1-α2 variant that selectively binds NKG2D.AF. In cases where multiple clones were identified representing the same sequence, the ELISA signal ratios were plotted and the concordance of the phage clones was confirmed by clustering the raw data (data not shown).

[0068] Thirty variants identified by ELISA were expanded in separate monocultures to generate high-titer phage microbatches. Purified phage concentrations were normalized to an _OD268 of 0.5 and then subjected to a 1:3 dilution series against plate-bound Fc-NKG2D.AF or Fc-NKG2D.wt, followed by phage detection and display in an ELISA assay performed as described above. All thirty variants analyzed in this manner consistently demonstrated selective binding to NKG2D.AF with little or no binding to NKG2D.wt (Figure 6), even at the highest phage concentrations analyzed. Selected phages also demonstrated a two or more logarithm shift in phage concentration to achieve half-maximal binding between NKG2D.AF and NKG2D.wt.

[0069] To confirm that the NKG2D.AF-selective α1-α2 domain variants retain specific binding to the hybrid antibodies, 21 variants (FIG. 22; e.g., SEQ ID NOs: 111-118) were cloned as C-terminal hybrids with the APTSSSGGGGS linker linked to the light chain of the rituximab antibody (SEQ ID NOs: 119-126). The resulting hybrids were cloned into the mammalian expression vector pD2610-V12 (ATUM, Newark, CA) via Gibson cloning (New England Biolabs Inc., Ipswich, MA) and co-expressed with the heavy chain of the parental antibody (SEQ ID NO: 99) as paired full-length IgG antibodies. Transient expression was performed in Expi293 ^™ cells (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's protocol and purified by standard Protein A affinity chromatography (catalog #20334, Pierce Biotechnology, Rockford, IL). ELISA binding of each ULBP2 α1-α2 antibody fusion variant to non-natural Fc-NKG2D.AF and to natural Fc-NKG2D.wt demonstrated significantly higher binding affinity to NKG2D.AF compared to natural NKG2D.wt (Fig. 22). Overall, these data demonstrated that the non-natural α1-α2 orthogonal domains of the invention had high binding affinity to the non-natural NKG2D.AF receptor and had significantly lower binding affinity to the natural NKG2D receptor. Furthermore, fusions of these orthogonal α1-α2 domains with antibody polypeptides retained their selective binding capacity and were used, for example, to generate chimeric antigen receptor (CAR) T cells to redirect non-natural NKG2D.AF receptors to specific antigens.

[0070] Example 5: Identification of non-natural NKG2D ligands that allows discrimination between non-natural NKG2D receptor variants by selective binding of one or the other ligand

[0071] Phage display to generate orthogonal non-natural α1-α2 domains with selective binding to NKG2D.Y152A (hereafter referred to as the NKG2D.YA receptor) was performed using the non-natural α1-α2 domain of ULBP2.R80W (SEQ ID NO: 108) as a starting point, as described above. The phage display α1-α2 libraries were panned for their high affinity binding to the non-natural receptor NKG2D.AF by selectively capturing phage clones bound to biotinylated Fc-NKG2D.YA protein (SEQ ID NO: 41) in the presence of non-biotinylated natural competitor protein Fc-NKG2D.wt (SEQ ID NO: 40). Further phage clone validation studies identified variants that preferentially bound Fc-NKG2D.YA over Fc-NKG2D.wt (Fig. 23). ULBP2.S3 (SEQ ID NO: 127), for example, thereby demonstrated selective binding to non-natural NKG2D.YA over natural NKG2D.wt as determined by ELISA and Octet assays (both in the monomeric His-tag and hybrid bispecific antibody forms). This form represents a unique form of the non-natural α1-α2 orthogonal domains of the invention that has high binding affinity for non-natural NKG2D receptors (in this case NKG2D.YA as opposed to NKG2D.AF, as described in Example 4). Furthermore, hybrids of the orthogonal α1-α2 domains with antibody polypeptides retained their selective binding capacity and were used to selectively retarget non-natural NKG2D receptors to specific molecules defined by hybrid heterologous peptides, such as antibodies.

[0072] To determine whether the non-natural α1-α2 domain that selectively binds to NKG2D.YA (ULBP2.S3, SEQ ID NO: 127) and the non-natural α1-α2 domains that selectively bind to NKG2D.AF differ between the two non-natural receptor variants, ELISA titration was performed. All 21 of the selected α1-α2 variants that bound to NKG2D.AF were directly compared for binding to NKG2D.AF compared to NKG2D.YA. Of these, four variants demonstrated an inability to bind to NKG2D.wt, high affinity for NKG2D.AF, and a significant reduction (15-20-fold) or inability to bind to NKG2D.YA compared to NKG2D.AF (Figure 7). These four non-naturally occurring ULBP2 α1-α2 variants, ULBP2.C, ULBP2.R, ULBP2.AA and ULBP2.AB (SEQ ID NOs: 111, 113, 115 and 117), were also assessed for alteration of the predicted immunogenicity profile relative to the wild-type ULBP2 peptide sequence (SEQ ID NO: 4) using the NetMHC4.0 server (for querying MHC class I peptide binding against HLA all supertypes using a 9-mer peptide assay; http://www.cbs.dtu.dk/services/NetMHC/) and the NetMHCII 2.3 server (for querying MHC class I peptide binding against HLA-DR, HLA-DQ, HLA-DP haplotypes using a 15-mer peptide assay; http://www.cbs.dtu.dk/services/NetMHCII/), where both of these algorithms were developed by the Technical University of Denmark (http://www.bioinformatics.dtu.dk/; Andreatta M and Nielsen M, Gapped sequence alignment using artificial neural networks: application to the MHC class I system, 2016 Bioinformatics, 32:511, PMID: 26515819; Jensen KK, Andreatta M, Marcatili P, Buus S, Greenbaum JA, Yan Z, Sette A, Peters B, and Nielsen M, Improved methods for predicting peptide binding affinity to MHC class I molecules, 2018 Immunology , PMID: 29315598). Mutations introduced into ULBP2.C, ULBP2.R, and ULBP2.AB did not increase the predicted immunogenicity, while the immunogenicity of ULPB2.AA was slightly increased for several haplotypes (Figs. 8 and 9). Due to the specificity of ULBP2.R for NKG2D.AF and the lack of predicted immunogenicity, ULBP2.R was selected for further ELISA analysis to directly compare its binding pattern with ULBP2.S3 (an unnatural orthogonal ligand selected for NKG2D.YA), ULBP2.R80W (an unnatural ligand with enhanced affinity for wild-type NKG2D), and wild-type ULBP2 (ULBP2.wt). Binding of four rituximab MicAbody reagents (SEQ ID NOs: 98 and 121, 98 and 129, 131 and 100, 98 and 106 as the heavy chain and light chain for ULBP2.R, ULBP2.S3, ULBP2.R80W, and ULBP2.wt, respectively) was analyzed in comparison with wild-type NKG2D (NKG2D.wt) and two inert non-native variants NKG2D.YA and NKG2D.AF (Fig. 10). The data demonstrated that the selected NKG2D.YA variant ULBP2.S3 as a MicAbody bound with high affinity to NKG2D.YA but did not interact with NKG2D.AF or native NKG2D. Furthermore, the selected NKG2D.AF MicAbody variant ULBP2.R bound with high affinity to NKG2D.AF but did not interact with NKG2D.YA or native NKG2D. These results demonstrate tremendous potential for exploring the NKG2D-MIC ligand system and developing unique pairs of novel selective non-natural NKG2D receptors and their corresponding cognate non-natural MIC ligand binding partners.

[0073] Example 6: Targeting and cytolytic activity of CAR-T cells expressing the non-native NKG2D.AF ectodomain is regulated by orthogonal α1-α2 domains associated with heterologous targeting polypeptides

[0074] Currently, means to selectively regulate CAR-T cell therapy are urgently needed to reduce toxicity and improve tumor efficacy (Gill and June, op cit). Previous attempts have been made to engineer CARs using the CD16 ectodomain, which can then be interacted with via the Fc domain of therapeutic monoclonal antibodies, allowing for antibody-based CAR-T targeting (Chang et al., op cit ). However, CD16-based CAR-T cells can recognize nearly all endogenous antibody molecules in the blood and tissues, and therapeutic antibodies used to kill these cells will face competition from endogenous CD16 receptors on NK cells, PMNs, monocytes, and macrophages. Both of these features contribute to problems with extratumor toxicity and poor pharmacokinetics, respectively.

[0075] Natural NKG2D ligands are present in some healthy tissues and many stressed tissues, posing an extreme risk of toxicity with current NKG2D CAR approaches (VanSeggelen et al. 2015). The unnatural NKG2D receptor Y152A, which specifically binds to unnatural NKG2D ligands of the α1-α2 domain, is an example of a means by which the activity of a unnatural NKG2D CAR can be selectively regulated using bispecific proteins consisting of unnatural α1-α2 domain NKG2D ligands of the invention.

[0076] We have engineered CAR-T cells with a receptor consisting of a modified Y152A/Y199F ("AF") ectodomain of NKG2D that does not bind to any natural NKG2D ligand or to the previously described non-natural α1-α2 domains orthogonal and related to Y152A-modified NKG2D (NKG2D.YA). The related non-natural α1-α2 domains of the invention bound with high affinity to the non-natural ectodomain of NKG2D.AF and did not bind to the natural NKG2D ectodomains or to the NKG2D.YA ectodomain. Thus, engineered α1-α2 domains that are highly selective for the unnatural NKG2D.AF ectodomain over natural NKG2D and unnatural NKG2D.YA represent an ideal system for selectively regulating unnatural NKG2D CAR receptors or any hybrid receptor or protein associated with unnatural NKG2D ectodomains that can selectively interact with the unnatural α1-α2 domains of the invention. The present invention also provides the ability to generate single cells expressing two different CARs, one composed of NKG2D.YA and the other of NKG2D.AF, each signaling with entirely different intracellular domains. These different CARs will have independent dual control over cellular activity through the extracellular action of the corresponding cognate orthogonal MicAbody or other non-antibody hybrid polypeptide.

[0077] To demonstrate the selective regulation of CAR T cells engineered with a chimeric receptor containing a non-native NKG2D.AF ectodomain, we engineered CARs containing native NKG2D.wt (SEQ ID NO: 135), non-native NKG2D.YA (SEQ ID NO: 137), or non-native NKG2D.AF ectodomains (SEQ ID NO: 139), based on previous work using 4-1BB/CD3-zeta CAR constructs (Campana patent 8399645) linking the corresponding NKG2D ectodomains to the CD8 CAR hinge region (SEQ ID NOs: 151, 153, 155). These constructs (SEQ ID NOs: 152, 154, 156) were cloned into a lentiviral vector and expressed in primary human CD8-positive T cells by lentiviral transduction. HeLa cells have constitutively elevated levels of MIC ligands on their surface, including MICA, MICB, ULBP3, and ULBP2/5/6 (the antibody used to confirm this cannot distinguish between these three ULBPs; human ULBP-2/5/6 antibody, R&D Systems, Minneapolis, MN)). HeLa cells were transfected to overexpress either native ULBP1 or the NKG2D.AF-selected ULBP2.R variant on their surface, and these cells were used as a target for an in vitro cytolysis assay. 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 after detergent treatment (Fig. 11). Due to elevated levels of MIC ligands normally expressed on the surface of HeLa cells, CD8 cells expressing native NKG2D (NKG2D.wt) as a CAR interact with HeLa cells through this overexpressed natural ligand and induce cytolysis. However, both NKG2D.YA and NKG2D.AF CAR-transduced CD8 cells showed very little lysis of native HeLa cells, even at high E:T ratios, with activity on par with that of untransduced CD8 T cells. When ULBP1 is overexpressed on the HeLa cell surface, only NKG2D.wt CAR CD8 T cells significantly lyse these cells. Some additional cell death was observed at high E:T ratios with NKG2D.YA CAR cells but not with NKG2D.AF CAR cells, indicating that the Y152A/Y199F double mutation renders NKG2D even more inert than the Y152A single mutation. In HeLa cells overexpressing the NKG2D.AF-selective non-native ULBP2.R, NKG2D.wt-CAR cells mediate lysis (due to recognition of endogenous MIC ligands), whereas NKG2D.AF-CAR cells mediate significant levels of lysis coupled with binding of the receptor and its selective ligand.

[0078] To demonstrate that NKG2D.YA or NKG2D.AF CAR cell lysis can be mediated by the respective cognate targeting MicAbody alone, Ramos cells were used as a target for cytolysis in combination with rituximab-based MicAbodies linked to the non-natural orthogonal ligands ULBP2.S3 or ULBP2.R. As shown in Fig. 12A, the rituximab-ULBP2.S3 MicAbody can mediate cellular cytolysis by NKG2D.YA CAR CD8 cells but not by NKG2D.AF CAR cells, whereas the rituximab-ULBP2.R MicAbody can mediate NKG2D.AF CAR cell activity but not by NKG2D.YA CAR cells. This also demonstrated the selectivity of the two unnatural ULBP2 variants over their cognate unnatural NKG2D variants for which they were engineered as preferred partners. To demonstrate the specificity of the MicAbody portion of the antibody, in vitro cytolysis assays were performed using NKG2D.AF-CAR-CD8 T cells that were preloaded by incubation with rituximab-ULBP2.R, trastuzumab-ULPB2.R (SEQ ID NOs: 95 and 133, heavy and light chains, respectively), or an equimolar combination of the two antibodies at a total saturating concentration of MicAbody. After removal of unbound MicAbody by washing, CD8 cells were applied to either Ramos cells (expressing CD20, the target of rituximab) or CT26-Her2 cells (a murine cell line transfected to express human Her2) that had been preloaded with calcein. After a 2-hour incubation at two different E:T ratios, the amount of released calcein was determined. As shown in Fig. 12B, when the cells were preloaded with rituximab-MicAbody, only Ramos cells were lysed, whereas trastuzumab-MicAbody provided cytolytic activity only against CT26-Her2 cells. However, when NKG2D.AF-CAR-CD8 cells were simultaneously preloaded with both rituximab- and trastuzumab-ULBP2.R MicAbodies, both target cell lines were lysed, indicating that these CAR cells, due to the selective and selective binding partnership that was established between the receptor and ligand, were readily multiplexed and thus provide simultaneous exposure to different tumor targets.

[0079] Example 7: Orthogonal α1-α2 domains as a means of selectively delivering cytokines to engineered T cells expressing NKG2D

[0080] Bispecific MicAbodies (Fig. 13B) that contain antigen-targeting Fv domains of antibodies and a preferential interaction between the orthogonal α1-α2 domains and engineered non-natural NKG2D.YA or NKG2D.AF (SEQ ID NOs: 137 and 139, respectively) can effectively mediate the cytolytic activity of T cells bearing NKG2D.YA or NKG2D.AF-CAR (SEQ ID NOs: 153 and 155, respectively) and thereby remove antigen-expressing target cells. Furthermore, the highly selective interaction between cognate orthogonal ligands and non-natural, modified (also referred to as “engineered”) NKG2D (eNKG2D) can be exploited to selectively deliver molecules to cells bearing the eNKG2D ectodomain (eNKG2D-ecd). If the heterologous atom or molecule (payload/cargo) to which the orthogonal ligand is bound is inherently bioactive and has potentially undesirable functions when delivered to a cell that does not express eNKG2D, then mutations in the bioactive molecule that reduce interactions with native receptors or targets can be explored. Consequently, the final molecule, when bound to the orthogonal ligand, will be effectively inert to all biological functions except when cells bearing the cognate eNKG2D receptor are present. Only when a high affinity interaction occurs between the orthogonal ligand and the eNKG2D receptor, is the interaction between the affinity-lowered mutant of the bioactive molecule and its natural target or receptor enabled and the residual function of the bioactive molecule activated. All fusions of orthogonal ligands with molecules that target them to eNKG2D-expressing cells are collectively referred to as MicAdaptors and, in principle, can take the form of direct protein fusions in the form of a single polypeptide between the payload and the orthogonal ligand at either the N- or C-terminus, with or without a linker or tag (e.g., a His-tag), where said linker or tag can be used for assays or purification (Fig. 13C). Additionally, if increased serum stability is desired, the Fc domain of the antibody can be included either as CH2-CH3 domains only or in a full-length antibody format, depending on the desired number of unique payloads, the valence of the orthogonal ligand or heterologous cargo, and the overall experimentally determined architecture that promotes maximum functionality of all components (Fig. 13D).

[0081] To determine whether cytokines can be selectively delivered to cells expressing NKG2D.YA-CAR (SEQ ID NO: 153), the cognate orthogonal ligand ULBP2.S3 (U2S3, SEQ ID NO: 127) was expressed as a fusion with the N-terminus of Fc1, which was substituted by two residues to express negatively charged aspartic acid residues at the Fc homodimerization interface (SEQ ID NO: 189). A mutant form of human IL2 (mutIL2) contains two R38A/F42K mutations that significantly reduce the affinity of the cytokine for the IL2R-alpha complex (K. M. Heaton, G. Ju, and E. A. Grimm, Human Interleukin 2 Analogues That Preferentially Bind the Intermediate-Affinity Interleukin 2 Receptor Lead to Reduced Secondary Cytokine Secretion: Implications for the Use of These Interleukin 2 Analogues in Cancer Immunotherapy, 1993 Cancer Res 53:2597, PMID: 8495422; K. Sauvé et al., Localization in Human Interleukin 2 of the Binding Site to the Alpha Chain (P55) of the Interleukin 2 Receptor, 1991 PNAS 88:4636, PMID: 2052547) was fused to the C terminus of Fc2, which was replaced by two residues to express positively charged lysine residues at the Fc homodimerization interface (SEQ ID NO: 183). Both of these mutants were independently cloned into the mammalian expression vector pD2610-V12 (ATUM, Newark, CA), cotransfected into Expi293 ^™ cells (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's protocol, and purified by standard Protein A affinity chromatography (catalog #20334, Pierce Biotechnology, Rockford, IL). Purified material was fractionated by size exclusion chromatography (SEC) on Akta Pur Superdex columns. Negatively charged residues on Fc1 and positive charges on Fc2 mediated an electrostatic interaction effect (Kannan Gunasekaran et al., Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects: Applications to Bispecific Molecules and Monovalent IgG, 2010 J Biol Chem 285:19637, PMID: 20400508) that stimulated heterodimeric assembly of a molecule that was monovalent to the orthogonal ligand U2S3 and mutIL2 (Fig. 13Da). In addition, MicAdaptors consisting of other combinations of orthogonal ULBP2 ligands with cytokine fusions were studied, which were generated as heterodimeric Fc1/Fc2 fusions or as a single polypeptide, where both components were directly linked to each other, and the DNA encoding them was similarly cloned, coexpressed and purified as detailed in Fig. 24 and as described above. Recombinant human IL2 (rhIL2, Peprotech) and recombinant human IL15 (rhIL15, Peprotech) were included in the assays as controls when appropriate.

[0082] Human CD8 T cells were transduced to express the NKG2D.wt-CAR construct (SEQ ID NO: 151) or the NKG2D.YA-CAR construct (SEQ ID NO: 153) and exposed to 30 IU/mL of control cytokine or various MicAdaptors for three days, and the level of cell proliferation was quantified using the WST-1 Cell Proliferation Reagent (Millipore Sigma). Control rhIL2 stimulated proliferation of CAR-expressing cells, while mutIL2 alone did not, as would be expected due to a reduced ability to interact with IL2R-alpha and hence a reduced ability to signal through IL2R-beta/gamma-C (Figure 15A). When the MicAdaptor consisted of a non-selective ligand U2R80W (U2R80W-mutIL2, SEQ ID NO: 177), which interacts with high affinity with both the wild-type and NKG2D.YA modified receptors, cells expressing either CAR responded by proliferating. However, when mutIL2 was attached to an orthogonal ligand such as ULBP2.S2 (SEQ ID NO: 179), which selectively interacts only with NKG2D.YA, only NKG2D YA CAR cells proliferated when the MicAdaptor, U2S2-mutIL2, interacted only with NKG2D.YA. Similar results were obtained when the cytokine associated with U2S2 was a mutant variant of IL15 with the V49D mutation (Bernard, J. et al., Identification of an Interleukin-15α Receptor-binding Site on Human Interleukin-15, 2004 Journal of Biological Chemistry, 279:24313, PMID: 15039446), which reduces interaction with IL15R-alpha (Fig. 15A). When NKG2D.YA-CAR T cells were co-cultured for seven days with these reagents, the % GFP ⁺ cells (indicating the percentage of CAR-expressing cells present compared to untransduced GFP-negative cells) increased, but only when the orthogonal ULBP2.S2 ligand (U2S2-mutIL2 or U2S2-Fc1/Fc2-mutIL2, Fig. 24 for SEQ ID NO) was present, and not in the case of the non-selective U2R80W variant (which bound to the natural wild-type NKG2D receptor constitutively present on human CD8 cells) (Fig. 15B). IL21 was also tested as a fusion of wild-type (IL21.wt) with individual mutations affecting IL21R-alpha binding (D18A or E109R), or as a variant with both mutations included (D18A/E109R) (Kang, L. et al., Rational Design of Interleukin-21 Antagonist through Selective Elimination of the γC Binding Epitope, 2010 Journal of Biological Chemistry, 285:12223, PMID: 20167599). When comparing untransduced CD8 cells or cells expressing the NKG2D.YA-CAR, only cells bearing NKG2D.YA responded to all IL21 fusion variants (see Fig. 24 for SEQ ID NO), including the IL21.wt fusion (Fig. 15C). Interestingly, although untransduced cells and NKG2D.YA-CAR cells proliferated in response to rhIL2 compared to the cytokine-free control, the extent of proliferation was higher for NKG2D.YA-CAR cells. The proliferative response of NKG2D.YA-CAR-expressing cells was not dependent on overall NKG2D.YA domain binding or interaction, as U2S3-Fc1/Fc2 (a heterodimeric Fc molecule with a single U2S3 domain and no attached cytokine or cytokine mutants) and ritixumab-MicAbody (bivalent with the U2S3 domain but lacking any cytokine component, Fig. 13Bb) did not induce proliferation over three days of culture even at IU/mL concentrations that were much higher than the rhIL2 control concentration (Fig. 16C).

[0083] Example 8: The presence of intracellular costimulatory domains in cis or trans orientation stimulates the response of non-native modified NKG2D-bearing cells to cytokines and cytokine MicAdaptors

[0084] Having demonstrated that the modified NKG2D.YA domain, when contained in a chimeric antigen receptor construct, did indeed serve as a highly selective docking site for the delivery of heterologous cargo linked to an orthogonal ligand, we sought to determine whether the NKG2D.YA receptor was not only necessary but also sufficient for the targeted delivery of cytokines that could then act on the recipient cell. The extracellular domain of NKG2D.YA (NKG2D.YA-ecd) was expressed as a transmembrane domain lacking all intracellular components except for the retained intracellular eGFP tag (FIG. 14, SEQ ID NO: 157). CD8 cells were transduced to express this “silenced CAR” and were shown to be unable to directly kill Ramos target cells in the presence of rituximab-ULBP2.S3 MicAbody (SEQ ID NOS: 98 and 129), as would be expected in the absence of costimulatory domains (Fig. 16A). Importantly, cells expressing this completely inert or silenced CAR did not proliferate when exposed to U2S3-Fc1/Fc2-mutIL2 (SEQ ID NOS: 189 and 193), but responded at a level comparable to that of untransduced cells (Fig. 16B). This observation, in addition to (a) the consistent observation of higher levels of proliferation of NKG2D.YA-CAR containing rhIL2 compared to untransduced cells (Figs. 15C, 16B, 16C) and (b) the observation that only NKG2D.YA-CAR cells responded to all forms of IL21-MicAdaptor, including IL21.wt, led the authors to speculate that the intracellular domains present in the NKG2D.YA-CAR construct enhance the response to these cytokines and cytokine MicAdaptors.

[0085] To test this, a series of CAR constructs were generated in which the signaling motifs of the CAR intracellular domains were mutated, either the two TRAF2 consensus binding sites of 4-1BB (SEQ ID NO: 161), the three pairs of ITAM motifs in CD3zeta (SEQ ID NO: 163), or the combined 4-1BB/CD3zeta mutants (SEQ ID NO: 165). These constructs (Figure 14) were transfected into CD8 cells, coincubated with the indicated cytokine reagents, and proliferation was quantified after three days (Figure 17A). NKG2D.YA-BB-CD3 _{∆ ITAM} -GFP (SEQ ID NO: 163) maintained a proliferative response similar to NKG2D.YA-CAR under all conditions tested, demonstrating that the CD3 zeta domain (SEQ ID NO: 145) is dispensable for the response to cytokines and cytokine-MicAdaptors in the presence of CAR. However, cells expressing the NKG2D.YA-BB _ΔTRAF2 -CD3zeta-GFP receptor (SEQ ID NO: 161) had significantly reduced sensitivity to all cytokines and cytokine-MicAdaptors, as did the NKG2D.YA-BB _ΔTRAF2 -CD3 _{∆ ITAM} -GFP receptor (SEQ ID NO: 165) carrying mutations in both intracellular domains. This indicates that the costimulatory 4-1BB (SEQ ID NO: 143) plays a role in the responsiveness of NKG2D.YA CAR cells to both cytokines and cytokine-MicAdaptors, and that the silent CAR expressed on the cell surface must also have an intracellular costimulatory domain capable of stimulating cytokine-MicAdaptor responsiveness. The inability of the silent NKG2D.YA-BB CAR (SEQ ID NO: 169, FIG. 14) to mediate MicAbody-mediated cell killing (FIG. 17B) demonstrated that this CAR architecture lacking CD3 zeta is suitable as a silent docking site for MicAdaptors and allows for cytokine-MicAdaptor signaling, but does not function as an agent for mediating MicAbody-targeted cell killing.

[0086] To further investigate how the 4-1BB domain is involved in cytokine and cytokine-MicAdaptor responses, a construct was generated in which the CD19scFv-CAR (based on the FMC63 Fv) contained the full complement of functional 4-1BB and CD3-zeta domains (SEQ ID NO: 173). The CD19scFv-CAR was co-expressed with NKG2D.YA-ecd (Figure 14). Both components were expressed as a single polypeptide with a self-cleaving T2A peptide motif separating the upstream CD19scFv-CAR construct from the downstream NKG2D.YA-ecd, which has independent GMCSFR alpha chain signal sequence, CD8a hinge region, and CD8a transmembrane domain. This construct was transfected into CD8 T cells and co-expression of CD19scFv-CAR and NKG2D.YA-ecd on the surface was confirmed by flow cytometry by assessing the GFP signal and staining for phycoerythrin-conjugated MicAbody, respectively. The cells were incubated for three days with cytokines and cytokine-adapter MicAdaptors and proliferation was quantified by WST assay. As expected, NKG2D.YA-CAR responded to both rhIL2 and U2S3-Fc1/Fc2-mutIL2, although control cells bearing only CD19scFv-CAR (SEQ ID NO: 171) were expanded by rhIL2 and to a lesser extent up to a maximum concentration (300 IU/mL) of U2S3-Fc1/Fc2-mutIL2 (Fig. 17C). Co-expression of NKG2D.YA-ecd on cells expressing CD19scFv-CAR (SEQ ID NO: 173) induced a greater proliferative response to the cytokine-MicAdaptor U2S3-Fc1/Fc2-mutIL2 than cells expressing CD19scFv-CAR (SEQ ID NO: 171) alone. Consistent with this, the 4-1BB domain was constitutively positioned in trans relative to NKG2D.YA-ecd. These data demonstrated that the response of NKG2D.YA-ecd expressing cells to cytokines and cytokine-MicAdaptors was stimulated by the 4-1BB domain in either cis or trans orientation, and that the NKG2D.YA-ecd domain can be co-expressed in a CAR cell containing the 4-1BB costimulatory domain to confer additional versatile functionality to engineered cells used in adoptive cell therapy strategies. This functionality is not limited to surface receptor interactions in ligand/silenced CAR interactions, but can be extended to intracellular delivery by incorporating cytoplasmic sequence motifs (K.N. Pandey, Functional roles of short sequence motifs in the endocytosis of membrane receptors, 2009 Front Biosci, 14:5339, PMID: 19482617) that stimulate the hydrolysis of non-native NKG2D variants, and therefore any bound MicAdaptor will be co-internalized and the heterologous cargo will be delivered to the cell interior.

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LIST OF SEQUENCES

<110> XYPHOS BIOSCIENCES INC.

<120> UNNATURAL NKG2D RECEPTORS THAT DIRECTLY

DO NOT TRANSMIT A SIGNAL TO THE CELLS WITH WHICH THEY ARE CONNECTED

<130> A251193

<150> US 62/755,776

<151> 2018-11-05

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<170> PatentIn version 3.5

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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> 5

<211> 187

<212> PRT

<213> Artificial sequence

<220>

<223> alpha1-alpha2 domain of the synthetic peptide ULBP3

<400> 5

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 Cys 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> 6

<211> 187

<212> PRT

<213> Artificial sequence

<220>

<223> alpha1-alpha2 domain of the synthetic peptide ULBP4

<400> 6

Ala Ala Glu Pro His Ser Leu Cys Phe Asn Phe Thr Ile Lys Ser Leu

1 5 10 15

Ser Arg Pro Gly Gln Pro Trp Cys Glu Ala Gln Val Phe Leu Asn Lys

20 25 30

Asn Leu Phe Leu Gln Tyr Asn Ser Asp Asn Asn Met Val Lys Pro Leu

35 40 45

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

50 55 60

Thr Gln Thr Leu Gly Glu Val Gly Arg Asp Leu Arg Met Leu Leu Cys

65 70 75 80

Asp Ile Lys Pro Gln Ile Lys Thr Ser Asp Pro Ser Thr Leu Gln Val

85 90 95

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

100 105 110

Gln Phe Ala Thr Asn Gly Glu Lys Ser Leu Leu Phe Asp Ala Met Asn

115 120 125

Met Thr Trp Thr Val Ile Asn His Glu Ala Ser Lys Ile Lys Glu Thr

130 135 140

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

145 150 155 160

Gly Asp Cys Asp His Trp Leu Arg Glu Phe Leu Gly His Trp Glu Ala

165 170 175

Met Pro Glu Pro Thr Val Ser Pro Pro Met Val

180 185

<210> 7

<211> 198

<212> PRT

<213> Artificial sequence

<220>

<223> Alpha1-alpha2 domain of the synthetic peptide ULBP5

<400> 7

Gly Leu Ala Asp Pro His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro

1 5 10 15

Lys Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp

20 25 30

Glu Lys Thr Phe Leu His Tyr Asp Cys Gly Ser Lys Thr Val Thr Pro

35 40 45

Val Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala

50 55 60

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

65 70 75 80

Leu Asp Ile Gln Leu Glu Asn Tyr Ile Pro Lys Glu Pro Leu Thr Leu

85 90 95

Gln Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Gly Ser Gly

100 105 110

Ser Trp Gln Leu Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser

115 120 125

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

130 135 140

Glu Lys Trp Glu Asn Asp Lys Asp Met Thr Met Ser Phe His Tyr Ile

145 150 155 160

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

165 170 175

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

180 185 190

Gly Thr Ala Gln Pro Arg

195

<210> 8

<211> 189

<212> PRT

<213> Artificial sequence

<220>

<223> alpha1-alpha2 domain of the synthetic peptide ULBP6

<400> 8

Ala Ala Glu Pro His Ser Leu Cys 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 Leu

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 Ile Asp Gly Gln Thr 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 Ile 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> 9

<211> 161

<212> PRT

<213> Artificial sequence

<220>

<223> alpha1-alpha2 domain of the synthetic peptide OMCP

<400> 9

Ala Ala Ala Glu Pro His Lys Leu Ala Phe Asn Phe Asn Leu Glu Ile

1 5 10 15

Asn Gly Ser Asp Thr His Ser Thr Val Asp Val Tyr Leu Asp Asp Ser

20 25 30

Gln Ile Ile Thr Phe Asp Gly Lys Asp Ile Arg Pro Thr Ile Pro Phe

35 40 45

Met Ile Gly Asp Glu Ile Phe Leu Pro Phe Tyr Lys Asn Val Phe Ser

50 55 60

Glu Phe Phe Ser Leu Phe Arg Arg Val Pro Thr Ser Thr Pro Tyr Glu

65 70 75 80

Asp Leu Thr Tyr Phe Tyr Glu Cys Asp Tyr Thr Asp Asn Lys Ser Thr

85 90 95

Phe Asp Gln Phe Tyr Leu Tyr Asn Gly Glu Glu Tyr Thr Val Lys Thr

100 105 110

Gln Glu Ala Thr Asn Lys Asn Met Trp Leu Thr Thr Ser Glu Phe Arg

115 120 125

Leu Lys Lys Trp Phe Asp Gly Glu Asp Cys Ile Met His Leu Arg Ser

130 135 140

Leu Val Arg Lys Met Glu Asp Ser Lys Arg Arg Thr Val Pro Pro Met

145 150 155 160

Val

<210> 10

<211> 543

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the alpha1-alpha2 domain of MICA

<400> 10

gagccccaca gtcttcgtta taacctcacg gtgctgtcct gggatggatc tgtgcagtca 60

gggtttctca ctgaggtaca tctggatggt cagcccttcc tgcgctgtga caggcagaaa 120

tgcagggcaa agccccaggg acagtgggca gaagatgtcc tgggaaataa gacatgggac 180

agagagacca gagacttgac agggaacgga aaggacctca ggatgaccct ggctcatatc 240

aaggaccaga aagaaggctt gcattccctc caggagatta gggtctgtga gatccatgaa 300

gacaacagca ccaggagctc ccagcatttc tactacgatg gggagctctt cctctcccaa 360

aacctggaga ctaaggaatg gacaatgccc cagtcctcca gagctcagac cttggccatg 420

aacgtcagga atttcttgaa ggaagatgcc atgaagacca agacacacta tcacgctatg 480

catgcagact gcctgcagga actacggcga tatctaaaat ccggcgtagt cctgaggaga 540

aca 543

<210> 11

<211> 561

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the alpha1-alpha2 domain of ULBP1

<400> 11

gctgctgagc cccactgtct ctgctacgac tttattataa ctcctaagtc aagaccagag 60

cctcagtggt gcgaagtaca aggtttggtt gacgaaaggc ctttccttca ctacgattgt 120

gtgaaccata aggcaaaggc tttcgccagc ctgggtaaga aggtaaacgt tactaagacg 180

tgggaggagc agacggaaac cctccgtgat gtggttgact ttcttaaggg tcagctcctc 240

gatatccaag tggagaattt aatccctatc gaaccgctca ctctgcaggc cagaatgtca 300

tgcgaacatg aagcacacgg tcatggaaga ggtagttggc aatttttatt taacggtcaa 360

aaattcctgc tgttcgactc aaacaaccgc aaatggactg cgctgcaccc tggagctaag 420

aagatgactg aaaaatggga gaagaacaga gacgttacca tgttcttcca gaagatttcc 480

ctgggagatt gtaagatgtg gttagaggag ttcttaatgt actgggaaca gatgctggac 540

cccacaaaac cccccatggt g 561

<210> 12

<211> 567

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the alpha1-alpha2 domain of ULBP2

<400> 12

gctgctgagc cccatagtct gtgttacgac atcacagtta ttcccaagtt caggcccgga 60

ccgcgctggt gtgccgtgca aggacaagtc gacgaaaaaa cctttcttca ttacgattgc 120

ggaaataaga ctgtaacgcc agtctctcct ttaggtaaga agttaaacgt cactacggcg 180

tggaaggcac aaaaccccgt cctgcgcgag gtcgtcgaca tcctgactga acaattgcgc 240

gacatccagc tcgagaatta cactccaaag gagcctctta ccctgcaggc tagaatgtct 300

tgcgagcaaa aggcagaggg ccactcctcc ggcagctggc agttcagttt cgacggacaa 360

atctttctgt tattcgattc agagaagaga atgtggacta cagttcaccc cggtgcccgt 420

aaaatgaagg agaagtggga aaacgacaaa gtggtggcga tgtcattcca ctatttctcg 480

atgggagact gcatcggttg gctggaagat ttcctcatgg gtatggactc cactttggag 540

ccacggctg gtgccccccc catggtg 567

<210> 13

<211> 561

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the alpha1-alpha2 domain of ULBP3

<400> 13

gctgctgagc cccacagctt gtggtacaac ttcaccatta tccacttgcc gagacatggc 60

cagcagtggt gcgaagtgca atcgcaagtc gaccaaaaaa acttcttatc atacgactgc 120

ggcagcgata aggtcttatc tatgggtcat ttggaggaac agctctacgc gaccgacgcc 180

tggggtaaac agctcgagat gctccgtgag gttggacaga ggctgagact ggaactggct 240

gacactgagc tggaagattt cacacctagt ggtccactca cattgcaagt acgcatgagc 300

tgcgagtgtg aggccgatgg atacattagg ggcagctggc agtttagctt cgacggaagg 360

aaattcctgc tcttcgacag taacaatagg aagtggactg ttgtgcatgc tggtgcgcgc 420

agaatgaagg aaaagtggga gaaagatagc ggcctgacga ccttcttcaa gatggtgtct 480

atgcgtgact gtaagagctg gctcagagat ttcctcatgc atcgcaagaa gaggttagaa 540

cctaccgctc cccccatggt g 561

<210> 14

<211> 561

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the alpha1-alpha2 domain of ULBP4

<400> 14

gctgctgagc cccactctct ttgcttcaac ttcaccatta aatccctgag caggcctggt 60

cagccgtggt gtgaggcgca ggtctttctt aacaagaatc tcttcctcca atacaactct 120

gataacaaca tggtaaagcc actgggtctc ctgggtaaaa aagtctatgc tacgagcact 180

tggggagaac tcacccagac tcttggcgag gtaggaagag acctgcgcat gctcctctgc 240

gatataaagc cccaaattaa gaccagtgat ccgtccactt tacaagtcga aatgttctgc 300

caaagggagg ctgaacgctg caccggagcc tcttggcagt tcgcgaccaa tggcgaaaag 360

tccctcttgt tcgatgccat gaatatgacc tggaccgtga tcaatcatga ggcctctaag 420

atcaaggaga cgtggaaaaa ggaccgcggc cttgaaaagt actttaggaa gttgtctaaa 480

ggagactgcg accattggtt acgcgagttc ctcggccatt gggaagcgat gcccgagcca 540

acggttagcc cccccatggt g 561

<210> 15

<211> 567

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the alpha1-alpha2 domain of ULBP6

<400> 15

gctgctgagc cccactcctt atgctatgat atcaccgtga ttccaaagtt ccgaccagga 60

ccccgatggt gcgccgtaca gggacaggtc gacgaaaaga cttttttaca ttacgactgc 120

ggtaacaaga cagtcacacc ggtaagtcct ttgggaaaaa agttaaacgt aaccactgct 180

tggaaggccc agaaccccgt ccttcgagaa gtagtggata ttttgactga acagctgctt 240

gacatccagc tggaaaacta cacacccaaa gagcccctga ctcttcaagc gcgtatgtcg 300

tgtgagcaaa aggccgaagg acacagctcc ggatcctggc agttcagtat cgacggtcag 360

accttcctcc tcttcgattc agaaaagcgc atgtggacta ctgtgcaccc cggcgctcgt 420

aagatgaagg aaaagtggga gaatgataag gacgttgcca tgagttttca ttacattagt 480

atgggagatt gcatcggttg gctggaagac ttcctgatgg gtatggatag tacccttgaa 540

cctagtgccg gagctccccc catggtg 567

<210> 16

<211> 483

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the alpha1-alpha2 domain of OMCP

<400> 16

gctgctgctg agccccacaa gcttgcgttc aacttcaatc tggaaataaa cggttcagat 60

acccattcaa ccgtggacgt ttatttagac gattcgcaga taatcacctt tgacggcaag 120

gacatccgcc caactatccc gttcatgata ggtgacgaaa tcttccttcc tttttataag 180

aatgtgttct ctgagttctt cagtttgttc cgccgcgtcc ctacctcaac cccctacgaa 240

gacttgactt atttctatga atgcgactac accgacaaca aatctacatt cgatcaattc 300

tacctgtaca acggtgaaga gtacaccgtg aagactcaag aggctactaa caagaacatg 360

tggctgacca cttccgagtt cagactgaag aagtggttcg acggcgagga ctgtatcatg 420

caccttagaa gtttagtgag gaaaatggaa gatagcaaga gaagaacagt gccccccatg 480

gtg 483

<210> 17

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the natural ectodomain of NKG2D

<400> 17

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 Tyr Ile Glu Asn Cys Ser Thr

115 120 125

Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 18

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the unnatural ectodomain of NKG2D Y152A

<400> 18

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> 19

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the unnatural ectodomain of NKG2D Y199A

<400> 19

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> 20

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the unnatural ectodomain of NKG2D Y152A/Y199A

<400> 20

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> 21

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of unnatural ectodomain NKG2D Y199F eNKG2D1

<400> 21

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> 22

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the unnatural ectodomain of NKG2D Y152S eNKG2D2

<400> 22

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> 23

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of unnatural ectodomain NKG2D Y152T eNKG2D3

<400> 23

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> 24

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the unnatural ectodomain of NKG2D Y152V eNKG2D4

<400> 24

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> 25

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the unnatural ectodomain of NKG2D Y152A/Y199F eNKG2D5

<400> 25

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> 26

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the unnatural ectodomain of NKG2D Y152L/Y199F eNKG2D6

<400> 26

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> 27

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the unnatural ectodomain of NKG2D Y152S/Y199F eNKG2D7

<400> 27

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> 28

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of unnatural ectodomain NKG2D Y152T/Y199F eNKG2D8

<400> 28

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> 29

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of unnatural ectodomain NKG2D Y152V/Y199F eNKG2D9

<400> 29

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> 30

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of unnatural ectodomain NKG2D Y199D eNKG2D10

<400> 30

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> 31

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of unnatural ectodomain NKG2D Y199E eNKG2D11

<400> 31

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> 32

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the unnatural ectodomain of NKG2D Y152D/Y199D eNKG2D12

<400> 32

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> 33

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of unnatural ectodomain NKG2D Y152E/Y199E eNKG2D13

<400> 33

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> 34

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of unnatural ectodomain NKG2D Y152L eNKG2D14

<400> 34

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> 35

<211> 139

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide of the unnatural ectodomain of NKG2D Y152F/Y199F eNKG2D15

<400> 35

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> 36

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence of the synthetic peptide MHCI

<400> 36

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> 37

<211> 69

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a signal

MHCI sequence

<400> 37

atgggccttg gcccagtgtt tctgctgttg gcaggcattt tcccttttgc tccgcccggc 60

gccgcagcc 69

<210> 38

<211> 237

<212> PRT

<213> Artificial sequence

<220>

<223> Fc human IgG1 synthetic peptide with IEGR linker

<400> 38

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> 39

<211> 711

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding human IgG1 Fc with

IEGR linker

<400> 39

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> 40

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion

<400> 40

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> 41

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Y152A Fc fusion

<400> 41

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> 42

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-ectodomain Fc fusion

NKG2D Y199A

<400> 42

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> 43

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-ectodomain Fc fusion

NKG2D Y152A/Y199A

<400> 43

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> 44

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y199F

eNKG2D1

<400> 44

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> 45

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y152S

eNKG2D2

<400> 45

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> 46

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide Fc fusion-NKG2D Y152T

eNKG2D3

<400> 46

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> 47

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y152V

eNKG2D4

<400> 47

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> 48

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y152A/Y199F

eNKG2D5

<400> 48

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> 49

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y152L/Y199F

eNKG2D6

<400> 49

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> 50

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y152S/Y199F

eNKG2D7

<400> 50

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> 51

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y152T/Y199F

eNKG2D8

<400> 51

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> 52

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide Fc fusion-NKG2D Y152V/Y199F

eNKG2D9

<400> 52

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> 53

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y199D

eNKG2D10

<400> 53

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> 54

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y199E

eNKG2D11

<400> 54

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> 55

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y152D/Y199D

eNKG2D12

<400> 55

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> 56

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y152E/Y199E

eNKG2D13

<400> 56

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> 57

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Y152L Fc fusion

eNKG2D14

<400> 57

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> 58

<211> 376

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 synthetic peptide-NKG2D Fc fusion Y152F/Y199F

eNKG2D15

<400> 58

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> 59

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1NKG2D

<400> 59

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> 60

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152A

<400> 60

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> 61

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc

IgG1-ectodomain NKG2D Y199A

<400> 61

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> 62

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc

IgG1-ectodomain-NKG2D Y152A/Y199A

<400> 62

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> 63

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1 -NKG2D Y199F eNKG2D1

<400> 63

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> 64

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152S eNKG2D2

<400> 64

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> 65

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152T eNKG2D3

<400> 65

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> 66

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152V eNKG2D4

<400> 66

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> 67

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152A/Y199F eNKG2D5

<400> 67

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> 68

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152L/Y199F eNKG2D6

<400> 68

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> 69

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152S/Y199F eNKG2D7

<400> 69

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> 70

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152T/Y199F eNKG2D8

<400> 70

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> 71

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152V/Y199F eNKG2D9

<400> 71

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> 72

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y199D eNKG2D10

<400> 72

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> 73

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y199E eNKG2D11

<400> 73

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> 74

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152D/Y199D eNKG2D12

<400> 74

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> 75

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152E/Y199E eNKG2D13

<400> 75

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> 76

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152L eNKG2D14

<400> 76

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> 77

<211> 1128

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding human Fc hybrid

IgG1-NKG2D Y152F/Y199F eNKG2D15

<400> 77

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> 78

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide MICwed alpha1-alpha2

<400> 78

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 Th

180

<210> 79

<211> 543

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding MICwed alpha1-alpha2

<400> 79

gagcctcaca gcctccggta taatttgact gtactctctt gggatggctc cgtgcagtcc 60

ggctttctga ctgaagttca tctcgacggt caacctttcc tgcgctgcga ccgacaaaaa 120

tgccgcgcca agccccaagg gcagtgggcc gaagatgtac tgggaaacaa gacctggggac 180

cggggagacac gagacctgac aggctggggc aaggacttgc gcatgacact cgcccatatc 240

aaggaccaga aggaaggatt gcactctttg caagagattc gcgtgtgtga aatccacgag 300

gacaattcaa cgaggagctc ccagcacttc tattacgatg gagaactctt cttgtcacag 360

aacttggaaa ccaaggaatg gactatgcct cagagctctc gggcacagac tctcgctatg 420

aacgttagaa acttccttaa ggaggatgct atggagaccg atactcacta ccacgccatg 480

cacgccgact gcctccagga actgcggaga tatctgaagt ccggcgtggt tttgagaaga 540

acc 543

<210> 80

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide MIC25 alpha1-alpha2

<400> 80

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 Th

180

<210> 81

<211> 543

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding MIC25 alpha1-alpha2

<400> 81

gagcctcaca gcctccggta taatttgact gtactctctt gggatggctc cgtgcagtcc 60

ggctttctga ctgaagttca tctcgacggt caacctttcc tgcgctgcga ccgacaaaaa 120

tgccgcgcca agccccaagg gcagtgggcc gaagatgtac tgggaaacaa gacctggggac 180

cggggagacac gagacctgac aggctggggc aaggacttgc gcatgacact cgcccatatc 240

aaggaccaga aggaaggatt gcactctttg caagagattc gcgtgtgtga aatccacgag 300

gacaattcaa cgaggagctc ccagcacttc tattacgatg gagaactctt cttgtcacag 360

aacttggaaa ccctcgaatg gactatgcct cagagctctc gggcacagac tctcgctatg 420

aacgttagaa acttccttaa ggaggatgct atggagaccg atactcacta ccacgccatg 480

cgcgccgact gcctctctga actgcggaga tatctgaagt ccggcgtggt tttgagaaga 540

acc 543

<210> 82

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain of human IgG1 synthetic peptide

CH1-CH2-CH3 D265A/N297A

<400> 82

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> 83

<211> 990

<212> DNA

<213> Artificial sequence

<220>

<223> Human IgG1 heavy chain CH1-CH2-CH3 D265A/N297A

<400> 83

gcgtcgacca agggcccgtc agtgttcccg ctggccccgt catccaagtc cacgtctggg 60

ggcacagcag ccctgggatg cttggtcaag gactacttcc ccgagcccgt gactgtgtcc 120

tggaactccg gagcactgac ctccggagtg cacacctttc ccgcggtgct gcagtcctcc 180

ggactgtact ccctgtcgtc ggtcgtgacc gtgccgagct cctcgctcgg aacccagacc 240

tacatctgca acgtgaacca caagccctcg aacaccaaag tggacaagaa ggtcgagccc 300

aaaagctgcg acaagactca cacttgtccg ccgtgccccg cccccgaact gctgggtggc 360

ccctccgtgt tcctgttccc gcctaagcct aaggacaccc ttatgatcag ccgcacccct 420

gaagtgacct gtgtcgtcgt ggcagtgtca cacgaggacc cggaggtcaa gttcaattgg 480

tacgtggacg gcgtggaagt gcataacgca aagaccaagc ctcgggagga acagtacgcc 540

tcgacctacc gcgtggtgtc agtcctgact gtgctgcacc aggactggct gaacgggaag 600

gagtacaagt gcaaagtgtc gaacaaggcc ctgccggctc caattgaaaa gaccatcagc 660

aaggccaagg gccagccaag ggaaccacag gtgtacaccc tccctccttc ccgggacgag 720

ctgaccaaaa accaagtgtc cctgacttgc cttgtgaagg ggttctaccc ttctgacatt 780

gccgtcgaat gggaatcgaa cggacagcct gaaaacaact ataagactac cccgcccgtg 840

ctggattccg acggaagctt cttcctgtac tccaagctga ccgtggacaa gtcgagatgg 900

cagcagggaa atgtgttcag ctgctccgtg atgcatgagg cgctgcacaa ccactacacc 960

cagaagtcac tgagcctctc ccccggaaag 990

<210> 84

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Human kappa light chain synthetic peptide

<400> 84

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> 85

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding the human kappa light chain

<400> 85

agaaccgtgg ccgccccgag cgtgttcatt ttccctccct ccgacgagca gttgaaatcg 60

ggcaccgcta gcgtggtctg ccttctcaac aatttctatc cacgggaagc caaagtgcag 120

tggaaggtcg acaacgcgct ccaatccggg aactcacagg aatccgtgac tgagcaggat 180

tccaaggact cgacctactc cctgtcatcc acgctgaccc tgagcaaggc agactacgag 240

aagcacaagg tctacgcctg cgaagtgaca caccagggac tgtccagccc cgtgaccaag 300

agcttcaaca gaggagaatg c 321

<210> 86

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> VH of trastuzumab synthetic peptide

<400> 86

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

115 120

<210> 87

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding VH of transtuzumab

<400> 87

gaagtccaat tggtcgaatc aggcggtgga ctcgtgcaac ctggaggttc gttacgctta 60

tcatgtgctg caagtggatt taatattaaa gatacctaca tccactgggt acgtcaagct 120

cccggcaagg gtctcgagtg ggtcgcacgc atttacccca ccaacggata cacgcgctac 180

gccgattcag tgaagggacg tttcacaatc tctgctgata ctagcaaaaa taccgcatac 240

ctccagatga actctcttag ggccgaggac acagctgtgt actactgtag ccgttgggga 300

ggagacggtt tttacgcaat ggattactgg ggccaaggaa ccctggtcac agtttcatcg 360

<210> 88

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of trastuzumab synthetic peptide

<400> 88

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

100 105

<210> 89

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding VL of trastuzumab

<400> 89

gatatccaaa tgactcaatc accatcttca ctctccgcga gcgtgggtga tcgggtcacc 60

atcacatgta gggcgagcca agatgtgaat accgccgtcg cgtggtatca acaaaagccg 120

ggaaaagcac caaaactgct tatatactct gcatccttcc tgtactctgg ggtgccaagc 180

cggttctccg gtagtagatc tggtactgac tttacactca ctatcagcag tctgcaacct 240

gaggactttg cgacatacta ttgccagcag cactacacaa ccccacctac atttggtcag 300

gggacaaagg tggagatcaa g 321

<210> 90

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> VH of rituximab synthetic peptide

<400> 90

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

115 120

<210> 91

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding VH of rituximab

<400> 91

caagttcagc ttcagcagcc gggggctgag ttggtgaaac ccggggccag tgtgaagatg 60

agctgtaaag cgagcggcta caccttcact tcttataata tgcattgggt taagcaaacg 120

caggaaggg ggctggagtg gatcggcgct atttacccag gtaacggtga cacatcatat 180

aaccaaaagt ttaagggaaa ggcaaccctc acagcggaca agagtagctc aaccgcatac 240

atgcaactgt caagccttac ctccgaagac agcgcagtgt actactgcgc cagaagcacc 300

tactatgggg gtgattggta cttcaacgtc tggggggctg gcaccacagt gactgtaagc 360

gca 363

<210> 92

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> VL of rituximab synthetic peptide

<400> 92

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

100 105

<210> 93

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding VL of rituximab

<400> 93

cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60

atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120

agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180

ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240

gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300

actaagttgg aaattaag 318

<210> 94

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Trastuzumab heavy chain synthetic peptide

<400> 94

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> 95

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the heavy chain of trastuzumab

<400> 95

gaagtccaat tggtcgaatc aggcggtgga ctcgtgcaac ctggaggttc gttacgctta 60

tcatgtgctg caagtggatt taatattaaa gatacctaca tccactgggt acgtcaagct 120

cccggcaagg gtctcgagtg ggtcgcacgc atttacccca ccaacggata cacgcgctac 180

gccgattcag tgaagggacg tttcacaatc tctgctgata ctagcaaaaa taccgcatac 240

ctccagatga actctcttag ggccgaggac acagctgtgt actactgtag ccgttgggga 300

ggagacggtt tttacgcaat ggattactgg ggccaaggaa ccctggtcac agtttcatcg 360

gcgtcgacca agggcccgtc agtgttcccg ctggccccgt catccaagtc cacgtctggg 420

ggcacagcag ccctgggatg cttggtcaag gactacttcc ccgagcccgt gactgtgtcc 480

tggaactccg gagcactgac ctccggagtg cacacctttc ccgcggtgct gcagtcctcc 540

ggactgtact ccctgtcgtc ggtcgtgacc gtgccgagct cctcgctcgg aacccagacc 600

tacatctgca acgtgaacca caagccctcg aacaccaaag tggacaagaa ggtcgagccc 660

aaaagctgcg acaagactca cacttgtccg ccgtgccccg cccccgaact gctgggtggc 720

ccctccgtgt tcctgttccc gcctaagcct aaggacaccc ttatgatcag ccgcacccct 780

gaagtgacct gtgtcgtcgt ggatgtgtca cacgaggacc cggaggtcaa gttcaattgg 840

tacgtggacg gcgtggaagt gcataacgca aagaccaagc ctcgggagga acagtacaac 900

tcgacctacc gcgtggtgtc agtcctgact gtgctgcacc aggactggct gaacgggaag 960

gagtacaagt gcaaagtgtc gaacaaggcc ctgccggctc caattgaaaa gaccatcagc 1020

aaggccaagg gccagccaag ggaaccacag gtgtacaccc tccctccttc ccgggacgag 1080

ctgaccaaaa accaagtgtc cctgacttgc cttgtgaagg ggttctaccc ttctgacatt 1140

gccgtcgaat gggaatcgaa cggacagcct gaaaacaact ataagactac cccgcccgtg 1200

ctggattccg acggaagctt cttcctgtac tccaagctga ccgtggacaa gtcgagatgg 1260

cagcagggaa atgtgttcag ctgctccgtg atgcatgagg cgctgcacaa ccactacacc 1320

cagaagtcac tgagcctctc ccccggaaag 1350

<210> 96

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> Trastuzumab synthetic peptide light chain

<400> 96

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> 97

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the light chain of trastuzumab

<400> 97

gatatccaaa tgactcaatc accatcttca ctctccgcga gcgtgggtga tcgggtcacc 60

atcacatgta gggcgagcca agatgtgaat accgccgtcg cgtggtatca acaaaagccg 120

ggaaaagcac caaaactgct tatatactct gcatccttcc tgtactctgg ggtgccaagc 180

cggttctccg gtagtagatc tggtactgac tttacactca ctatcagcag tctgcaacct 240

gaggactttg cgacatacta ttgccagcag cactacacaa ccccacctac atttggtcag 300

gggacaaagg tggagatcaa gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360

gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420

cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480

tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540

agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccaggactg 600

tccagccccg tgaccaagag cttcaacaga ggagaatgct ag 642

<210> 98

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> Rituximab heavy chain synthetic peptide

<400> 98

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> 99

<211> 1353

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the heavy chain of rituximab

<400> 99

caagttcagc ttcagcagcc gggggctgag ttggtgaaac ccggggccag tgtgaagatg 60

agctgtaaag cgagcggcta caccttcact tcttataata tgcattgggt taagcaaacg 120

caggaaggg ggctggagtg gatcggcgct atttacccag gtaacggtga cacatcatat 180

aaccaaaagt ttaagggaaa ggcaaccctc acagcggaca agagtagctc aaccgcatac 240

atgcaactgt caagccttac ctccgaagac agcgcagtgt actactgcgc cagaagcacc 300

tactatgggg gtgattggta cttcaacgtc tggggggctg gcaccacagt gactgtaagc 360

gcagcgtcga ccaagggccc gtcagtgttc ccgctggccc cgtcatccaa gtccacgtct 420

gggggcacag cagccctggg atgcttggtc aaggactact tccccgagcc cgtgactgtg 480

tcctggaact ccggagcact gacctccgga gtgcacacct ttcccgcggt gctgcagtcc 540

tccggactgt actccctgtc gtcggtcgtg accgtgccga gctcctcgct cggaacccag 600

acctacatct gcaacgtgaa ccacaagccc tcgaacacca aagtggacaa gaaggtcgag 660

cccaaaagct gcgacaagac tcacacttgt ccgccgtgcc ccgcccccga actgctgggt 720

ggcccctccg tgttcctgtt cccgcctaag cctaaggaca cccttatgat cagccgcacc 780

cctgaagtga cctgtgtcgt cgtggatgtg tcacacgagg acccggaggt caagttcaat 840

tggtacgtgg acggcgtgga agtgcataac gcaaagacca agcctcggga ggaacagtac 900

aactcgacct accgcgtggt gtcagtcctg actgtgctgc accaggactg gctgaacggg 960

aaggagtaca agtgcaaagt gtcgaacaag gccctgccgg ctccaattga aaagaccatc 1020

agcaaggcca agggccagcc aagggaacca caggtgtaca ccctccctcc ttcccgggac 1080

gagctgacca aaaaccaagt gtccctgact tgccttgtga aggggttcta cccttctgac 1140

attgccgtcg aatgggaatc gaacggacag cctgaaaaca actataagac taccccgccc 1200

gtgctggatt ccgacggaag cttcttcctg tactccaagc tgaccgtgga caagtcgaga 1260

tggcagcagg gaaatgtgtt cagctgctcc gtgatgcatg aggcgctgca caaccactac 1320

acccagaagt cactgagcct ctcccccgga aag 1353

<210> 100

<211> 212

<212> PRT

<213> Artificial sequence

<220>

<223> Rituximab light chain synthetic peptide

<400> 100

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> 101

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the light chain of rituximab

<400> 101

cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60

atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120

agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180

ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240

gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300

actaagttgg aaattaagac cgtggccgcc ccgagcgtgt tcattttccc tccctccgac 360

gagcagttga aatcgggcac cgctagcgtg gtctgccttc tcaacaattt ctatccacgg 420

gaagccaaag tgcagtggaa ggtcgacaac gcgctccaat ccgggaactc acaggaatcc 480

gtgactgagc aggattccaa ggactcgacc tactccctgt catccacgct gaccctgagc 540

aaggcagact acgagaagca caaggtctac gcctgcgaag tgacacacca gggactgtcc 600

agccccgtga ccaagagctt caacagagga gaatgctag 639

<210> 102

<211> 634

<212> PRT

<213> Artificial sequence

<220>

<223> Trastuzumab synthetic peptide HC_MICwed

<400> 102

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 Ala 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 Ala 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 Gly Gly Gly Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Asp Arg Glu Thr Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met

515 520 525

Thr Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln

530 535 540

Glu Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser

545 550 555 560

Gln His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu

565 570 575

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

580 585 590

Met Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr

595 600 605

His Tyr His Ala Met His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr

610 615 620

Leu Lys Ser Gly Val Val Leu Arg Arg Thr

625 630

<210> 103

<211> 1902

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding trastuzumab HC_MICwed

<400> 103

gaagtccaat tggtcgaatc aggcggtgga ctcgtgcaac ctggaggttc gttacgctta 60

tcatgtgctg caagtggatt taatattaaa gatacctaca tccactgggt acgtcaagct 120

cccggcaagg gtctcgagtg ggtcgcacgc atttacccca ccaacggata cacgcgctac 180

gccgattcag tgaagggacg tttcacaatc tctgctgata ctagcaaaaa taccgcatac 240

ctccagatga actctcttag ggccgaggac acagctgtgt actactgtag ccgttgggga 300

ggagacggtt tttacgcaat ggattactgg ggccaaggaa ccctggtcac agtttcatcg 360

gcgtcgacca agggcccgtc agtgttcccg ctggccccgt catccaagtc cacgtctggg 420

ggcacagcag ccctgggatg cttggtcaag gactacttcc ccgagcccgt gactgtgtcc 480

tggaactccg gagcactgac ctccggagtg cacacctttc ccgcggtgct gcagtcctcc 540

ggactgtact ccctgtcgtc ggtcgtgacc gtgccgagct cctcgctcgg aacccagacc 600

tacatctgca acgtgaacca caagccctcg aacaccaaag tggacaagaa ggtcgagccc 660

aaaagctgcg acaagactca cacttgtccg ccgtgccccg cccccgaact gctgggtggc 720

ccctccgtgt tcctgttccc gcctaagcct aaggacaccc ttatgatcag ccgcacccct 780

gaagtgacct gtgtcgtcgt ggcagtgtca cacgaggacc cggaggtcaa gttcaattgg 840

tacgtggacg gcgtggaagt gcataacgca aagaccaagc ctcgggagga acagtacgcc 900

tcgacctacc gcgtggtgtc agtcctgact gtgctgcacc aggactggct gaacgggaag 960

gagtacaagt gcaaagtgtc gaacaaggcc ctgccggctc caattgaaaa gaccatcagc 1020

aaggccaagg gccagccaag ggaaccacag gtgtacaccc tccctccttc ccgggacgag 1080

ctgaccaaaa accaagtgtc cctgacttgc cttgtgaagg ggttctaccc ttctgacatt 1140

gccgtcgaat gggaatcgaa cggacagcct gaaaacaact ataagactac cccgcccgtg 1200

ctggattccg acggaagctt cttcctgtac tccaagctga ccgtggacaa gtcgagatgg 1260

cagcagggaa atgtgttcag ctgctccgtg atgcatgagg cgctgcacaa ccactacacc 1320

cagaagtcac tgagcctctc ccccggagga ggtggcagcg agcctcacag cctccggtat 1380

aatttgactg tactctcttg ggatggctcc gtgcagtccg gctttctgac tgaagttcat 1440

ctcgacggtc aacctttcct gcgctgcgac cgacaaaaat gccgcgccaa gccccaaggg 1500

cagtgggccg aagatgtact gggaaacaag acctgggacc gggagacacg agacctgaca 1560

ggctggggca aggacttgcg catgacactc gcccatatca aggaccagaa ggaaggattg 1620

cactctttgc aagagattcg cgtgtgtgaa atccacgagg acaattcaac gaggagctcc 1680

cagcacttct attacgatgg agaactcttc ttgtcacaga acttggaaac caaggaatgg 1740

actatgcctc agagctctcg ggcacagact ctcgctatga acgttagaaa cttccttaag 1800

gaggatgcta tggagaccga tactcactac cacgccatgc acgccgactg cctccaggaa 1860

ctgcggagat atctgaagtc cggcgtggtt ttgagaagaa cc 1902

<210> 104

<211> 634

<212> PRT

<213> Artificial sequence

<220>

<223> Trastuzumab synthetic peptide HC_MIC25

<400> 104

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 Ala 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 Ala 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 Gly Gly Gly Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Asp Arg Glu Thr Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met

515 520 525

Thr Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln

530 535 540

Glu Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser

545 550 555 560

Gln His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu

565 570 575

Thr Leu Glu Trp Thr Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala

580 585 590

Met Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr

595 600 605

His Tyr His Ala Met Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr

610 615 620

Leu Lys Ser Gly Val Val Leu Arg Arg Thr

625 630

<210> 105

<211> 1902

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding trastuzumab HC_MIC25

<400> 105

gaagtccaat tggtcgaatc aggcggtgga ctcgtgcaac ctggaggttc gttacgctta 60

tcatgtgctg caagtggatt taatattaaa gatacctaca tccactgggt acgtcaagct 120

cccggcaagg gtctcgagtg ggtcgcacgc atttacccca ccaacggata cacgcgctac 180

gccgattcag tgaagggacg tttcacaatc tctgctgata ctagcaaaaa taccgcatac 240

ctccagatga actctcttag ggccgaggac acagctgtgt actactgtag ccgttgggga 300

ggagacggtt tttacgcaat ggattactgg ggccaaggaa ccctggtcac agtttcatcg 360

gcgtcgacca agggcccgtc agtgttcccg ctggccccgt catccaagtc cacgtctggg 420

ggcacagcag ccctgggatg cttggtcaag gactacttcc ccgagcccgt gactgtgtcc 480

tggaactccg gagcactgac ctccggagtg cacacctttc ccgcggtgct gcagtcctcc 540

ggactgtact ccctgtcgtc ggtcgtgacc gtgccgagct cctcgctcgg aacccagacc 600

tacatctgca acgtgaacca caagccctcg aacaccaaag tggacaagaa ggtcgagccc 660

aaaagctgcg acaagactca cacttgtccg ccgtgccccg cccccgaact gctgggtggc 720

ccctccgtgt tcctgttccc gcctaagcct aaggacaccc ttatgatcag ccgcacccct 780

gaagtgacct gtgtcgtcgt ggcagtgtca cacgaggacc cggaggtcaa gttcaattgg 840

tacgtggacg gcgtggaagt gcataacgca aagaccaagc ctcgggagga acagtacgcc 900

tcgacctacc gcgtggtgtc agtcctgact gtgctgcacc aggactggct gaacgggaag 960

gagtacaagt gcaaagtgtc gaacaaggcc ctgccggctc caattgaaaa gaccatcagc 1020

aaggccaagg gccagccaag ggaaccacag gtgtacaccc tccctccttc ccgggacgag 1080

ctgaccaaaa accaagtgtc cctgacttgc cttgtgaagg ggttctaccc ttctgacatt 1140

gccgtcgaat gggaatcgaa cggacagcct gaaaacaact ataagactac cccgcccgtg 1200

ctggattccg acggaagctt cttcctgtac tccaagctga ccgtggacaa gtcgagatgg 1260

cagcagggaa atgtgttcag ctgctccgtg atgcatgagg cgctgcacaa ccactacacc 1320

cagaagtcac tgagcctctc ccccggagga ggtggcagcg agcctcacag cctccggtat 1380

aatttgactg tactctcttg ggatggctcc gtgcagtccg gctttctgac tgaagttcat 1440

ctcgacggtc aacctttcct gcgctgcgac cgacaaaaat gccgcgccaa gccccaaggg 1500

cagtgggccg aagatgtact gggaaacaag acctgggacc gggagacacg agacctgaca 1560

ggctggggca aggacttgcg catgacactc gcccatatca aggaccagaa ggaaggattg 1620

cactctttgc aagagattcg cgtgtgtgaa atccacgagg acaattcaac gaggagctcc 1680

cagcacttct attacgatgg agaactcttc ttgtcacaga acttggaaac cctcgaatgg 1740

actatgcctc agagctctcg ggcacagact ctcgctatga acgttagaaa cttccttaag 1800

gaggatgcta tggagaccga tactcactac cacgccatgc gcgccgactg cctctctgaa 1860

ctgcggagat atctgaagtc cggcgtggtt ttgagaagaa cc 1902

<210> 106

<211> 404

<212> PRT

<213> Artificial sequence

<220>

<223> Rituximab synthetic peptide LC_ULBP2.wt

<400> 106

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> 107

<211> 1212

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding rituximab LC_ULBP2.wt

<400> 107

cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60

atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120

agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180

ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240

gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300

actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360

gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420

cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480

tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540

agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccaggactg 600

tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660

ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720

cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780

gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840

acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900

ttgcgcgaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960

atgtcttgcg agcaaaaggc agaggccac tcctccggca gctggcagtt cagtttcgac 1020

ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080

gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgatgtc attccactat 1140

ttctcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200

ttggagccat cg 1212

<210> 108

<211> 187

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide variant ULPB2 alpha1-alpha2 R80W

<400> 108

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> 109

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding a variant of ULPB2

alpha1-alpha2 R80W

<400> 109

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 gcgatgtcat tccactattt ctcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

<210> 110

<211> 513

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding a library of NNK variants

ULPB2 alpha1-alpha2

R80W

<220>

<221> misc_feature

<222> (457)..(458)

<223> n stands for a, c, g, or t

<220>

<221> misc_feature

<222> (460)..(461)

<223> n stands for a, c, g, or t

<220>

<221> misc_feature

<222> (463)..(464)

<223> n stands for a, c, g, or t

<220>

<221> misc_feature

<222> (466)..(467)

<223> n stands for a, c, g, or t

<220>

<221> misc_feature

<222> (469)..(470)

<223> n stands for a, c, g, or t

<400> 110

gagccccata gcctcagcta tgacattacg gtgattccca aatttcgccc aggaccacgt 60

tggtgcgccg tccagggtca ggtagatgaa aagactttcc tgcattatga ttgcggcaat 120

aaaaccgtga cgccggtatc gccgttaggc aaaaaattga atgtcacgac agcgtggaaa 180

gcacagaacc cggtgttgcg cgaggtagtc gatattttga cggaacaact ctgggacatt 240

cagctcgaga attacacccc aaaagaaccg ctgacgctgc aagcgcgtat gtcgtgcgaa 300

caaaaagcag aaggtcactc tagcgggagt tggcagtttt ccttcgatgg gcagattttt 360

ctgctgtttg attcggagaa acgcatgtgg actacagtcc acccgggtgc ccggaaaatg 420

aaagagaagt gggagaatga taaagtggtg gccactnnkn nknnknnknn ktccatgggc 480

gattgcattg gctggttaga ggattttctc atg 513

<210> 111

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> ULBP2.C variant of the synthetic peptide ULBP2 alpha1-alpha2

<400> 111

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> 112

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding a variant of ULBP2.C

ULBP2 alpha1-alpha2 peptide

<400> 112

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> 113

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> ULBP2.R variant of the synthetic peptide ULBP2 alpha1-alpha2

<400> 113

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> 114

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding a variant of ULBP2.R

ULBP2 alpha1-alpha2 peptide

<400> 114

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> 115

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> ULBP2.AA variant of the synthetic peptide ULBP2 alpha1-alpha2

<400> 115

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> 116

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding a variant of ULBP2.AA

ULBP2 alpha1-alpha2 peptide

<400> 116

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> 117

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> ULBP2.AB variant of the synthetic peptide ULBP2 alpha1-alpha2

<400> 117

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> 118

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the ULBP2.AB variant

ULBP2 alpha1-alpha2 peptide

<400> 118

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> 119

<211> 404

<212> PRT

<213> Artificial sequence

<220>

<223> Rituximab synthetic peptide LC_ULBP2.C

<400> 119

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 Ile Leu Trp Gln Thr 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> 120

<211> 1212

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding rituximab LC_ULBP2.C

<400> 120

cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60

atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120

agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180

ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240

gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300

actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360

gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420

cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480

tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540

agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccaggactg 600

tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660

ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720

cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780

gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840

acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900

ttgtgggaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960

atgtcttgcg agcaaaaggc agaggccac tcctccggca gctggcagtt cagtttcgac 1020

ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080

gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgactat tctgtggcag 1140

acttcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200

ttggagccat cg 1212

<210> 121

<211> 404

<212> PRT

<213> Artificial sequence

<220>

<223> Rituximab synthetic peptide LC_ULBP2.R

<400> 121

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 Leu Leu Trp Gly 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> 122

<211> 1212

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding rituximab LC_ULBP2.R

<400> 122

cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60

atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120

agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180

ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240

gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300

actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360

gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420

cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480

tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540

agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccaggactg 600

tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660

ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720

cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780

gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840

acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900

ttgtgggaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960

atgtcttgcg agcaaaaggc agaggccac tcctccggca gctggcagtt cagtttcgac 1020

ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080

gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgacttt gttgtggggg 1140

tggtcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200

ttggagccat cg 1212

<210> 123

<211> 404

<212> PRT

<213> Artificial sequence

<220>

<223> Rituximab synthetic peptide LC_ULBP2.AA

<400> 123

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 Met Phe Trp Ser 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> 124

<211> 1212

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding rituximab LC_ULBP2.AA

<400> 124

cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60

atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120

agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180

ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240

gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300

actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360

gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420

cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480

tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540

agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccaggactg 600

tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660

ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720

cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780

gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840

acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900

ttgtgggaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960

atgtcttgcg agcaaaaggc agaggccac tcctccggca gctggcagtt cagtttcgac 1020

ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080

gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgactat gttttggagt 1140

tggtcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200

ttggagccat cg 1212

<210> 125

<211> 404

<212> PRT

<213> Artificial sequence

<220>

<223> Rituximab synthetic peptide LC_ULBP2.AB

<400> 125

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 Leu Met Trp Gln 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> 126

<211> 1212

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding rituximab LC_ULBP2.AB

<400> 126

cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60

atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120

agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180

ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240

gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300

actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360

gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420

cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480

tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540

agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccaggactg 600

tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660

ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720

cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780

gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840

acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900

ttgtgggaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960

atgtcttgcg agcaaaaggc agaggccac tcctccggca gctggcagtt cagtttcgac 1020

ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080

gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgactct tatgtggcag 1140

tggtcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200

ttggagccat cg 1212

<210> 127

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> ULBP2.S3 variant of the synthetic peptide ULBP2 alpha1-alpha2

<400> 127

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> 128

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding a variant of the ULBP2.S3 peptide

ULBP2 alpha1-alpha2

<400> 128

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 gcgactaagc tttatctttg gtcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

<210> 129

<211> 404

<212> PRT

<213> Artificial sequence

<220>

<223> Rituximab synthetic peptide LC_ULBP2.S3

<400> 129

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> 130

<211> 1212

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding rituximab LC_ULBP2.S3

<400> 130

cagatcgtgt tgtcccaatc acccgcaatt ctctctgcga gcccagggga gaaggtgacc 60

atgacttgcc gcgcttcaag ttccgtatcc tacattcact ggttccagca gaagcccgga 120

agttccccta agccctggat ctatgctaca tccaatctgg caagcggtgt tcccgttaga 180

ttttccggaa gcgggtctgg aaccagttac agtctgacta tttccagggt cgaggccgaa 240

gatgcggcta cttattattg ccaacagtgg acctctaacc cacccacatt cggcggcggc 300

actaagttgg aaattaagcg gaccgtggcc gccccgagcg tgttcatttt ccctccctcc 360

gacgagcagt tgaaatcggg caccgctagc gtggtctgcc ttctcaacaa tttctatcca 420

cgggaagcca aagtgcagtg gaaggtcgac aacgcgctcc aatccgggaa ctcacaggaa 480

tccgtgactg agcaggattc caaggactcg acctactccc tgtcatccac gctgaccctg 540

agcaaggcag actacgagaa gcacaaggtc tacgcctgcg aagtgacaca ccaggactg 600

tccagccccg tgaccaagag cttcaacaga ggagaatgcg cacctacctc aagctctgga 660

ggaggtggca gcgagcccca tagtctgagc tacgacatca cagttattcc caagttcagg 720

cccggaccgc gctggtgtgc cgtgcaagga caagtcgacg aaaaaacctt tcttcattac 780

gattgcggaa ataagactgt aacgccagtc tctcctttag gtaagaagtt aaacgtcact 840

acggcgtgga aggcacaaaa ccccgtcctg cgcgaggtcg tcgacatcct gactgaacaa 900

ttgtgggaca tccagctcga gaattacact ccaaaggagc ctcttaccct gcaggctaga 960

atgtcttgcg agcaaaaggc agaggccac tcctccggca gctggcagtt cagtttcgac 1020

ggacaaatct ttctgttatt cgattcagag aagagaatgt ggactacagt tcaccccggt 1080

gcccgtaaaa tgaaggagaa gtgggaaaac gacaaagtgg tggcgactaa gctttatctt 1140

tggtcgatgg gagactgcat cggttggctg gaagatttcc tcatgggtat ggactccact 1200

ttggagccat cg 1212

<210> 131

<211> 634

<212> PRT

<213> Artificial sequence

<220>

<223> Rituximab synthetic peptide HC_ULBP2.R80W

<400> 131

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> 132

<211> 1902

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding rituximab HC_ULBP2.R80W

<400> 132

caagttcagc ttcagcagcc gggggctgag ttggtgaaac ccggggccag tgtgaagatg 60

agctgtaaag cgagcggcta caccttcact tcttataata tgcattgggt taagcaaacg 120

caggaaggg ggctggagtg gatcggcgct atttacccag gtaacggtga cacatcatat 180

aaccaaaagt ttaagggaaa ggcaaccctc acagcggaca agagtagctc aaccgcatac 240

atgcaactgt caagccttac ctccgaagac agcgcagtgt actactgcgc cagaagcacc 300

tactatgggg gtgattggta cttcaacgtc tggggggctg gcaccacagt gactgtaagc 360

gcagcgtcga ccaagggccc gtcagtgttc ccgctggccc cgtcatccaa gtccacgtct 420

gggggcacag cagccctggg atgcttggtc aaggactact tccccgagcc cgtgactgtg 480

tcctggaact ccggagcact gacctccgga gtgcacacct ttcccgcggt gctgcagtcc 540

tccggactgt actccctgtc gtcggtcgtg accgtgccga gctcctcgct cggaacccag 600

acctacatct gcaacgtgaa ccacaagccc tcgaacacca aagtggacaa gaaggtcgag 660

cccaaaagct gcgacaagac tcacacttgt ccgccgtgcc ccgcccccga actgctgggt 720

ggcccctccg tgttcctgtt cccgcctaag cctaaggaca cccttatgat cagccgcacc 780

cctgaagtga cctgtgtcgt cgtggcagtg tcacacgagg acccggaggt caagttcaat 840

tggtacgtgg acggcgtgga agtgcataac gcaaagacca agcctcggga ggaacagtac 900

gcctcgacct accgcgtggt gtcagtcctg actgtgctgc accaggactg gctgaacggg 960

aaggagtaca agtgcaaagt gtcgaacaag gccctgccgg ctccaattga aaagaccatc 1020

agcaaggcca agggccagcc aagggaacca caggtgtaca ccctccctcc ttcccgggac 1080

gagctgacca aaaaccaagt gtccctgact tgccttgtga aggggttcta cccttctgac 1140

attgccgtcg aatgggaatc gaacggacag cctgaaaaca actataagac taccccgccc 1200

gtgctggatt ccgacggaag cttcttcctg tactccaagc tgaccgtgga caagtcgaga 1260

tggcagcagg gaaatgtgtt cagctgctcc gtgatgcatg aggcgctgca caaccactac 1320

acccagaagt cactgagcct ctcccccgga ggaggtggca gcgagcccca tagtctgagc 1380

tacgacatca cagttattcc caagttcagg cccggaccgc gctggtgtgc cgtgcaagga 1440

caagtcgacg aaaaaacctt tcttcattac gattgcggaa ataagactgt aacgccagtc 1500

tctcctttag gtaagaagtt aaacgtcact acggcgtgga aggcacaaaa ccccgtcctg 1560

cgcgaggtcg tcgacatcct gactgaacaa ttgtgggaca tccagctcga gaattacact 1620

ccaaaggagc ctcttaccct gcaggctaga atgtcttgcg agcaaaaggc agaggccac 1680

tcctccggca gctggcagtt cagtttcgac ggacaaatct ttctgttatt cgattcagag 1740

aagagaatgt ggactacagt tcaccccggt gcccgtaaaa tgaaggagaa gtgggaaaac 1800

gacaaagtgg tggcgatgtc attccactat ttctcgatgg gagactgcat cggttggctg 1860

gaagatttcc tcatgggtat ggactccact ttggagccat cg 1902

<210> 133

<211> 405

<212> PRT

<213> Artificial sequence

<220>

<223> Trastuzumab synthetic peptide LC_ULBP2.R

<400> 133

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 Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly Gly Gly Gly

210 215 220

Ser Glu Pro His Ser Leu Ser Tyr Asp Ile Thr Val Ile Pro Lys Phe

225 230 235 240

Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys

245 250 255

Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser

260 265 270

Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn

275 280 285

Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp Asp

290 295 300

Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala

305 310 315 320

Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp

325 330 335

Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys

340 345 350

Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys

355 360 365

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

370 375 380

Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser

385 390 395 400

Thr Leu Glu Pro Ser

405

<210> 134

<211> 1215

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding trastuzumab LC_ULBP2.R

<400> 134

gatatccaaa tgactcaatc accatcttca ctctccgcga gcgtgggtga tcgggtcacc 60

atcacatgta gggcgagcca agatgtgaat accgccgtcg cgtggtatca acaaaagccg 120

ggaaaagcac caaaactgct tatatactct gcatccttcc tgtactctgg ggtgccaagc 180

cggttctccg gtagtagatc tggtactgac tttacactca ctatcagcag tctgcaacct 240

gaggactttg cgacatacta ttgccagcag cactacacaa ccccacctac atttggtcag 300

gggacaaagg tggagatcaa gcggaccgtg gccgccccga gcgtgttcat tttccctccc 360

tccgacgagc agttgaaatc gggcaccgct agcgtggtct gccttctcaa caatttctat 420

ccacgggaag ccaaagtgca gtggaaggtc gacaacgcgc tccaatccgg gaactcacag 480

gaatccgtga ctgagcagga ttccaaggac tcgacctact ccctgtcatc cacgctgacc 540

ctgagcaagg cagactacga gaagcacaag gtctacgcct gcgaagtgac acaccaggga 600

ctgtccagcc ccgtgaccaa gagcttcaac agaggagaat gcgcacctac ctcaagctct 660

ggaggaggtg gcagcgagcc ccatagtctg agctacgaca tcacagttat tcccaagttc 720

aggcccggac cgcgctggtg tgccgtgcaa ggacaagtcg acgaaaaaac ctttcttcat 780

tacgattgcg gaaataagac tgtaacgcca gtctctcctt taggtaagaa gttaaacgtc 840

actacggcgt ggaaggcaca aaaccccgtc ctgcgcgagg tcgtcgacat cctgactgaa 900

caattgtggg acatccagct cgagaattac actccaaagg agcctcttac cctgcaggct 960

agaatgtctt gcgagcaaaa ggcagagggc cactcctccg gcagctggca gttcagtttc 1020

gacggacaaa tctttctgtt attcgattca gagaagagaa tgtggactac agttcacccc 1080

ggtgcccgta aaatgaagga gaagtgggaa aacgacaaag tggtggcgac tttgttgtgg 1140

gggtggtcga tgggagactg catcggttgg ctggaagatt tcctcatggg tatggactcc 1200

actttggagc catcg 1215

<210> 135

<211> 156

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide, ectodomain NKG2D.wt

<400> 135

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

145 150 155

<210> 136

<211> 468

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the ectodomain of NKG2D.wt

<400> 136

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 ggaccgtg 468

<210> 137

<211> 156

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide, ectodomain NKG2D.YA

<400> 137

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

145 150 155

<210> 138

<211> 468

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the ectodomain of NKG2D.YA

<400> 138

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 ggaccgtg 468

<210> 139

<211> 156

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide, ectodomain NKG2D.AF

<400> 139

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> 140

<211> 468

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding the ectodomain of NKG2D.AF

<400> 140

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 ggaccgtg 468

<210> 141

<211> 69

<212> PRT

<213> Artificial sequence

<220>

<223> CD8-alpha synthetic peptide hinge region and transmembrane domain

<400> 141

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

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

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 142

<211> 207

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding the hinge region of CD8-alpha and

transmembrane domain

<400> 142

accaccacac cagctcctag acctccaact cctgctccta caatcgccag ccagcctctg 60

tctctgaggc cagaagcttg tagacctgct gcaggcggag ccgtgcatac aagaggactg 120

gatttcgcct gcgacatcta catctgggcc cctctggctg gaacatgtgg cgtgctgctg 180

ctgagcctgg tcatcaccct gtactgc 207

<210> 143

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide 4-1BB

<400> 143

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 144

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding 4-1BB

<400> 144

aagcggggca gaaagaagct gctgtacatc tttaagcagc ccttcatgcg gcccgtgcag 60

accacacaag aggaagatgg ctgctcctgc agattccccg aggaagaaga aggcggctgc 120

gagctg 126

<210> 145

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide CD3zeta

<400> 145

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 146

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding CD3zeta

<400> 146

agagtgaagt tcagccgttc tgccgacgct cccgcctata agcagggaca gaaccagctg 60

tacaacgagc tgaacctggg gagaagagaa gagtacgacg tgctggacaa gcggagaggc 120

agagatcctg agatgggcgg caagcccaga cggaagaatc ctcaagaggg cctgtataat 180

gagctgcaga aagacaagat ggccgaggcc tacagcgaga tcggaatgaa gggcgagcgc 240

agaagaggca agggacacga tggactgtac cagggcctga gcaccgccac caaggatacc 300

tatgatgccc tgcacatgca ggccctgcct ccaaga 336

<210> 147

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide EGFP

<400> 147

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

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

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 148

<211> 717

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding EGFP

<400> 148

atggtgtcta aaggcgagga actgttcacc ggcgtggtgc ccattctggt ggaactggac 60

ggggatgtga acggccacaa gtttagcgtt agcggcgaag gcgaagggga tgccacatac 120

ggaaagctga ccctgaagtt catctgcacc accggcaagc tgcctgtgcc ttggcctaca 180

ctggtcacca cactgacata cggcgtgcag tgctttagca gataccccga ccatatgaag 240

cagcacgact tcttcaagtc cgccatgcct gagggctacg tgcaagagcg gaccatcttc 300

tttaaggacg acggcaacta caagaccagg gccgaagtga agtttgaggg cgacaccctg 360

gtcaaccgga tcgagctgaa gggcatcgac ttcaaagagg atggcaacat cctgggccac 420

aagctcgagt acaactacaa cagccacaac gtgtacatca tggccgacaa gcagaagaac 480

ggcatcaagg ccaacttcaa gatccggcac aacatcgagg acggcagcgt tcagctggcc 540

gatcactacc agcagaacac ccctatcgga gatggccctg tgctgctccc cgacaatcac 600

tacctgagca cacagagcgc cctgagcaag gaccccaacg agaagaggga tcacatggtg 660

ctgctggaat ttgtgaccgc cgcaggcatc accctcggca tggacgaact gtacaaa 717

<210> 149

<211> 473

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide CD8hingeTM_4-1BB_CD3zeta_EGFP

<400> 149

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

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

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys Ser Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr

65 70 75 80

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

85 90 95

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

100 105 110

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln

115 120 125

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

130 135 140

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

145 150 155 160

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

165 170 175

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

180 185 190

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

195 200 205

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

210 215 220

Arg Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu

225 230 235 240

Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp

245 250 255

Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala

260 265 270

Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu

275 280 285

Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln

290 295 300

Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys

305 310 315 320

Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys

325 330 335

Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp

340 345 350

Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp

355 360 365

Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn

370 375 380

Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe

385 390 395 400

Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His

405 410 415

Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp

420 425 430

Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu

435 440 445

Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile

450 455 460

Thr Leu Gly Met Asp Glu Leu Tyr Lys

465 470

<210> 150

<211> 1419

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding CD8hingeTM_4-1BB_CD3zeta_EGFP

<400> 150

accaccacac cagctcctag acctccaact cctgctccta caatcgccag ccagcctctg 60

tctctgaggc cagaagcttg tagacctgct gcaggcggag ccgtgcatac aagaggactg 120

gatttcgcct gcgacatcta catctgggcc cctctggctg gaacatgtgg cgtgctgctg 180

ctgagcctgg tcatcaccct gtactgcagc ctgaagcggg gcagaaagaa gctgctgtac 240

atctttaagc agcccttcat gcggcccgtg cagaccacac aagaggaaga tggctgctcc 300

tgcagattcc ccgaggaaga agaaggcggc tgcgagctga gagtgaagtt cagccgttct 360

gccgacgctc ccgcctataa gcagggacag aaccagctgt acaacgagct gaacctgggg 420

agaagagaag agtacgacgt gctggacaag cggagaggca gagatcctga gatgggcggc 480

aagcccagac ggaagaatcc tcaagagggc ctgtataatg agctgcagaa agacaagatg 540

gccgaggcct acagcgagat cggaatgaag ggcgagcgca gaagaggcaa gggacacgat 600

ggactgtacc agggcctgag caccgccacc aaggatacct atgatgccct gcacatgcag 660

gccctgcctc caagatcagg ctctggttct ggcagcggca gcatggtgtc taaaggcgag 720

gaactgttca ccggcgtggt gcccattctg gtggaactgg acggggatgt gaacggccac 780

aagtttagcg ttagcggcga aggcgaaggg gatgccacat acggaaagct gaccctgaag 840

ttcatctgca ccaccggcaa gctgcctgtg ccttggccta cactggtcac cacactgaca 900

tacggcgtgc agtgctttag cagatacccc gaccatatga agcagcacga cttcttcaag 960

tccgccatgc ctgagggcta cgtgcaagag cggaccatct tctttaagga cgacggcaac 1020

tacaagacca gggccgaagt gaagtttgag ggcgacaccc tggtcaaccg gatcgagctg 1080

aagggcatcg acttcaaaga ggatggcaac atcctgggcc acaagctcga gtacaactac 1140

aacagccaca acgtgtacat catggccgac aagcagaaga acggcatcaa ggccaacttc 1200

aagatccggc acaacatcga ggacggcagc gttcagctgg ccgatcacta ccagcagaac 1260

acccctatcg gagatggccc tgtgctgctc cccgacaatc actacctgag cacacagagc 1320

gccctgagca aggaccccaa cgagaagagg gatcacatgg tgctgctgga atttgtgacc 1380

gccgcaggca tcaccctcgg catggacgaa ctgtacaaa 1419

<210> 151

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length chimeric antigen receptor synthetic peptide

NKG2D.wt_CD8hingeTM_4-1BB_CD3zeta_EGFP

<400> 151

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> 152

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a full-length chimeric

antigen receptor NKG2D.wt_CD8hingeTM_4-1BB_CD3zeta_EGFP

<400> 152

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> 153

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length chimeric antigen receptor synthetic peptide

NKG2D.YA_CD8hingeTM_4-1BB_CD3zeta_EGFP

<400> 153

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> 154

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a full-length chimeric

antigen receptor NKG2D.YA_CD8hingeTM_4-1BB_CD3zeta_EGFP

<400> 154

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> 155

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length chimeric antigen receptor synthetic peptide

NKG2D.AF_CD8hingeTM_4-1BB_CD3zeta_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 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> 156

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a full-length

chimeric antigen receptor NKG2D.AF_CD8hingeTM_4-1BB_CD3zeta_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 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> 157

<211> 473

<212> PRT

<213> Artificial sequence

<220>

<223> Silent chimeric antigen receptor synthetic peptide

NKG2D.YA_CD8hingeTM_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 Gly Ser Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu

225 230 235 240

Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp

245 250 255

Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala

260 265 270

Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu

275 280 285

Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln

290 295 300

Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys

305 310 315 320

Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys

325 330 335

Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp

340 345 350

Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp

355 360 365

Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn

370 375 380

Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe

385 390 395 400

Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His

405 410 415

Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp

420 425 430

Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu

435 440 445

Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile

450 455 460

Thr Leu Gly Met Asp Glu Leu Tyr Lys

465 470

<210> 158

<211> 1419

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a silent chimeric

antigen receptor NKG2D.YA_CD8hingeTM_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 actgctcagg ctctggttct ggcagcggca gcatggtgtc taaaggcgag 720

gaactgttca ccggcgtggt gcccattctg gtggaactgg acggggatgt gaacggccac 780

aagtttagcg ttagcggcga aggcgaaggg gatgccacat acggaaagct gaccctgaag 840

ttcatctgca ccaccggcaa gctgcctgtg ccttggccta cactggtcac cacactgaca 900

tacggcgtgc agtgctttag cagatacccc gaccatatga agcagcacga cttcttcaag 960

tccgccatgc ctgagggcta cgtgcaagag cggaccatct tctttaagga cgacggcaac 1020

tacaagacca gggccgaagt gaagtttgag ggcgacaccc tggtcaaccg gatcgagctg 1080

aagggcatcg acttcaaaga ggatggcaac atcctgggcc acaagctcga gtacaactac 1140

aacagccaca acgtgtacat catggccgac aagcagaaga acggcatcaa ggccaacttc 1200

aagatccggc acaacatcga ggacggcagc gttcagctgg ccgatcacta ccagcagaac 1260

acccctatcg gagatggccc tgtgctgctc cccgacaatc actacctgag cacacagagc 1320

gccctgagca aggaccccaa cgagaagagg gatcacatgg tgctgctgga atttgtgacc 1380

gccgcaggca tcaccctcgg catggacgaa ctgtacaaa 1419

<210> 159

<211> 225

<212> PRT

<213> Artificial sequence

<220>

<223> Silent chimeric antigen receptor synthetic peptide

NKG2D.YA_CD8hingeTM

<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 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

225

<210> 160

<211> 675

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a silent chimeric

antigen receptor NKG2D.YA_CD8hingeTM

<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 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 actgc 675

<210> 161

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length chimeric antigen receptor synthetic peptide

NKG2D.YA_CD8hingeTM_4-1BBtraf2_CD3zeta_EGFP

<400> 161

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 Ala Ala Ala Gly Cys Ser

245 250 255

Cys Arg Phe Pro Glu Ala Ala Ala 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> 162

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a full-length chimeric

antigen receptor NKG2D.YA_CD8hingeTM_4-1BBtraf2_CD3zeta_EGFP

<400> 162

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 gccgctgcag gctgctcctg cagattcccc 780

gaggctgccg caggcggctg 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> 163

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length chimeric antigen receptor synthetic peptide

NKG2D.YA_CD8hingeTM_4-1BB_CD3itam_EGFP

<400> 163

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 Phe Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Phe 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 Phe Asn Glu Leu Gln Lys Asp Lys Met

325 330 335

Ala Glu Ala Phe Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

340 345 350

Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala Thr Lys Asp

355 360 365

Thr Phe 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> 164

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a full-length chimeric

antigen receptor NKG2D.YA_CD8hingeTM_4-1BB_CD3itam_EGFP

<400> 164

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 ccagctgttc aacgagctga acctggggag aagagaagag 900

ttcgacgtgc tggacaagcg gagaggcaga gatcctgaga tgggcggcaa gccgacgg 960

aagaatcctc aagaggggcct gtttaatgag ctgcagaaag acaagatggc cgaggccttc 1020

agcgagatcg gaatgaaggg cgagcgcaga agaggcaagg gacacgatgg actgttccag 1080

ggcctgagca ccgccaccaa ggataccttt 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> 165

<211> 629

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length chimeric antigen receptor synthetic peptide

NKG2D.YA_CD8hingeTM_4-1BBtraf2_CD3itam_EGFP

<400> 165

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 Ala Ala Ala Gly Cys Ser

245 250 255

Cys Arg Phe Pro Glu Ala Ala Ala 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 Phe Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Phe 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 Phe Asn Glu Leu Gln Lys Asp Lys Met

325 330 335

Ala Glu Ala Phe Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

340 345 350

Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala Thr Lys Asp

355 360 365

Thr Phe 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> 166

<211> 1887

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a full-length chimeric

antigen receptor NKG2D.YA_CD8hingeTM_4-1BBtraf2_CD3itam_EGFP

<400> 166

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 gccgctgcag gctgctcctg cagattcccc 780

gaggctgccg caggcggctg cgagctgaga gtgaagttca gccgttctgc cgacgctccc 840

gcctataagc agggacagaa ccagctgttc aacgagctga acctggggag aagagaagag 900

ttcgacgtgc tggacaagcg gagaggcaga gatcctgaga tgggcggcaa gccgacgg 960

aagaatcctc aagaggggcct gtttaatgag ctgcagaaag acaagatggc cgaggccttc 1020

agcgagatcg gaatgaaggg cgagcgcaga agaggcaagg gacacgatgg actgttccag 1080

ggcctgagca ccgccaccaa ggataccttt 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> 167

<211> 517

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length chimeric antigen receptor synthetic peptide

NKG2D.YA_CD8hingeTM_4-1BB_EGFP

<400> 167

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 Ser Gly Ser

260 265 270

Gly Ser Gly Ser Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr

275 280 285

Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His

290 295 300

Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys

305 310 315 320

Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp

325 330 335

Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg

340 345 350

Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro

355 360 365

Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn

370 375 380

Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn

385 390 395 400

Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu

405 410 415

Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met

420 425 430

Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His

435 440 445

Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn

450 455 460

Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu

465 470 475 480

Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His

485 490 495

Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met

500 505 510

Asp Glu Leu Tyr Lys

515

<210> 168

<211> 1551

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a full-length chimeric

antigen receptor NKG2D.YA_CD8hingeTM_4-1BB_EGFP

<400> 168

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 cgagctgtca ggctctggtt ctggcagcgg cagcatggtg 840

tctaaaggcg aggaactgtt caccggcgtg gtgcccattc tggtggaact ggacggggat 900

gtgaacggcc acaagtttag cgttagcggc gaaggcgaag gggatgccac atacggaaag 960

ctgaccctga agttcatctg caccaccggc aagctgcctg tgccttggcc tacactggtc 1020

accacactga catacggcgt gcagtgcttt agcagatacc ccgaccatat gaagcagcac 1080

gacttcttca agtccgccat gcctgagggc tacgtgcaag agcggaccat cttctttaag 1140

gacgacggca actacaagac cagggccgaa gtgaagtttg agggcgacac cctggtcaac 1200

cggatcgagc tgaagggcat cgacttcaaa gaggatggca acatcctggg ccacaagctc 1260

gagtacaact acaacagcca caacgtgtac atcatggccg acaagcagaa gaacggcatc 1320

aaggccaact tcaagatccg gcacaacatc gaggacggca gcgttcagct ggccgatcac 1380

taccagcaga acacccctat cggagatggc cctgtgctgc tccccgacaa tcactacctg 1440

agcacacaga gcgccctgag caaggacccc aacgagaaga gggatcacat ggtgctgctg 1500

gaatttgtga ccgccgcagg catcaccctc ggcatggacg aactgtacaa a 1551

<210> 169

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length chimeric antigen receptor synthetic peptide

NKG2D.YA_CD8hingeTM_4-1BB

<400> 169

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

260 265

<210> 170

<211> 807

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a full-length chimeric

antigen receptorNKG2D.YA_CD8hingeTM_4-1BB

<400> 170

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 cgagctg 807

<210> 171

<211> 736

<212> PRT

<213> Artificial sequence

<220>

<223> Full-length chimeric antigen receptor synthetic peptide

CD19scFv_CD8hingeTM_4-1BB_CD3zeta_EGFP

<400> 171

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 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

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

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

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

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Ser Leu Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

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

485 490 495

Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile

500 505 510

Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser

515 520 525

Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe

530 535 540

Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr

545 550 555 560

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

565 570 575

Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln

580 585 590

Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala

595 600 605

Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys

610 615 620

Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu

625 630 635 640

Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys

645 650 655

Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly

660 665 670

Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp

675 680 685

Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala

690 695 700

Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu

705 710 715 720

Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

725 730 735

<210> 172

<211> 2208

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding a full-length chimeric

antigen receptor CD19scFv_CD8hingeTM_4-1BB_CD3zeta_EGFP

<400> 172

atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60

cctgacatcc agatgaccca gacaaccagc agcctgtctg ccagcctggg cgatagagtg 120

accatcagct gtagagccag ccaggacatc agcaagtacc tgaactggta tcagcagaaa 180

cccgacggca ccgtgaagct gctgatctac cacaccagca gactgcacag cggcgtgcca 240

agcagatttt ctggcagcgg ctctggcacc gactacagcc tgacaatcag caacctggaa 300

caagaggata tcgctaccta cttctgccag caaggcaaca ccctgcctta cacctttggc 360

ggaggcacca agctggaaat cacaggcggc ggaggaagcg gaggcggagg atctggtggt 420

ggtggatctg aagtgaaact gcaagagtct ggccctggcc tggtggcccc atctcaatct 480

ctgagcgtga cctgtaccgt cagcggagtg tccctgcctg attatggcgt gtcctggatc 540

cggcagcctc ctagaaaagg cctggaatgg ctgggcgtga tctggggcag cgagacaacc 600

tactacaaca gcgccctgaa gtcccggctg accatcatca aggacaactc caagagccag 660

gtgttcctga agatgaacag cctgcagacc gacgacaccg ccatctacta ttgcgccaag 720

cactactact acggcggcag ctacgccatg gattattggg gccagggcac cagcgtgacc 780

gtttcttcta ccaccacacc agctcctaga cctccaactc ctgctcctac aatcgccagc 840

cagcctctgt ctctgaggcc agaagcttgt agacctgctg caggcggagc cgtgcataca 900

agaggactgg atttcgcctg cgacatctac atctgggccc ctctggctgg aacatgtggc 960

gtgctgctgc tgagcctggt catcaccctg tactgcagcc tgaagcgggg cagaaagaag 1020

ctgctgtaca tctttaagca gcccttcatg cggcccgtgc agaccacaca agaggaagat 1080

ggctgctcct gcagattccc cgaggaagaa gaaggcggct gcgagctgag agtgaagttc 1140

agccgttctg ccgacgctcc cgcctataag cagggacaga accagctgta caacgagctg 1200

aacctgggga gaagagaaga gtacgacgtg ctggacaagc ggagaggcag agatcctgag 1260

atgggcggca agcccagacg gaagaatcct caagagggcc tgtataatga gctgcagaaa 1320

gacaagatgg ccgaggccta cagcgagatc ggaatgaagg gcgagcgcag aagaggcaag 1380

ggacacgatg gactgtacca gggcctgagc accgccacca aggataccta tgatgccctg 1440

cacatgcagg ccctgcctcc aagatcaggc tctggttctg gcagcggcag catggtgtct 1500

aaaggcgagg aactgttcac cggcgtggtg cccattctgg tggaactgga cggggatgtg 1560

aacggccaca agtttagcgt tagcggcgaa ggcgaagggg atgccacata cggaaagctg 1620

accctgaagt tcatctgcac caccggcaag ctgcctgtgc cttggcctac actggtcacc 1680

acactgacat acggcgtgca gtgctttagc agataccccg accatatgaa gcagcacgac 1740

ttcttcaagt ccgccatgcc tgagggctac gtgcaagagc ggaccatctt ctttaaggac 1800

gacggcaact acaagaccag ggccgaagtg aagtttgagg gcgacaccct ggtcaaccgg 1860

atcgagctga agggcatcga cttcaaagag gatggcaaca tcctgggcca caagctcgag 1920

tacaactaca acagccacaa cgtgtacatc atggccgaca agcagaagaa cggcatcaag 1980

gccaacttca agatccggca caacatcgag gacggcagcg ttcagctggc cgatcactac 2040

cagcagaaca cccctatcgg agatggccct gtgctgctcc ccgacaatca ctacctgagc 2100

acacagagcg ccctgagcaa ggaccccaac gagaagaggg atcacatggt gctgctggaa 2160

tttgtgaccg ccgcaggcat caccctcggc atggacgaac tgtacaaa 2208

<210> 173

<211> 1115

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide CD19scFv_CD8hingeTM_4-1BB_CD3zeta_EGFP-

T2A-NKG2D.YA_CD8hingeTM

<400> 173

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 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

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

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

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

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Ser Leu Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

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

485 490 495

Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile

500 505 510

Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser

515 520 525

Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe

530 535 540

Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr

545 550 555 560

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

565 570 575

Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln

580 585 590

Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala

595 600 605

Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys

610 615 620

Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu

625 630 635 640

Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys

645 650 655

Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly

660 665 670

Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp

675 680 685

Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala

690 695 700

Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu

705 710 715 720

Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

725 730 735

Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

740 745 750

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

755 760 765

Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Leu Ile Pro Leu Phe

770 775 780

Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys

785 790 795 800

Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp

805 810 815

Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn

820 825 830

Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys

835 840 845

Leu Val Lys Ser Ala His Trp Met Gly Leu Val His Ile Pro Thr Asn

850 855 860

Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu

865 870 875 880

Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser

885 890 895

Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys

900 905 910

Met Gln Arg Thr Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro

915 920 925

Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe

930 935 940

Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln

945 950 955 960

Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu

965 970 975

Lys Leu Val Lys Ser Ala His Trp Met Gly Leu Val His Ile Pro Thr

980 985 990

Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu

995 1000 1005

Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala

1010 1015 1020

Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

1025 1030 1035

Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro Ala Pro Arg

1040 1045 1050

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

1055 1060 1065

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

1070 1075 1080

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

1085 1090 1095

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

1100 1105 1110

Tyr Cys

1115

<210> 174

<211> 3345

<212> DNA

<213> Artificial sequence

<220>

<223> A synthetic polynucleotide encoding

CD19scFv_CD8hingeTM_4-1BB_CD3zeta_EGFP-T2A-NKG2D.YA_CD8hingeTM

<400> 174

atggcattgc ctgttacagc tctgctgctg cccctggctc tgcttctgca tgctgccaga 60

cctgacatcc agatgaccca gacaaccagc agcctgtctg ccagcctggg cgatagagtg 120

accatcagct gtagagccag ccaggacatc agcaagtacc tgaactggta tcagcagaaa 180

cccgacggca ccgtgaagct gctgatctac cacaccagca gactgcacag cggcgtgcca 240

agcagatttt ctggcagcgg ctctggcacc gactacagcc tgacaatcag caacctggaa 300

caagaggata tcgctaccta cttctgccag caaggcaaca ccctgcctta cacctttggc 360

ggaggcacca agctggaaat cacaggcggc ggaggaagcg gaggcggagg atctggtggt 420

ggtggatctg aagtgaaact gcaagagtct ggccctggcc tggtggcccc atctcaatct 480

ctgagcgtga cctgtaccgt cagcggagtg tccctgcctg attatggcgt gtcctggatc 540

cggcagcctc ctagaaaagg cctggaatgg ctgggcgtga tctggggcag cgagacaacc 600

tactacaaca gcgccctgaa gtcccggctg accatcatca aggacaactc caagagccag 660

gtgttcctga agatgaacag cctgcagacc gacgacaccg ccatctacta ttgcgccaag 720

cactactact acggcggcag ctacgccatg gattattggg gccagggcac cagcgtgacc 780

gtttcttcta ccaccacacc agctcctaga cctccaactc ctgctcctac aatcgccagc 840

cagcctctgt ctctgaggcc agaagcttgt agacctgctg caggcggagc cgtgcataca 900

agaggactgg atttcgcctg cgacatctac atctgggccc ctctggctgg aacatgtggc 960

gtgctgctgc tgagcctggt catcaccctg tactgcagcc tgaagcgggg cagaaagaag 1020

ctgctgtaca tctttaagca gcccttcatg cggcccgtgc agaccacaca agaggaagat 1080

ggctgctcct gcagattccc cgaggaagaa gaaggcggct gcgagctgag agtgaagttc 1140

agccgttctg ccgacgctcc cgcctataag cagggacaga accagctgta caacgagctg 1200

aacctgggga gaagagaaga gtacgacgtg ctggacaagc ggagaggcag agatcctgag 1260

atgggcggca agcccagacg gaagaatcct caagagggcc tgtataatga gctgcagaaa 1320

gacaagatgg ccgaggccta cagcgagatc ggaatgaagg gcgagcgcag aagaggcaag 1380

ggacacgatg gactgtacca gggcctgagc accgccacca aggataccta tgatgccctg 1440

cacatgcagg ccctgcctcc aagatcaggc tctggttctg gcagcggcag catggtgtct 1500

aaaggcgagg aactgttcac cggcgtggtg cccattctgg tggaactgga cggggatgtg 1560

aacggccaca agtttagcgt tagcggcgaa ggcgaagggg atgccacata cggaaagctg 1620

accctgaagt tcatctgcac caccggcaag ctgcctgtgc cttggcctac actggtcacc 1680

acactgacat acggcgtgca gtgctttagc agataccccg accatatgaa gcagcacgac 1740

ttcttcaagt ccgccatgcc tgagggctac gtgcaagagc ggaccatctt ctttaaggac 1800

gacggcaact acaagaccag ggccgaagtg aagtttgagg gcgacaccct ggtcaaccgg 1860

atcgagctga agggcatcga cttcaaagag gatggcaaca tcctgggcca caagctcgag 1920

tacaactaca acagccacaa cgtgtacatc atggccgaca agcagaagaa cggcatcaag 1980

gccaacttca agatccggca caacatcgag gacggcagcg ttcagctggc cgatcactac 2040

cagcagaaca cccctatcgg agatggccct gtgctgctcc ccgacaatca ctacctgagc 2100

acacagagcg ccctgagcaa ggaccccaac gagaagaggg atcacatggt gctgctggaa 2160

tttgtgaccg ccgcaggcat caccctcggc atggacgaac tgtacaaact cgaggcggc 2220

ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctagg 2280

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 2340

atcccactgt tcaaccagga ggttcagatt ccgctgaccg aaagctactg tggaccatgt 2400

cccaagaact ggatctgcta caaaaacaat tgttatcaat ttttcgacga aagcaagaat 2460

tggtacgaaa gccaggcttc atgcatgagc caaaatgcat ccctgctgaa ggtgtacagc 2520

aaggaggacc aggacctgct gaaacttgtc aaatccgctc actggatggg actggtccat 2580

attcctacaa atggatcctg gcagtgggag gatggctcta tcctgtctcc caatctcctg 2640

actatcatag agatgcagaa gggagattgt gcattgtatg ctagttcatt caagggatac 2700

attgaaaact gctcaacacc taatacctac atatgtatgc agcgcacagt gcagatccct 2760

ctgaccgaga gctactgtgg cccctgtcct aagaactgga tctgctacaa gaacaactgc 2820

taccagttct tcgacgagag caagaattgg tacgagagcc aggccagctg catgagccag 2880

aatgccagcc tgctgaaggt gtacagcaaa gaggaccagg atctgctgaa gctggtcaag 2940

agcgcccact ggatgggact cgtgcacatc cctacaaacg gcagctggca gtgggaggac 3000

ggctctatcc tgtctcctaa cctgctgacc atcatcgaga tgcagaaggg cgactgcgcc 3060

ctgtacgcca gcagctttaa gggctacatc gagaactgca gcacccctaa cacctacatc 3120

tgtatgcagc ggaccgtgac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 3180

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 3240

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 3300

acttgtgggg tccttctcct gtcactggtt atcacccttt actgc 3345

<210> 175

<211> 161

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide FLAG_6xHis_mutIL2

<400> 175

Gly Ser Ser Gly Ser Ser Asp Tyr Lys Asp Asp Asp Asp Lys His His

1 5 10 15

His His His His His Gly Ser Ser Gly Ser Ser Ala Pro Thr Ser

20 25 30

Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp

35 40 45

Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu

50 55 60

Thr Ala Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu

65 70 75 80

Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu

85 90 95

Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp

100 105 110

Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu

115 120 125

Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu

130 135 140

Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu

145 150 155 160

Th

<210> 176

<211> 483

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding FLAG_6xHis_mutIL2

<400> 176

ggaagtagcg gtagtagtga ttacaaggac gatgacgaca agcaccacca tcatcatcat 60

caccacggta gcagcggcag cagtgccccc acctctagca gcacaaagaa gacccagctg 120

caactggaac acctcctgct ggacctgcag atgatcctga acggcatcaa caactacaag 180

aaccccaagc tgaccgccat gctgaccaaa aagttttaca tgcccaagaa ggccaccgag 240

cttaaacacc tgcaatgcct tgaggaggag ctgaagcccc tggaggaggt actgaacctg 300

gccccagagca agaactttca tctgaggccc agggacctga ttagcaacat caacgtgatc 360

gtgttggagt tgaagggcag cgagaccacg ttcatgtgcg agtacgccga cgagacggcc 420

accatagtgg agtttcttaa caggtggatc accttctcac agtctatcat cagcaccctg 480

acc 483

<210> 177

<211> 343

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP2.R80W_FLAG_6xHis_mutIL2

<400> 177

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 Gly Thr Gly Ser Ser Gly Ser Ser Asp Tyr Lys Asp

180 185 190

Asp Asp Asp Lys His His His His His His His Gly Ser Ser Gly

195 200 205

Ser Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu

210 215 220

Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn

225 230 235 240

Tyr Lys Asn Pro Lys Leu Thr Ala Met Leu Thr Lys Lys Phe Tyr Met

245 250 255

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

260 265 270

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe

275 280 285

His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu

290 295 300

Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu

305 310 315 320

Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln

325 330 335

Ser Ile Ile Ser Thr Leu Thr

340

<210> 178

<211> 1029

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding ULBP2.R80W_FLAG_6xHis_mutIL2

<400> 178

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 gcgatgtcat tccactattt ctcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

ggtaccggta gtagcggtag tagtgattac aaggacgatg acgacaagca caccatcat 600

catcatcacc acggtagcag cggcagcagt gcccccacct ctagcagcac aaagaagacc 660

cagctgcaac tggaacacct cctgctggac ctgcagatga tcctgaacgg catcaacaac 720

tacaagaacc ccaagctgac cgccatgctg accaaaaagt tttacatgcc caagaaggcc 780

accgagctta aacacctgca atgccttgag gaggagctga agcccctgga ggaggtactg 840

aacctggccc agagcaagaa ctttcatctg aggcccaggg acctgattag caacatcaac 900

gtgatcgtgt tggagttgaa gggcagcgag accacgttca tgtgcgagta cgccgacgag 960

acggccacca tagtggagtt tcttaacagg tggatcacct tctcacagtc tatcatcagc 1020

accctgacc 1029

<210> 179

<211> 343

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP2.S2_FLAG_6xHis_mutIL2

<400> 179

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 Arg Ile 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 Gly Thr Gly Ser Ser Gly Ser Ser Asp Tyr Lys Asp

180 185 190

Asp Asp Asp Lys His His His His His His His Gly Ser Ser Gly

195 200 205

Ser Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu

210 215 220

Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn

225 230 235 240

Tyr Lys Asn Pro Lys Leu Thr Ala Met Leu Thr Lys Lys Phe Tyr Met

245 250 255

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

260 265 270

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe

275 280 285

His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu

290 295 300

Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu

305 310 315 320

Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln

325 330 335

Ser Ile Ile Ser Thr Leu Thr

340

<210> 180

<211> 1029

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding ULBP2.S2_FLAG_6xHis_mutIL2

<400> 180

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 gcgactctga tgaggatttg gtcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

ggtaccggta gtagcggtag tagtgattac aaggacgatg acgacaagca caccatcat 600

catcatcacc acggtagcag cggcagcagt gcccccacct ctagcagcac aaagaagacc 660

cagctgcaac tggaacacct cctgctggac ctgcagatga tcctgaacgg catcaacaac 720

tacaagaacc ccaagctgac cgccatgctg accaaaaagt tttacatgcc caagaaggcc 780

accgagctta aacacctgca atgccttgag gaggagctga agcccctgga ggaggtactg 840

aacctggccc agagcaagaa ctttcatctg aggcccaggg acctgattag caacatcaac 900

gtgatcgtgt tggagttgaa gggcagcgag accacgttca tgtgcgagta cgccgacgag 960

acggccacca tagtggagtt tcttaacagg tggatcacct tctcacagtc tatcatcagc 1020

accctgacc 1029

<210> 181

<211> 412

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP2.R80W_Fc1

<400> 181

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 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

180 185 190

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

195 200 205

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

210 215 220

Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe

225 230 235 240

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

245 250 255

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

260 265 270

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

275 280 285

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

290 295 300

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

305 310 315 320

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

325 330 335

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

340 345 350

Glu Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

355 360 365

Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

370 375 380

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

385 390 395 400

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

405 410

<210> 182

<211> 1236

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding ULBP2.R80W_Fc1

<400> 182

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 gcgatgtcat tccactattt ctcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 600

ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 660

acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 720

aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 780

tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 840

gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 900

atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 960

gacgagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 1020

gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactatga cactaccccg 1080

cccgtgctgg attccgacgg aagcttcttc ctgtactccg acctgaccgt ggacaagtcg 1140

agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 1200

tacacccaga agtcactgag cctctccccc ggaaag 1236

<210> 183

<211> 368

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide Fc2_mutIL2

<400> 183

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

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

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser

225 230 235 240

Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu

245 250 255

Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr

260 265 270

Ala Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu

275 280 285

Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val

290 295 300

Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu

305 310 315 320

Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr

325 330 335

Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe

340 345 350

Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 184

<211> 1104

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding Fc2_mutIL2

<400> 184

gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60

ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120

acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180

aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240

tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300

gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360

atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420

aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480

gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540

cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600

agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660

tacacccaga agtcactgag cctctccccc ggaggaggtg gcagcgcccc cacctctagc 720

agcacaaaga agacccagct gcaactggaa cacctcctgc tggacctgca gatgatcctg 780

aacggcatca acaactacaa gaaccccaag ctgaccgcca tgctgaccaa aaagttttac 840

atgcccaaga aggccaccga gcttaaacac ctgcaatgcc ttgaggagga gctgaagccc 900

ctggaggagg tactgaacct ggccgagc aagaactttc atctgaggcc cagggacctg 960

attagcaaca tcaacgtgat cgtgttggag ttgaagggca gcgagaccac gttcatgtgc 1020

gagtacgccg acgagacggc caccatagtg gagtttctta acaggtggat caccttctca 1080

cagtctatca tcagcaccct gacc 1104

<210> 185

<211> 349

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide Fc2_mutIL15

<400> 185

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

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

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Asn Trp Val Asn Val

225 230 235 240

Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile

245 250 255

Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val

260 265 270

Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu

275 280 285

Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu

290 295 300

Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys

305 310 315 320

Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln

325 330 335

Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

340 345

<210> 186

<211> 1047

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding Fc2_mutIL15

<400> 186

gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60

ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120

acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180

aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240

tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300

gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360

atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420

aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480

gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540

cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600

agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660

tacacccaga agtcactgag cctctccccc ggaggaggtg gcagcaattg ggtgaacgtg 720

atttctgact tgaagaaaat cgaggacttg attcaaagta tgcacataga cgcaacgctc 780

tatactgaga gtgatgttca tccctcttgt aaagttaccg ctatgaagtg tttcttgctc 840

gaactccaag ttatcagtct ggagagcggt gatgcctcca tacacgatac agtcgaaaac 900

cttatcattc tcgcaaataa ctcattgagt agcaatggca atgttacaga gtctgggtgt 960

aaggaatgcg aagaacttga agagaaaaac ataaaggagt tcctccaatc attcgtgcat 1020

attgtccaga tgtttatcaa cactagc 1047

<210> 187

<211> 412

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP2.S2_Fc1

<400> 187

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 Arg Ile 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 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

180 185 190

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

195 200 205

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

210 215 220

Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe

225 230 235 240

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

245 250 255

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

260 265 270

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

275 280 285

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

290 295 300

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

305 310 315 320

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

325 330 335

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

340 345 350

Glu Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

355 360 365

Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

370 375 380

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

385 390 395 400

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

405 410

<210> 188

<211> 1236

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding ULBP2.S2_Fc1

<400> 188

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 gcgactctga tgaggatttg gtcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 600

ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 660

acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 720

aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 780

tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 840

gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 900

atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 960

gacgagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 1020

gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactatga cactaccccg 1080

cccgtgctgg attccgacgg aagcttcttc ctgtactccg acctgaccgt ggacaagtcg 1140

agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 1200

tacacccaga agtcactgag cctctccccc ggaaag 1236

<210> 189

<211> 412

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide ULBP2.S3_Fc1

<400> 189

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 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

180 185 190

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

195 200 205

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

210 215 220

Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe

225 230 235 240

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

245 250 255

Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

260 265 270

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

275 280 285

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

290 295 300

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

305 310 315 320

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

325 330 335

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

340 345 350

Glu Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

355 360 365

Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

370 375 380

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

385 390 395 400

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

405 410

<210> 190

<211> 1236

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding ULBP2.S3_Fc1

<400> 190

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 gcgactaagc tttatctttg gtcgatggga 480

gactgcatcg gttggctgga agatttcctc atgggtatgg actccacttt ggagccatcg 540

gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 600

ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 660

acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 720

aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 780

tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 840

gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 900

atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 960

gacgagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 1020

gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactatga cactaccccg 1080

cccgtgctgg attccgacgg aagcttcttc ctgtactccg acctgaccgt ggacaagtcg 1140

agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 1200

tacacccaga agtcactgag cctctccccc ggaaag 1236

<210> 191

<211> 368

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide Fc2_wtIL2 (wild type)

<400> 191

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

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

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser

225 230 235 240

Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu

245 250 255

Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr

260 265 270

Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu

275 280 285

Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val

290 295 300

Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu

305 310 315 320

Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr

325 330 335

Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe

340 345 350

Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 192

<211> 1104

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding Fc2_wtIL2

<400> 192

gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60

ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120

acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180

aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240

tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300

gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360

atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420

aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480

gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540

cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600

agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660

tacacccaga agtcactgag cctctccccc ggaggaggtg gcagcgctcc aacctctagc 720

tctacgaaga aaactcaact ccagcttgag catttgcttt tggaccttca aatgatattg 780

aacgggatta acaattacaa gaaccccaaa ctgacgcgaa tgcttacatt caaattttac 840

atgccaaaga aggccaccga gctgaaacac ttgcaatgtc ttgaggaaga gctgaagcca 900

ttggaggagg tcctgaattt ggctcaaagt aagaacttcc acctgcggcc aagggatctc 960

ataagcaaca taaatgtcat tgtattggaa ctgaaaggga gcgaaaccac ctttatgtgc 1020

gagtatgcag atgaaactgc aaccatagtc gagttcctca atcgctggat aactttcgcg 1080

cagtcaatta tatcaacact cacc 1104

<210> 193

<211> 368

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide Fc2_IL21.wt (wild type)

<400> 193

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

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

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gln Gly Gln Asp Arg

225 230 235 240

His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys

245 250 255

Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp

260 265 270

Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala

275 280 285

Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val

290 295 300

Leu Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg

305 310 315 320

Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys

325 330 335

Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys

340 345 350

Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser

355 360 365

<210> 194

<211> 1104

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding Fc2_IL21.wt (wild type)

<400> 194

gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60

ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120

acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180

aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240

tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300

gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360

atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420

aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480

gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540

cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600

agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660

tacacccaga agtcactgag cctctccccc ggaggaggtg gcagccaggg tcaagaccgc 720

catatgatcc gaatgcgaca gctcattgat attgtcgatc aattgaaaaa ttacgtgaat 780

gatcttgtac cggagttcct ccccgcaccg gaggacgttg aaacgaattg tgagtggtca 840

gcattttctt gctttcagaa ggctcaactc aagagtgcaa acacgggtaa caacgagcgc 900

attatcaatg ttctcatcaa aaagctgaaa cgaaaaccgc ctagcaccaa cgcaggcaga 960

cgacagaagc accggctcac gtgcccaagt tgcgattctt atgagaaaaa gccaccgaaa 1020

gaattcctgg agcggttcaa gtccctcttg cagaaaatga ttcatcagca tctctccagc 1080

aggacacacg gctccgagga ctcc 1104

<210> 195

<211> 368

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide Fc2_IL21.D18A

<400> 195

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

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

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gln Gly Gln Asp Arg

225 230 235 240

His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Ala Gln Leu Lys

245 250 255

Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp

260 265 270

Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala

275 280 285

Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val

290 295 300

Leu Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg

305 310 315 320

Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys

325 330 335

Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys

340 345 350

Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser

355 360 365

<210> 196

<211> 1104

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding Fc2_IL21.D18A

<400> 196

gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60

ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120

acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180

aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240

tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300

gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360

atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420

aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480

gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540

cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600

agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660

tacacccaga agtcactgag cctctccccc ggaggaggtg gcagccaggg tcaagaccgc 720

catatgatcc gaatgcgaca gctcattgat attgtcgcac aattgaaaaa ttacgtgaat 780

gatcttgtac cggagttcct ccccgcaccg gaggacgttg aaacgaattg tgagtggtca 840

gcattttctt gctttcagaa ggctcaactc aagagtgcaa acacgggtaa caacgagcgc 900

attatcaatg ttctcatcaa aaagctgaaa cgaaaaccgc ctagcaccaa cgcaggcaga 960

cgacagaagc accggctcac gtgcccaagt tgcgattctt atgagaaaaa gccaccgaaa 1020

gaattcctgg agcggttcaa gtccctcttg cagaaaatga ttcatcagca tctctccagc 1080

aggacacacg gctccgagga ctcc 1104

<210> 197

<211> 368

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide Fc2_IL21.E109R

<400> 197

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

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

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gln Gly Gln Asp Arg

225 230 235 240

His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys

245 250 255

Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp

260 265 270

Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala

275 280 285

Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val

290 295 300

Leu Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg

305 310 315 320

Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys

325 330 335

Lys Pro Pro Lys Glu Phe Leu Arg Arg Phe Lys Ser Leu Leu Gln Lys

340 345 350

Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser

355 360 365

<210> 198

<211> 1104

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding Fc2_IL21.E109R

<400> 198

gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60

ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120

acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180

aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240

tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300

gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360

atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420

aaggagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480

gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540

cccgtgctga agtccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600

agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660

tacacccaga agtcactgag cctctccccc ggaggaggtg gcagccaggg tcaagaccgc 720

catatgatcc gaatgcgaca gctcattgat attgtcgatc aattgaaaaa ttacgtgaat 780

gatcttgtac cggagttcct ccccgcaccg gaggacgttg aaacgaattg tgagtggtca 840

gcattttctt gctttcagaa ggctcaactc aagagtgcaa acacgggtaa caacgagcgc 900

attatcaatg ttctcatcaa aaagctgaaa cgaaaaccgc ctagcaccaa cgcaggcaga 960

cgacagaagc accggctcac gtgcccaagt tgcgattctt atgagaaaaa gccaccgaaa 1020

gaattcctgc ggcggttcaa gtccctcttg cagaaaatga ttcatcagca tctctccagc 1080

aggacacacg gctccgagga ctcc 1104

<210> 199

<211> 368

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide Fc2_IL21.D18A.E109R

<400> 199

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

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

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gln Gly Gln Asp Arg

225 230 235 240

His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Ala Gln Leu Lys

245 250 255

Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp

260 265 270

Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala

275 280 285

Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val

290 295 300

Leu Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg

305 310 315 320

Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys

325 330 335

Lys Pro Pro Lys Glu Phe Leu Arg Arg Phe Lys Ser Leu Leu Gln Lys

340 345 350

Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser

355 360 365

<210> 200

<211> 1104

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding Fc2_IL21.D18A.E109R

<400> 200

gagcccaaaa gctgcgacaa gactcacact tgtccgccgt gccccgcccc cgaactgctg 60

ggtggcccct ccgtgttcct gttcccgcct aagcctaagg acacccttat gatcagccgc 120

acccctgaag tgacctgtgt cgtcgtggca gtgtcacacg aggacccgga ggtcaagttc 180

aattggtacg tggacggcgt ggaagtgcat aacgcaaaga ccaagcctcg ggaggaacag 240

tacgcctcga cctaccgcgt ggtgtcagtc ctgactgtgc tgcaccagga ctggctgaac 300

gggaaggagt acaagtgcaa agtgtcgaac aaggccctgc cggctccaat tgaaaagacc 360

atcagcaagg ccaagggcca gccaagggaa ccacaggtgt acaccctccc tccttcccgg 420

gacgagctga ccaaaaacca agtgtccctg acttgccttg tgaaggggtt ctacccttct 480

gacattgccg tcgaatggga atcgaacgga cagcctgaaa acaactataa gactaccccg 540

cccgtgctgg attccgacgg aagcttcttc ctgtactcca agctgaccgt ggacaagtcg 600

agatggcagc agggaaatgt gttcagctgc tccgtgatgc atgaggcgct gcacaaccac 660

tacacccaga agtcactgag cctctccccc ggaggaggtg gcagccaggg tcaagaccgc 720

catatgatcc gaatgcgaca gctcattgat attgtcgcac aattgaaaaa ttacgtgaat 780

gatcttgtac cggagttcct ccccgcaccg gaggacgttg aaacgaattg tgagtggtca 840

gcattttctt gctttcagaa ggctcaactc aagagtgcaa acacgggtaa caacgagcgc 900

attatcaatg ttctcatcaa aaagctgaaa cgaaaaccgc ctagcaccaa cgcaggcaga 960

cgacagaagc accggctcac gtgcccaagt tgcgattctt atgagaaaaa gccaccgaaa 1020

gaattcctgc ggcggttcaa gtccctcttg cagaaaatga ttcatcagca tctctccagc 1080

aggacacacg gctccgagga ctcc 1104

<210> 201

<211> 233

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide Fc2

<400> 201

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 Ala 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 Ala 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 Lys 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 Lys 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

225 230

<210> 202

<211> 699

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide encoding Fc2

<400> 202

atggacccca aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 60

ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 120

cgcacccctg aagtgacctg tgtcgtcgtg gcagtgtcac acgaggaccc ggaggtcaag 180

ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 240

cagtacgcct cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 300

aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 360

accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 420

cggaaggagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 480

tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 540

ccgcccgtgc tgaagtccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 600

tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 660

cactacaccc agaagtcact gagcctctcc cccggaaag 699

<---

Claims (6)

1. A non-naturally occurring modified NKG2D receptor that binds to non-naturally occurring, modified α1-α2 domains of NKG2D ligands but not to natural NKG2D ligands comprising SEQ ID NO: 4, wherein said non-naturally occurring modified NKG2D receptor comprises an amino acid sequence of any one of SEQ ID NOs: 18-35. 2. The non-naturally occurring modified NKG2D receptor of claim 1, wherein the modified NKG2D receptor is attached to a mammalian T cell, macrophage, or NK cell, wherein the modified NKG2D receptor has an attached intracellular costimulatory domain that does not contain an active CD3-zeta domain. 3. The modified NKG2D receptor of claim 2, wherein the costimulatory domain is a 4-1BB costimulatory domain. 4. A method for delivering a payload to a mammalian T cell, macrophage or NK cell, wherein the method comprises delivering to a mammalian T cell, macrophage or NK cell comprising a modified NKG2D receptor according to any one of claims 1-3, a payload attached to a non-natural modified α1-α2 domain of an NKG2D ligand that binds to the modified NKG2D receptor. 5. The method of claim 4, wherein the payload activates said cell or transmits a signal to it via corresponding receptor subunits on or within the cell. 6. The method according to claim 4 or 5, wherein the payload is a cytokine, chemokine, lymphokine or cytotoxin.

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