KR20190019091A - The insertable variable fragment of the antibody and the modified A1-A2 domain of the NKG2D ligand - Google Patents

Abstract

The present application relates generally to the production of polypeptides having specific antigen-binding properties of the Fv domain, such as insertable variable fragments of antibodies, and modified α1-α2 domains of NKG2D ligands. The present application also relates to a modified α1-α2 domain of a NKG2D ligand attached to a polypeptide, in some embodiments an antibody or a fragment of an antibody. The present application also relates to antigen-binding peptides derived from light and heavy chain antibody variable domains, which contain two linker sites and a split variable domain.

Description

The insertable variable fragment of the antibody and the modified A1-A2 domain of the NKG2D ligand

The present application relates generally to the production of polypeptides having specific antigen-binding properties of the Fv domain, such as insertable variable fragments of antibodies, and modified α1-α2 domains of NKG2D ligands.

The antibody (Ab) (Figure 1), also known as immunoglobulin (Ig) in many mammals, including humans, is a large, Y-shaped (Charles Janeway (2001), Immunobiology (5th ed.), Chapter 3. Garland Publishing, ISBN 0-8153-3642-X. Antibodies recognize a unique portion of an exogenous target referred to as an antigen. Each end of the two arms of the " Y " of the antibody contains a paratope (lock-like structure) specific for the antigen binding site, or for a particular epitope of the antigen (similarly similar to a key) These two structures are precisely combined together. Using such a binding mechanism, the antibody can tag microorganisms or infected cells for attack by other parts of the immune system, or can block the portion of the microorganism that is essential for the infestation and survival of the microorganism, for example, . The production of antibodies is a major function of the humoral, or "adaptive", immune system. Antibodies are secreted by plasma cells. Indeed, antibodies can occur in two physical forms, a soluble form secreted from cells, and a membrane-bound form attached to the surface of B cells through the " stem "

Antibodies are glycoproteins belonging to the immunoglobulin superfamily and usually consist of basic structural units - each having two large heavy chains and two small light chains. There are several different types of antibodies that are grouped into different isotypes based on different types of antibody heavy chains, and heavy chains they have. Five different antibody homologs are known in mammals (Market E, Papavasiliou FN (October 2003). "V (D) J recombination and the evolution of the adaptive immune system". PLoS Biol . 1 (1): E16. doi: 10.1371 / journal.pbio.0000016. PMC 212695. PMID 14551913). The general structure of all antibodies is very similar, but the small portion at the end of each of the Y-shaped proteins is highly variable, resulting in the presence of millions of antibodies with slightly different end structures, or antigen-binding sites. Such a region is known as a hypervariable or variable region. Each of these natural variants can bind to different antigens. This huge diversity of antibodies is adapted to a wide range of the immune system is equivalent to an antigen, and recognizes (Hozumi N, Tonegawa S (1976 ). "Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions". Proc. Natl. Acad. . Sci USA 73 (10): . 3628-3632 doi:.. 10.1073 / pnas.73.10.3628 PMC 431171. PMID 824647.)

The natural " Y " -shaped Ig molecule consists of four polypeptide chains; Two identical heavy chains connected by a disulfide bond and two identical light chains (Figure 1). Each heavy chain has two main parts, an invariant part (CH) and a variable part (VH). Invariants are essentially the same for all antibodies of the same isotype but different for different isotype antibodies. The light chain also has two consecutive domains: a smaller constant region (CL) and a variable region (VL) (Woof J, Burton D (2004). "Human antibody-Fc receptor interactions illuminated by crystal structures." Nat Rev Immunol 4 (2): 89-99, doi: 10.1038 / nril266, PMID 15040582).

Some parts of the antibody have the same function. Each of the two arms of Y contains, for example, a site capable of binding to an antigen, and thus recognizes a specific foreign body. The site of such an antibody is referred to as Fv (fragment, variable) portion. It consists of a variable domain (V _H ) from the heavy chain of the antibody and a variable domain (V _L ) from the light chain (Hochman J, Inbar D, Givol D (1973) variable portions of heavy and light chains. Biochemistry 12 (6): 1130-1135, doi: 10.1021 / bi00730a018.PMID 4569769). The paratope is shaped at one end of the Fv and is the site for antigen binding. It consists of variable loops of the [beta] -string, 3 of which are on V _L and V _H and responsible for binding to the antigen (Fig. 2). Shows these six loop as a complementarity determining region (CDR) (North B, Lehmann A, Dunbrack RL (2010). "A new clustering of antibody CDR loop conformations". J Mol Biol 406 (2): 228-256. doi: 10.1016 / j.jmb.2010.10.030. PMC 3065967. PMID 21035459).

Useful polypeptides having specific antigen binding function can be derived from the CDRs of the variable regions of the antibody. These two antibody variable domains (one from the light chain with three CDRs each (V _L ) and one from the heavy chain (V _H )) can be generated using either a single, short linker peptide of 10 to about 25 amino acids (Fig. 3) (Bird, RE, Hardman, KD, Jacobson, JW < RTI ID = 0.0 > Single-chain antigen-binding proteins, Science 242 , 423-426 (1988), Johnson, S., Kaufman, BM, Lee, SM, Lee, T., Pope, SH, Riordan, GS, and Whitlow, M. Huston, JS, Levinson, D, Mudgett-Hunter, M, Tai, MS, Novotny, J, Margolies, MN, Ridge, R., Bruccoleri, RE, News, E., Crea, R., and Opperman, H (1988) Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli PNAS 85: 5879-5883).

The linker is usually rich in glycine for flexible, and that not only rich in serine, threonine, or charged amino acids for solubility, and it is possible to connect the C- terminal of the N- terminus of the V _H and V _L, and Conversely, it is possible. These proteins retain the intrinsic immunoglobulin specificity, despite the elimination of invariants and introduction of a single linker. This format allows a person skilled in the art of recombinant DNA technology to genetically fuse a linear scFv at the N- or C-terminus of the parent protein to confer antigen binding properties of the scFv to the parent protein. There are a number of other proposed or generated sequences of multivalent and tandem scFv sites, but all have importantly all at least two spatially remote ends as described below (Fig. 4) (Le Gall, F .; (1999). "Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding." FEBS Letters 453 (1): 164-168 doi: 10.1016 / S0014-5793 (99) 00713-9, PMID 10403395).

Summary of the Invention

The disclosure relates to a modified α1-α2 domain of a NKG2D ligand attached to a polypeptide, in some embodiments an antibody or a fragment of an antibody. In some embodiments, the disclosure relates to antigen-binding peptides derived from light and heavy chain antibody variable domains that contain two linker sites and a split variable domain.

The patent or application file contains at least one colored drawing. A copy of this patent or patent application publication with colored drawing (s) will be provided by the office upon request and payment of the required fee.
FIG. A sketch of a typical mammalian antibody showing its Y-shaped structure and structural components.
FIG. Sketch of the Fv region structure of a natural mammalian antibody that shows three unlabeled loops of three labeled (complementarity determining regions) CDRs and V _L CDRs of V _H , forming a paratope or antigen binding site.
FIG. Sketch of two possible structures of a single-chain variable fragment (scFv), with an antigen-binding site comprising the N-terminus and the C-terminus on the left. A single linker moiety, or linker peptide, in each scFv is indicated as an arrow.
FIG. (scFv) multivalent single-chain variable fragment. Structure of the divalent (top) and tridentate (bottom) scFv, tandem (left) and dimerization / trimerization format (right). Each having two or more spatially free ends.
5A and 5B . Insertable variable fragment, illustration of iFv. Insertable variable fragment, illustration of iFv. Figure 5a shows the structure of the variable light (VL) and variable heavy (VH) domains from FGFR3-binding antibodies that represent the domain topology of the iFv format. The gray arrows represent two linker sites (LR), one and only one of which is traditionally used to link the ends of VL and VH to generate scFv. The LR with the dashed line connects the C-terminus of VL to the N-terminus of VH (shown behind the molecule). The LR with the solid line border connects the C-terminus of VH to the N-terminus of VL. The segment of the segmented VL domain is labeled Nt and Ct as described in the text. VL is divided into an N-terminal segment (VLN) and a C-terminal segment (VLC) as a result of the non-natural pairing of the N- and C-termini between strand 1 (S1) and strand 2 (S2). The six CDRs of VL and VH are displayed as loops in the upper part of the figure. Fig. 5B shows a schematic diagram of a domain layout for inserting iFv into loop 1 (L1) of MICA-? 3 with or without a spacer site SR. iFv may also be inserted similarly to loop 2 (L2) and / or loop 3 (L3).
FIG. Titration curve for modified sMICA molecules binding to FGFR3 coated wells. The bound sMICA was detected by ELISA using NKG2D-Fc to ensure bispecific activity. Both versions of the inserted variable fragment (MICA-a3-iFv.1 and MICA-a3-iFv.2) bound to FGFR3 similar to the C-terminal fusion of scFv (MICA-scFv).
Figures 7a and 7b . Thermal stability of MICA-α3-iFv.2. ELISA Titration Curve of MICA-scFv (FIG. 7A) or MICA-α3-iFv.2 (FIG. 7B) binding to FGFR3-coated wells after exposure to the indicated temperature (Celsius temperature) for 1 hour. MICA-α3-iFv showed strong binding to FGFR3 after exposure to 80 ° C, while MICA-scFv lost significant activity after exposure to 70 ° C.
Figure 8 . NK-mediated target cell lysis assay. Effector: At a target ratio of 15: 1, NKL effector cells were co-incubated with Kaltsin-loaded, FGFR3-expressing P815 target cells. Increased concentrations of negative control MICA (sMICA) did not have an effect on target cell lysis, while the indicated FGFR3-linked MICA-a3-iFv mutants stimulated target cell lysis. Compared to MICA-scFv, all of the MICA-a3-iFv variants induced greater target cell lysis.
Figures 9a and 9b . Target binding and cytolytic activity of CD20-specific sMICA mutants. MICA- [alpha] 3-iFv.3 exhibited titratable binding (Fig. 9a) to CD20-coated wells in ELISA and also enhanced NK-mediated cell lysis of CD20-expressing Ramos cells (Fig. 9b) . In the experiment shown in Figure 9b, NKL effector cells were co-cultured with callus-loaded CD20-expressing Ramos cells at increasing concentrations of effector: target ratio of 15: 1, and negative control (sMICA) or MICA-a3-iFv.3 - < / RTI > incubation.
10. Titration curve for NKG2DL-a3-iFv.2 protein binding to FGFR3-coated wells. The bound protein was detected by ELISA using NKG2D-Fc to ensure bispecific activity. All versions of the tested NKG2DL-α3-iFv.2 proteins (OMCP, ULBP1, 2, 3, 4, 6) similarly bound to FGFR3.
11. NK-mediated target cell lysis assay. Effector: At a target ratio of 15: 1, NKL effector cells were co-incubated with Kaltsin-loaded, FGFR3-expressing P815 target cells. Increased concentrations of negative control MICA (sMICA) did not have an effect on target cell lysis, while the indicated NKG2DL-a3-iFv.2 proteins stimulated target cell lysis.
12A and 12B . Structure-directed mutagenesis of the α1-α2 domain of MICA for enhanced NKG2D affinity. Figure 12a shows the structure (PDB 1HYR) of the α1-α2 domain of MICA with an NKG2D-binding surface mapped to 57 residues in the dark gray color. Figure 12b shows six positions identified as key sites for the NKG2D affinity mutation. The wild-type amino acid residue is labeled and its side chain is represented by a dark gray color.
13A and 13B . Competitive ELISA for NKG2D-Fc to rank α1-α2 variant affinity. Figure 13a shows titration data for a panel of alpha 1-alpha2 affinity variants (15-18), wild-type (WT), or WED soluble MICA protein (inhibiting human NKG2D-Fc binding to plate-coated MICA) . Figure 13b shows the same set of proteins in Figure 13a titrated for mouse NKG2D-Fc. In both assays, variants 15, 16, 17 and 18 show significantly lower IC ₅₀ values than in both WT and WED proteins. The equilibrium IC ₅₀ values are shown in Table 3.
14. Analysis of binding and dissociation rates for α1-α2 variants binding to NKG2D, as measured by biolayer interferometry on Octet instruments. Reaction rate trajectories for α1-α2 mutants on panels. The bond and dissociation images were fitted using a single exponential 1: 1 bond equation and the on- and off-rate constants from the pits are shown in Table 3.
15. NK-mediated target cell death assay for [alpha] 1- [alpha] 2 variants targeting FGFR3-expressing target cells. Effector: At a target ratio of 15: 1, NKL effector cells were co-incubated with Kaltsin-loaded, FGFR3-expressing P815 target cells. Increased concentrations of negative control MICA (sMICA) did not have an effect on target cell lysis, while the indicated α1-α2 mutants stimulated target cell lysis. For WT and WED-MICA, mutants 16, 17 and 18 showed significantly increased mortality at low concentrations.
Fig. Analysis of binding and dissociation rates for α1-α2 variants 20, 25 and 48 binding to NKG2D, as determined by biological shielding interferometry on Octet instruments. The bond and dissociation phases were fitted using a single exponential 1: 1 bond equation and the on-and off-rate constants resulting from the pits are shown in Table 5.
Fig. NK-mediated target cell death, calcein-based assays for α1-α2 variants 16, 25 and WED targeting FGFR3-expressing P815 target cells.
Fig. Protein sequence alignment of the [alpha] 1- [alpha] 2 domain from MICA and ULBP (SEQ ID NO: 77-83). Gray highlighted amino acids were selected for NNK mutagenesis in ULBP2 (60 amino acids) and ULBP3 (36 amino acids). The black highlighted residues were identified as identified and identified key positions as mutations controlling binding affinity for NKG2D (Tables 6 and 7).
19a and 19b . ELISA titration of ULBP variants binding to NKG2D. Figure 19A shows an experiment in which the ULBP2 variant displayed on the phage was titrated against NKG2D and relative binding affinity measured for congenital ULBP2 (WT, black circle). Figure 19b shows an experiment in which the ULBP3 variant displayed on the phage was titrated against NKG2D and relative binding affinity measured for congenital ULBP3 (WT, black circle).
20a- d . Conjugates (WT), modified variant WED, 25 or 48 a1-a2 domain fusion to the heavy chain (Figure 20a) or light chain (Figure 20b) of FGFR3-specific antibodies affected NK-dependent target cell death . Fusion of variants 25 and 48 to the heavy chain (Figure 20c) or light chain (Figure 20d) significantly enhanced the extent of killing and killing efficacy compared to WED variants and congenital (WT) fusions.
21a- c . Fusion of the variant 25 alpha 1-alpha 2 domain to the heavy or light chain of antibodies targeting human EGFR (Figure 21a), HER2 (Figure 21b) or PDLl (Figure 21c) enhanced NKL cell-mediated target cell apoptosis, respectively. (Figure 21a), trastuzumab (Figure 21b) and anti-PDL1 (Figure 21c), respectively.
22A and 22B . In vivo variants 25 [alpha] -la2 domain Tritusum-based fusion of NK cells. Parent trastuzumab, trastuzumab HC_25 fusions and trastuzumab LC_25 fusions were conjugated with Alexa Flour. Groups of 3 C57BL / 6 mice were injected with a single dose of 100 [mu] g of parent, HC fusion or LC fusion; Blood was drawn from each animal at the times indicated for plasma PK ELISA (Figure 22a) and flow cytometry analysis of fluorescently labeled molecules bound to peripheral NK cells (Figure 22b).
23a- c . (Fig. 23A), trastuzumab-based HC (Fig. 23B) and trastuzumab-LC (Fig. 23C) fusions for mutant 25 were administered The resulting anti-drug antibody. Control (Ctrl) plasma was from untreated mice with any antibody containing system.
24A and 24B . A variant 25 alpha 1-alpha 2 domain for trastuzumab-HC and -LC as described in Example 7 and Figures 21-22, which binds to both the parent antibody (Figure 24a) and the alpha 1- alpha 2 domain (Figure 24b) An antibody produced in an animal to which a fusion has been administered.
25. Anti-tumor activity of anti-PDL1 fusion against variant 25. Winter MC38 tumors were subcutaneously transplanted in C57BL / 6 mice and tumors were grown to an average of 100 ㎣ before initiation of treatment. At the onset of treatment, four cohorts of 10 mice per group on days 1, 4 and 7 were injected intraperitoneally with vehicle, 100 μg ip, parent anti-PDL1 (300 ug iv) or anti-PDL1 HC_25 fusion 300 ug iv). Tumor volume (㎣) was measured in each animal at the indicated times.
26A and 26B . Fusion of the ULBP2 and ULBP3 [alpha] 1- [alpha] 2 domain variants to the heavy chain of the HER2-specific antibody showed enhanced NKG2D binding affinity. The modified ULBP2 alpha 1-alpha 2 domain variants R80W (SEQ ID NO: 84) and V151D (SEQ ID NO: 85) exhibited enhanced NKG2D binding relative to native ULBP2 (SEQ ID NO: 16) fusion (WT) 26A). The modified ULBP3 variant R162G (SEQ ID NO: 86) exhibited enhanced NKG2D binding relative to native ULBP3 (SEQ ID NO: 17) fusion (WT) (Fig. 26B).
Figures 27a and 27b . Fusion of the ULBP2 and ULBP3 [alpha] 1- [alpha] 2 domain variants to the heavy chain of HER2-specific antibodies showed specific lysis of SKBR3 target cells by NKL cells. The modified ULBP2 a1-a2 domain variants R80W (SEQ ID NO: 84) and V151D (SEQ ID NO: 85) exhibited enhanced target cell apoptosis relative to native ULBP2 (SEQ ID NO: 16) fusion (WT) (Fig. 27A). The modified ULBP3 variant R162G (SEQ ID NO: 86) exhibited enhanced target cell apoptosis relative to native ULBP3 (SEQ ID NO: 17) fusion (WT) (Figure 27b).

In some embodiments, the invention relates to an insertable variable fragment (iFv) peptide. Since the C-terminus and the N-terminus of the scFv molecule containing the multivalent scFv structure are spatially remote, the scFv structure does not destroy the Fv framework required for proper positioning of the CDR or overhangs so that the antigen- Nor can it be inserted into the loop region that is embedded in the protein fold of the parent or recipient protein without destroying or destabilizing its fold (s).

A variable fragment of an antibody containing up to six CDRs can be attached to an antigen derived from a light chain and heavy chain antibody variable domain to insert at one or more loop sites of the parent parent protein molecule without destroying the structural folds of the parental or variable fragment Lt; RTI ID = 0.0 > of-binding-peptide < / RTI > The new construct contained two linker sites, as well as a split variable domain, rather than the traditional single linker of the scFv structure. Conceptually the canonical ends of the variable (VL) and middle (VH) domains were fused to continuous or " circular " peptides. This circular peptide structure containing all six CDRs of Fv can be conceptually segmented from one of several possible new sites to produce insertable Fv (iFv). The non-natural cleavage site is generated within the light or heavy chain variable domain at or near the apex or bend of the loop so that it can be inserted into the loop of another (parent or recipient) protein or polypeptide without destruction of structure, stability or desirable function , New, unique N- and C-termini located spatially close together, preferably within 0.5 to 1.5 nm, can be produced. This new class of peptides is referred to as insertable variable fragment (iFv). The binding or targeting specificity delivered by the iFv to the recipient molecule can be altered by inserting another or different iFV into the recipient based on different antibodies or scFvs, or by replacing one or more of the CDRs of an existing insertable iFv.

Insertion of one or more iFv polypeptides that exhibit specific antigen-binding properties of the Fv domain into other proteins and thereby imparting novel binding properties thereto will have multiple usefulness. Such applications include, but are not limited to, allowing the parent protein to bind to a specific antigen, allowing the antigen to be targeted, allowing the presence of the antigen to be detected, allowing the antigen to be removed, Allowing access to the antigen, allowing delivery of the payload to the antigen or antigen-expressing cells, inducing the antigen, and imaging the presence of the antigen. The payload can be directly conjugated to one or both of the amino-terminal and carboxy-termini of the iFv or indirectly conjugated to the iFv via the parent protein or peptide. Examples of payloads include, but are not limited to, chromophore, fluorophore, pharmacodynamic chromophore, atom, middle eastern or radioactive isotope, imaging agent, chemotherapeutic agent or toxin. A payloaded iFv can be used to identify or locate the presence of a target molecule that specifically binds to iFv and is itself a small and stable in vitro or in vivo imaging or diagnostic agent Can act. In addition, chemotherapeutic agents or toxin molecules may be conjugated to one or both of the amino-terminal and carboxy-termini of the iFv peptide to produce an iFv-drug conjugate as a therapeutic agent for malignancy or infection, for example. A single payload may be conjugated to both the amino-terminal and carboxy-terminal ends of the iFv peptide so that the two ends are stretched or linked; These kidneys can further stabilize the iFv by blocking the end from exopeptidase degradation or protecting the iFv from degradation or unfolding.

Examples of parent or recipient proteins or polypeptides that are candidates for insertion of iFv peptides include, but are not limited to, proteins composed of antibodies, Igfolds or Ig domains, globulins, albumin, fibronectin and fibronectin domains, integrins, fluorescent proteins, enzymes, (MHC) family of proteins, receptor proteins, T-cell receptors, chimeric antigen receptors, viral antigens, viral capsids, viral ligands to cell receptors, high molecular weight bacteriocins, histones, hormones, nortins, cyclic peptides or polypeptides, Proteins, MIC proteins, ligands for lectins and lectins. The iFv structure may also be inserted into non-protein recipient molecules such as polysaccharides, dendrimers, polyglycols, peptidoglycans, antibiotics and polyketides.

NK cells and specific (CD8 + αβ and γδ) T-cells of the immune system have an important role in human and other mammals as first line, congenital defenses for neoplastic and virus-infected cells (Cerwenka, A., and LL Lanier, 2001. NK cells, viruses and cancer, Nat. Rev. Immunol., 1: 41-49). NK cells and specific T-cells exhibit NKG2D, an important and homodimeric surface immunoreceptor, on their surface, responsible for recognizing target cells and activating innate defense against pathologic cells (Lanier, LL, 1998. NK cell receptors, Ann. Rev. Immunol. 16: 359-393; Houchins JP et al., 1991. DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human NK cells. 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 comprises a C-type lectin-like extracellular domain, 84% sequence identical or homologous, monomeric MICA and MICB, a major histocompatibility complex (MHC) class I chain-associated glycoprotein (MIC) binding to its cognate ligand, (1987), and the second lineage of mammalian MHC class I genes, PNAS, has been reported to have a polymorphic homologue of PNAS (Weis et al., 1998. The C-type lectin superfamily of the immune system, Immunol. Rev. 163: 19-34; Immunogenetics 44: 80-81; Bahram and Spies TA 1996. Nucleotide sequence of human MHC class I MICB cDNA. Immunogenetics 43: 230-233). Non-pathological expression of MICA and MICB is restricted to the epithelial, keratinocyte, endothelial and mononuclear cells, but abnormal surface expression of these MIC proteins occurs in response to many types of cellular stresses such as proliferation, oxidation and heat shock And the cells are marked as pathological (Groh et al., 1996. Cell stress-regulated human MHC class I gene expressed in GI epithelium. PNAS 93: 12445-12450; Groh et al 1998. Recognition of stress-induced MHC molecules Intestinal γδ T cells. Science 279: 1737-1740; Zwirner et al 1999. Differential expression of MICA by endothelial cells, fibroblasts, keratinocytes and monocytes. Human Immunol 60: 323-330). Pathological expression of MIC proteins also appears to be involved in some autoimmune diseases (Ravetch, JV and Lanier LL 2000. Immune Inhibitory Receptors. Science 290: 84-89; Burgess, SJ. 2008. Immunol. Res. 40: 18-34). Polymorphism Differential control of NKG2D ligands such as MICA and MICB is important in providing the immune system with a means to identify and respond to a wide range of emergent signals while protecting healthy cells from unwanted attacks (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).

Viral infection is a common inducer of MIC protein expression and identifies virus-infected cells for NK or T-cell attack (Groh et al. 1998; Groh et al. 2001. Co-stimulation of CD8 + αβ T cells by NKG2D Immunol. 2: 255-260; Cerwenka, A., and LL Lanier, 2001). In fact, to prevent such attacks in host cells, cytomegaloviruses and other viruses developed a mechanism to prevent the expression of MIC proteins on the surface of the cells they infected so as to escape the connexion of the innate immune system (Lodoen, M. et al. , 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. Stern-Ginossar et al., (2007) Human microRNAs regulate stress-induced immune responses mediated by the receptor NKG2D. Nature Immunology 9: 1065-73; Slavuljica, IA Busche, M Babic, M Mitrovic, I Gasparovic, D Cekinovic, E Markova Car, EP Pugel, A Cikovic, VJ Lisnic, WJ Britt, U Koszinowski, M Messerle, A Krpotic and S Jonjic. 2010. Recombinant mouse cyt Omegalovirus expressing a ligand for the NKG2D receptor is attenuated and has improved vaccine properties. J. Clin. Invest. 120: 4532-4545).

Despite his stress, many malignant cells, such as malignant cells of lung cancer and glioblastoma brain cancer, also prevent the expression of MIC proteins and, as a result, they can be particularly aggressive due to their deviation from the innate immune system (Busche, A Doubrovina, ES, MM Doubrovin, E Vider, RB Sisson, 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; R. 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. / NKG2D-mediated immunogenic therapy of experimental gliomas. Cancer Research 63: 8996-9006; Fuertes, MB, MV Girart, LL Molinero, CI Domaica, LE Rossi, MM Barrio, J Mordoh, GA Rabinovich and NW Zwirner. 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).

The high-resolution structure of human MICA bound to NKG2D has been resolved and it has been demonstrated that the α3 domain of MICA has no direct interaction with NKG2D (Li et al. 2001. Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA Nature Immunol 2: 443-451; Protein Data Bank accession code 1HYR). The [alpha] 3 domain of MICA, e.g., the [alpha] 3 domain of MICB, is linked to the [alpha] 1- [alpha] 2 platform domain by a short, flexible linker peptide, which itself is naturally located as a " spacer " between the surface of the MIC- expressing cell and the platform. The three-dimensional structure of the human MICA and MICB alpha 3 domains are nearly identical (functionally exchangeable) (mean square root distance < 1 A at 94 C-alpha a's) (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).

As used herein, " soluble MIC protein ", " soluble MICA " and " soluble MICB " refer to MIC proteins containing the alpha 1, alpha 2 and alpha 3 domains of the MIC protein but without membrane penetration or intracellular domains.

The α1-α2 platform domain of the soluble MIC protein is moored in the α3 domain and is diffusible in the intracellular or intravascular space of the mammal. Preferably, the α1-α2 platform domain of the non-natural MIC protein of the invention is at least 80% identical or homologous to the native or natural α1-α2 domain of the human MICA or MICB protein and binds to NKG2D. In some embodiments, the α1-α2 platform domain is 85% identical to the native or natural α1-α2 platform domain of a human MICA or MICB protein and binds to NKG2D. In another embodiment, the α1-α2 platform domain is 90%, 95%, 96%, 97%, 98% or 99% identical to NKG2D with the native or natural α1-α2 platform domain of human MICA or MICB protein.

In some embodiments, the heterologous peptide tag can be fused to the soluble MIC protein or the N-terminus or C-terminus of the [alpha] 1- [alpha] 2 domain to aid in the purification of soluble MIC proteins. The tag sequence includes peptides such as poly-histidine, myc-peptide or FLAG tag. Such tags can be removed after isolation of the MIC molecule by methods known to those skilled in the art.

As used herein, " peptide ", " polypeptide " and " protein " are used interchangeably; A "heterologous molecule", "heterologous peptide", "heterologous sequence" or "heterologous atom" is a molecule, peptide, nucleic acid or amino acid sequence, or atom, which is physically linked to a subject molecule naturally or not usually found, respectively.

The word "comprising" is used interchangeably with "comprising", "comprising", or "characterized by" is a generic or open language and does not exclude additional, unrecited elements or method steps. The phrase " consisting of " excludes any element, step or component not explicitly stated in the claims. The phrase " consisting essentially of " restricts the scope of the claims to those that do not materially affect the specified material or step, and the basic and novel features of the claimed invention. The present disclosure contemplates embodiments of inventive compositions and methods corresponding to the respective categories of these phrases. Thus, a method or composition comprising the recited element or step contemplates certain embodiments in which the method or composition consists of or consists essentially of these elements or steps.

All references cited herein are incorporated by reference in their entirety, whether specifically incorporated or not previously included. As used herein, the singular form " and " optional " are intended to include both singular and plural.

It will now be appreciated by those skilled in the art that the present invention has been fully described, and that the invention may be practiced within a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is based on and claims the benefit of the inventors that this invention is not limited to the particular embodiments of the invention as defined by the appended claims, Use, or adaptation of the present invention.

iFv , And NKG2D  Modified ligand alpha 1 - α2  Of the domain Example

Example 1 ( iFv ) . As specific examples, 1126 bp and 1144 bp DNA fragments (SEQ ID NOs: 1 and 2, respectively) encoding in the following order were synthesized: α3 domain of human MICA (as parent peptide) amino acid 182 to amino acid 194 (MAbR3; Qing, J., Du, X. et al.) Based on the structure of a free spacer or GGS amino acid spacer site (SR), fibroblast growth factor receptor 3 (FGFR3) -binding antibody. , Chen, Y., Chan, P., Li, H., Wu, P., Marsters, S., Stawicki, S., Tien, J., Totpal, K., Ross, S., Stinson, S. (2009) Antibody-based targeting of FGFR3 in bladder carcinoma and t (4; Doranan, D., French, D., Wang, QR, Stephan, JP, Wu, Y., Wiesmann, C and Ashkenazi, A. 14) -positive multiple myeloma in mice, the Journal of clinical investigation 119, 1216-1229), non-GGS spacer spacer or another site, starting at amino acid 196 of the α3 domain, and the remaining carboxy-terminal part availability at MICA Party moped of the loop 1 of the α3 domain that includes amino acids to 276. Each synthetic, double-stranded DNA polynucleotide encodes a polypeptide containing six CDRs in the form of an iFv inserted into loop 1 of the α3 domain of MICA.

This iFv peptide itself (SEQ ID NO: 3) encoded by SEQ ID NO: 4 contained two identical, typical linker sites (LR) corresponding to residues GGSSRSSSSGGGGSGGGG (SEQ ID NO: 5) (1998) Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, Cold Spring Harbor protocols 2011 ). One LR linked the C-terminus of VL to the N-terminus of the VH domain, and the second LR linked the C-terminus of the VH domain to the N-terminus of VL. Conceptually, this new structure was the continuous or " circular " peptide shown above and contained the six CDRs of the starting Fv. The variable VL chain of the antibody was effectively split within the loop region between beta-strand 1 and 2 (S1 and S2), thereby resulting in a new, non-natural C- and N- (VLN) and a new C-terminal segment (VLC) (Fig. 5A). This pair of ends has created a unique site for attachment or conjugation of iFv to a recipient molecule such as a protein. A schematic diagram of the inserted iFv in the parent alpha 3 domain is shown in Figure 5b.

(SEQ ID NO: 6) and [alpha] 3-iFv.2 (SEQ ID NO: 7), respectively, to produce soluble MICA proteins with heterologous iFv peptides inserted into the [alpha] SEQ ID NOS: 1 and 2). ≪ / RTI > The DNA fragment was amplified by PCR, digested with NcoI and EcoRI restriction enzymes, and subcloned into the baculovirus expression vector, SW403, replacing the wild-type? 3 domain. SW403 is a baculovirus expression vector derived from pVL1393 (Invitrogen, Inc.) in which wild-type sMICA (residue 1-276) was previously cloned using the 5 'BamHI and 3' EcoRI sites. The new expression vector was co-transfected with baculovirus DNA in SF9 insect cells, the baculovirus was grown during two amplification cycles, and the His-tagged < RTI ID = 0.0 > RTI ID = 0.0 > MICA-a3-iFv < / RTI > Expression was performed in 100 ml volumes for 3 days and the growth medium was harvested for the purification of secreted soluble proteins using Ni-affinity chromatography. Monomeric MICA-α3-iFv was purified to> 90% purity with an expected molecular weight of 60.9 kDa as determined by SDS-PAGE. Functional assays were performed using binding ELISA and in vitro target cell death assays.

Purified MICA- [alpha] 3-iFv protein was tested in FGFR3-binding ELISA to confirm simultaneous binding to FGFR3 target and NKG2D receptor. FGFR3 in phosphate buffered saline (PBS) was coated on a Maxisorp plate at a concentration of 2 ug / ml. Each MICA protein was titrated, bound to FGFR3 for 1 hour, and washed to remove unbound sMICA protein. The bound MICA-α3-iFv protein was detected using NKG2D-Fc and anti-Fc-HRP conjugates. Figure 6 shows that the binding of both MICA-α3-iFv.1 and MICA-α3-iFv.2 to FGFR3 is similar to that of MICA-scFv produced by fusion of a conventional scFv constructed from MAbR3 at the C-terminus of soluble MICA . These ELISA results also showed that both the FGFR3 and NKG2D binding specificities of the scFv and < RTI ID = 0.0 > a1-a2 < / RTI > domains were maintained by modified MICA and demonstrated that the inserted iFv peptides were functional using different spacer formats.

The thermal stability of sMICA-a3-iFv.2 against sMICA-scFv was tested and compared. Both proteins were increased in temperature from 60-90 ° C for 1 hour and then equilibrated to room temperature for 1 hour before assaying for binding properties by ELISA. The results in Figures 7a and 7b show that MICA- [alpha] 3-iFv.2 can be treated at temperatures as high as 80 [deg.] C without loss of specific binding to FGFR3. Traditional MICA-scFv loses binding activity at 70 < 0 > C. These results indicated that the soluble MICA containing the invented iFv format was considerably more stable than the end fusions of traditional scFvs (Miller, BR, Demarest, SJ, Lugovskoy, A., Huang, F., (2002) Stability engineering of scFvs for the development of bispecific and multivalent antibodies, Protein engineering, design & selection (2002), Snyder, WB, Croner, LJ, Wang, N., Amatucci, A., Michaelson, JS, and Glaser : PEDS 23 , 549-557; Weatherill, EE, Cain, KL, Heywood, SP, Compson, JE, Heads, JT, Adams, R., and Humphreys, DP (2012) Towards a universal disulphide stabilized single chain Fv format: orientation of the interchain disulphide bond location and vL-vH orientation, Protein engineering, design & selection: PEDS 25 , 321-329).

The ability of MICA-α3-iFv to re-induce NK cell-mediated dissolution of FGFR3-expressing target cells was demonstrated in vitro in a calcine-emission assay. The natural killer (NK) cell line, NKL, was co-cultured with callus-loaded P815 target cells expressing FGFR3 ectopically. The results in Figure 8 show that the two MICA-a3-iFv molecules induced significantly greater NK-mediated dissolution compared to the traditional MICA-scFv fusion while the non-targeted soluble MICA control had no killing activity . These results confirmed that the inventive iFv bound to FGFR3 in the target cells and that soluble MICA in the context of the entire parental protein molecule induced strong NK cell-mediated dissolution.

The α3-iFv.3 (SEQ ID NO: 8) containing the iFv derived from the CD20-specific antibody was similarly constructed to demonstrate the applicability of the iFv format to other antibody variable domains (Du, J., (2007) Structural basis for recognition of CD20 by therapeutic Jang, Wang, H., Zhong, C, Peng, B., Zhang, M., Li, B., Huo, S., Guo, Y., antibody Rituximab, The Journal of Biochemistry 282 , 15073-15080). Figures 9a and 9b show that MICA- [alpha] 3-iFv.3 can specifically bind to CD20 coated wells in plate-based ELISAs as described above and also inhibit CD20 expressing Ramos cells Lt; RTI ID = 0.0 > NK-mediated < / RTI >

Example 2 ( NKG2D Lt; / RTI > domain of the ligand ) . Human proteins designated ULBP-1 to ULBP-6 are naturally occurring, stress-induced, cell surface ligands, such as MICA and MICB, that bind to NKG2D receptors and activate human NK cells and specific T-cells 15, Cerwenkaa, 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). In addition, the vaccinia virus protein OMCP is a secreted domain as the [alpha] 1- [alpha] 2 domain of the MIC protein binds to NKG2D. OMCP clearly shows a very high affinity for NKG2D to block the recognition of the NKG2D of the natural stress ligand induced by the virus in his infected host cell (Eric Lazear, Lance W. Peterson, Chris A. Nelson, David H. Fremont, J Virol, 2013 January; 87 (2): 840-850, doi: 10.1128 / JV.01948-12). While ULBP and OMCP are considered as NKG2D ligands (NKG2DL) sharing a canonical α1-α2 domain structure, the sequence homology with MICA α1-α2 is less than 27%, and all of the α3 domains for mating the targeting domain are naturally deficient . FGFR3-expressing cells were specifically targeted as a result of fusion of the modified [alpha] 3 domain of MICA inserted with the targeted iFv into each of ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-6 and OMCP Attachment of the killed heterologous polypeptides constituted a series of non-natural ULB and OMCP proteins. In addition, the α1-α2 domain of MICA was modified to enhance the affinity of the α1-α2 domain for NKG2D, and then a heterologous molecule such as a polypeptide was attached to the modified α1-α2 domain. ULBP-2, ULBP-3, ULBP-4, ULBP-6, and OMCP (to which the α1-α2 domain is attached in the modified α3-iFv domain) Baculovirus expression vectors were generated to accommodate DNA fragments (SEQ ID NO: 9-14) encoding the different [alpha] 1- [alpha] 2 domains of SEQ ID NO: 15-20. DNA fragments were amplified by PCR, digested with BlpI and NcoI restriction enzymes, and subcloned individually into the baculovirus expression vector, KLM44, replacing the MICA alpha 1-alpha 2 domain. KLM44 was a baculovirus expression vector derived from SW403 in which MICA-α3-iFv.2 was previously cloned (Example 1). ULBP and OMCP α1-α2 domain fusions (ULBP1-α3-iFv.2, ULBP2-α3-iFv.2, ULBP3-α3-iFv.2, ULBP4-α3-iFv.2, ULBP6 - 3-iFv.2 and OMCP- [alpha] 3-iFv.2, SEQ ID NO: 21-26, respectively) were co-transfected with baculovirus DNA in SF9 insect cells Respectively. Baculovirus was grown for two cycles of amplification and used to express this His-tagged NKG2DL-a3-iFv.2 protein in T. nis insect cells according to the manufacturer's protocol (Invitrogen). Expression was performed in 100 ml volumes for 3 days and the growth medium was harvested for the purification of secreted soluble proteins using Ni-affinity chromatography. Monomelic proteins of the correct molecular weight were purified to> 90% purity, as determined by SDS-PAGE. Functional assays were performed using binding ELISA and in vitro target cell death assays.

Six purified NKG2DL-a3-iFv.2 proteins were tested in an FGFR3-binding ELISA to confirm simultaneous binding to FGFR3 targets and NKG2D receptors. FGFR3 in phosphate buffered saline (PBS) was coated on a Maxisorp plate at a concentration of 2 ug / ml. Each NKG2DL-α3-iFv.2 protein was titrated, bound to FGFR3 for 1 hour, and washed to remove unbound proteins. The bound NKG2DL-α3-iFv.2 protein was detected using NKG2D-Fc and anti-Fc-HRP conjugates. Figure 10 strongly binds to FGFR3 through interaction with the iFv.2 domain, as expected for all six NKG2DL-a3-iFv.2 proteins, and by NKG2DL alpha 1-alpha 2 domain with NKG2D binding activity , Demonstrating that the attached [alpha] 3-iFv domain conferred functional FGFR3 binding activity to the ULBP and OMPC proteins as the MIC protein binds to NKG2D.

The ability of the NKG2DL- [alpha] 3-iFv.2 protein to re-induce NK cell-mediated dissolution of FGFR3-expressing target cells was demonstrated in vitro in calcein-emission assays. The natural killer (NK) cell line, NKL, was co-cultured with callus-loaded P815 target cells expressing FGFR3 ectopically. The results in Fig. 11 show that while OMCP-a3-iFv.2 induced the largest NK-mediated dissolution, all other NKG2DL-a3-iFv.2 proteins exhibited a specific killing activity . These results confirm that the inventive iFv retains its unique functional properties and confers specific binding activity to other proteins that have induced different levels of cell-mediated dissolution of iFv-targeted cells.

Example 3 ( modified α1 - α2 of NKG2D ligand Domain) . This is an embodiment in which the polypeptide is attached to modified NKG2DL so that the binding affinity for the human NKG2D receptor is significantly enhanced. The α1-α2 domain of the MIC protein is NKG2DL for the NKG2D receptor. This affinity is sufficient to stimulate the physiological activation of NK cells and the lysis of cells expressing the innate full-length MIC protein irreversibly moored on the surface of the two-dimensional plasma membrane of the " target cell " (Bauer S, Groh V, Wu J, Steinlee, Phillips JH, Lanier LL, Spies T., Science 1999 Jul 30; 285 (5428): 727-9.). However, since the engineered soluble MIC protein of the present invention reversibly binds to the specific target antigen on the surface of the target cell, the binding affinity of the engineered soluble MIC protein to NKG2D is dependent on the soluble MIC formed between the NK cell and the target antigen expressing cell Lt; RTI ID = 0.0 > dependency < / RTI > In particular, if the affinity between sMICA and NKG2D is increased by a substantially slower dissociation rate or off-rate of modified sMICA from NKG2D, then NK cell-based killing may be induced at a lower density of soluble MIC molecules bound to the target cell It is expected to be larger. Prior to the present invention, no [alpha] 1- [alpha] 2 mutation was found to significantly alter the killing offactivity of the soluble MIC protein or significantly reduce the binding off-rate to enhance the affinity of the MIC protein for NKG2D. The computational design results show that the stability of MICA with three mutations in the α1-α2 domain of the combined wild-type MICA: N69W, K152E and K154D (WED-MICA) and thus the on-rate of binding to NKG2D (B), Berker D, Kortemme T, Strong RK, McFarland BJ., J Biol Chem. (1986) 2007 Oct 19; 282 (42): 30658-66, Epub 2007 Aug 8); Morris DL, Willcox BE, Steinlee A, Spies T, and Lynn A. (1985), "Identification of NKG2D by Nucleotide Sequence Analysis" Strong RK., Nat Immunol. 2001 May; 2 (5): 443-451), combined with the three initially designed changes, added a rational, iterative computational design of MICA to a total of seven combined mutations (Kd = 2.5 nM) to NKG2D (Henger, Samuel H., Melissa A. Hale, Nicholas J. et al. 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, rational affinity maturation of the MICA-NKG2D interface. Protein Science 21: 1396-1 402). Conversely, the experimental approach described in the present invention is based on the method of Lengyel et al (Lengyel CS, Willis LJ, Mann P, Baker D, Kortemme T, Strong RK, McFarland BJ, J Biol Chem. 2007 Oct 19; Starting with MICA stabilized by 3 WED changes of 30658-66. Epub 2007 Aug 8, amino acid modification of MICA in which the off-rate between MICA and the α1-α2 domains of NKG2D was slowed down was experimentally selected.

This embodiment of the present invention is based on the finding that at a selected amino acid position within the < RTI ID = 0.0 > ai-a2 < / RTI > domain, which affects the off-rate binding kinetics and thus changes the NK cell- mediated killing activity of the invented non-natural, Lt; RTI ID = 0.0 > of MIC < / RTI > proteins through specific mutagenesis of the NKG2D binding affinity of the soluble MIC protein.

To manipulate the soluble non-natural? 1-? 2 domain with altered affinity for NKG2D, 57 residues in the? 1-α2 domain were selected for large-scale mutagenesis (FIG. 12a). Synthetic DNA libraries encoding the α1-α2 domain and containing the NNK mutation-inducing codon at each of the 57 amino acid positions were synthesized and cloned individually as a fusion to the pIII minor coat protein of M13 phage and the mutated α1- The phage particles displaying the? 2 variants were prepared in SS320 E. coli cells according to standard methodology (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C, and Barbas, CF, (2011) Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, Cold Spring Harbor protocols 2011 ). The α1-α2 phage library was classified for increased binding affinity using recombinant biotinylated NKG2D as the target antigen and cyclically treated through repeated rounds of intentionally extended binding, extended washing and phage cloning elongation, High-affinity mutants enhanced at off-rate were selected. A specific set of amino acid mutations occurred at a high frequency in the six positions at a1-a2 and were selected as preferred amino acid substitutions with enhanced NKG2D binding affinity (Figure 12b, Table 1).

Table 1 . The selected affinity mutation at the indicated six amino acid positions of the alpha 1-alpha 2 domain of the MIC. The amino acids of SEQ ID NO: 35 at each of the six positions are shown in bold in the first row of the table. List the identified affinity mutations at a frequency that decreases from top to bottom. All amino acids are indicated by the single-letter IUPAC abbreviation.

DNA polynucleotides (SEQ ID NO: 27-30) encoding the α1-α2 domain of four representative variants 15, 16, 17 and 18 containing different combinations of specific mutations were synthesized (Table 2).

Table 2 . Sequence of specific α1-α2 domain variants. Specific amino acid substitutions for variants 15, 16, 17 and 18 (SEQ ID NO: 31-34, respectively) are listed in bold for the amino acids of SEQ ID NO: 35. All amino acids are indicated by the single-letter IUPAC abbreviation.

For the NKG2DL in this example, a heterologous molecule such as a polypeptide was directly attached to each of these four modified α1-α2 NKG2DL using a linker peptide. Four His-tagged proteins (SEQ ID NO: 31-34) consisting of modified NKG2DL with attached heterologous molecules were expressed in insect cells and purified to analyze their NKG2D binding affinity and kinematic binding parameters . Competitive Binding ELISA was used to measure relative NKG2D binding affinity of the four modified α1-α2 variants. Soluble wildtype (WT) NKG2DL, sMICA protein was coated in all wells of maxisorp ELISA plates to provide binding partners for human NKG2D-Fc reagent. A solution of WT and WED- alpha 1-alpha2 domains (SEQ ID NO: 35) as well as four alpha 1-alpha2 variants were competitively inhibited in 2 nM human NKG2D-Fc binding to WT sMICA titrated in ELISA wells . The level of human NKG2D-Fc bound to WT NKG2DL in the plate was detected using anti-Fc-HRP antibody. Figure 13a shows that mutants 16, 17 and 18 showed IC ₅₀ values of 0.7, 0.6 and 0.5 nM, while variant 15 showed IC ₅₀ values of 1.7 nM, all with fairly good binding to NKG2D, 32-, 38- and 11-fold better than WT NKG2DL, respectively, and substantially better than WED-MICA (Table 3).

Table 3 . Equilibrium and kinematic coupling parameters for α1-α2 variants. The IC ₅₀ values were derived from a 4-parameter pit for competitive binding titrations (Figures 12a and 12b) and kinematic binding parameters were derived from a single exponential fit to the binding kinetics (Figures 13a and 13b ). The equilibrium coupling constant (K _d ) is given by the equation K _d = k _OFF / k _ON .

Significantly, relative IC ₅₀ differences were transferred to better binding to murine and NKG2D-Fc (Fig. 13b), and the availability for human and non-human NKG2D receptors, important properties for preclinical drug development, Domain < / RTI > binding.

To understand the kinematical basis for the altered affinity, both the on-rate and off-rate for the a1-a2 variant NKG2DL that binds to the surface coated biotinylated human NKG2D were compared to those of each of the modified < RTI ID = lt; / RTI > were measured using a biological shielding interferometry (Octet). Consistent with the results from the IC ₅₀ ELISA, each of variants 16, 17 and 18 showed a significant decrease in off-rate (18-fold compared to WT), which is a major cause of increased affinity (WT α1- 30) (Fig. 14: Table 3). Variant 15 exhibited similar slow off-rates as in variants 16, 17 and 18, but its on-rate decreased, resulting in a weaker affinity than mutants 16, 17 and 18, although stronger than WT. Since the only difference between variant 15 (SEQ ID NO: 31) and 16 (SEQ ID NO: 32) was K125N versus K125L, mutations at position 125 evidently altered the on-rate while reduced off- . Thus, while a selected set of NKG2DL mutations (Table 1) was used to increase the affinity of < RTI ID = 0.0 > a1-a2 < / RTI > for NKG2D through significant off-rate reduction, certain substitutions also altered the on- , Which in the present invention has been shown to have differential activity in NK cell-mediated killing assays as described below.

The ability of the [alpha] 1- [alpha] 2 affinity variants to re-induce NK cell-mediated dissolution of FGFR3-expressing target cells was demonstrated in vitro in a Carlin-Emission assay. The human natural killer (NK) cell line, NKL, was co-cultured with calcein-loaded P815 target cells expressing FGFR3 ectopically and titrated with soluble modified MIC protein. The results in Figure 15 showed that the killing activity of the FGFR3-specific soluble MIC variant correlates with its engineered alpha-alpha 2 affinity. Specifically, variants 16, 17 and 18 showed ~ 15-fold more death at 0.78 nM than WT. WED-MICA (SEQ ID NO: 35) was only slightly better than WT. Thus, the present invention describes that amino acid substitution in the [alpha] 1- [alpha] 2 domain reduces the off-rate of soluble MIC protein binding to human NKG2D, thereby increasing NKG2D binding affinity and consequently resulting in increased killing efficacy . WED-MICA (Fig. 13A), which exhibited an affinity somewhat greater than WT MICA for NKG2D (Fig. 14) by increasing on-rate rather than decreasing off-rate, did not exhibit any substantial improvement in target cell apoptosis 15). 13A and 13B), WED-MICA showed greater affinity for both murine and NKG2D than WT MICA, as shown in FIG. 13B, while variants 15, 16, 17 and 18 exhibited greater affinity for both human and murine NKG2D And exhibited substantially poorer bonding.

These α1-α2 NKG2DL affinity variants 15, 16, 17 and 18 enhanced the binding affinity of the attached polypeptides to the NKG2D receptor and thereby promoted NK cell-mediated dissolution of the target cells (FIG. 15).

Example 4 (Modified α1-α2 domain of NKG2D ligand) . This embodiment of the invention is characterized by the additional α1-α2 NKG2DL affinity derived from the specific mutagenesis at selected amino acid positions in the α1-α2 domain of the soluble MIC molecule as described in Example 3 (Table 1) Mutant, which also affects the off-rate binding kinetics and thus the NK cell-mediated killing activity of the non-natural a1-a2 domain. Mutants 15-18 were focused on the specific mutations found in positions S20, G68, K125 and H161, but another set of mutants were isolated with additional mutations in E152, H158 and Q166 (Table 4).

Table 4 . Sequence of specific α1-α2 domain variants. Specific amino acid substitutions for mutants 20, 25 and 48 are listed for the amino acids of SEQ ID NO: 35, which are indicated in bold in the first row of the table. All amino acids are indicated by the single-letter IUPAC abbreviation.

DNA polynucleotides encoding the alpha 1-alpha 2 domain of three representative variants 20, 25 and 48 (SEQ ID NO: 39-41, respectively) containing different combinations of the specific mutations found (Table 4) 36-38). For NKG2DL in this example, a heterologous molecule such as an FGFR3-binding polypeptide was directly attached to each of these three modified alpha 1-alpha 2 NKG2DL using a linker peptide. Constructs were cloned into the XbaI and BamHI sites of pD2509, a CMV-based mammalian cell expression vector. Three His-tagged proteins (SEQ ID NO: 39-41) consisting of modified NKG2DL with attached heterologous molecules that bind to FGFR3 were cloned into HEK293 cells using the Expi293 expression system according to the manufacturer's protocol (Life Technologies) And purified using Ni-affinity chromatography to obtain isolated proteins for biochemical and activity-based analysis.

To analyze the NKG2D binding affinity, both the on-rate and off-rate for the three alpha 1-alpha2 variants NKG2DL binding to the surface-coated biotinylated human NKG2D were determined using the Biological Shield Interference (Octet) Respectively. Coupling titrations were performed on each protein using an appropriate range of 1-100 nM and kinetic data were fitted to obtain on-rate, off-rate, and equilibrium binding constants.

Variant 25 (SEQ ID NO: 40) contained only the addition of the Q166S mutation with respect to variant 16 (SEQ ID NO: 32) (Table 2) and showed an NKG2D binding affinity of 62 pM mainly due to reduced off- (Fig. 16 and Table 5). This indicated an 8-fold enhancement in equilibrium binding affinity due to the Q166S mutation (compare Table 3 and Table 5), demonstrating that the specific mutation at Q166 affected the binding affinity via decreased off-rate .

Table 5 . Kinetic binding parameters for α1-α2 variants. Kinematic coupling parameters were derived from a single exponential fit to the binding reaction rate (Figure 16). The equilibrium coupling constant (K _d ) is given by the equation K _d = k _OFF / k _ON .

Variant 20 (SEQ ID NO: 39) contained the specific mutations G68A, E152Q, H158R and Q166F and retained similar binding parameters as variant 25 (Table 5), indicating that this unique combination of specific mutations is also present, Has improved NKG2D binding affinity due to reduced off-rate.

Mutant 48 (SEQ ID NO: 41) contained the K125L and H161R mutations found in variant 16 (Table 2); Addition of the mutations E152A, H158I and Q166A (Table 4) significantly increased the off-rate, resulting in a 250-fold reduction in NKG2D binding affinity (Figure 16 and Table 5). The Q166A mutation is not one of the preferred affinity enhancing mutations selected for position Q166 (Table 1) and can contribute to a reduction in the observed off-rate. This data clearly demonstrates that the unique combination of identified manipulated mutations was selected at defined positions in the [alpha] l- [alpha] 2 domain and adjusted the NKG2D binding affinity through off-rate modulation.

Non-natural α1-α2 affinity variants with attached polypeptides re-induced NK cell-mediated dissolution of FGFR3-expressing target cells, as demonstrated in vitro in Carlthine-Exit Test. The human natural killer (NK) cell line, NKL, was co-cultured with calcein-loaded P815 target cells expressing FGFR3 ectopically and titrated with soluble modified NKG2D ligand α1-α2 protein. The results in Figure 17 showed that the killing efficacy of the FGFR3-targeted soluble MIC variant correlates with its engineered α1-α2 affinity. Specifically, variant 25 exhibited ~ 3-fold greater death at 0.2 nM than variant 16, indicating ~ 5-fold improvement in EC ₅₀ for apoptosis. In addition, the data clearly demonstrated the desirable killing activity against representative soluble MIC variants in the order of mutant 25 > 16 > WED (Fig. 17).

Example 5 (Modified α1-α2 domain of NKG2D ligand) . This embodiment relates to an additional α1-α2 NKG2DL affinity variant derived through the manipulation of the α1-α2 domain of the ULBP protein. The ULBP protein contains the α1-α2 domain, an NKG2D ligand capable of binding to the NKG2D receptor (Cerwenka A, Lanier LL (2004).) NKG2D ligands: unconventional MHC class I-like molecules exploited by viruses and cancer. Tissue Antigens 61 5): 335-43 doi: 10.1034 / J.L399-0039.2003.00070.x.PMID 12753652). This affinity of NKG2D binding is sufficient to stimulate the physiological activation of NK cells and the lysis of cells expressing innate, full-length ULBP proteins naturally irreversibly mapped on the two-dimensional plasma membrane surface of " target cells " (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.l399-0039.2003.00070.x.PMID 12753652) . However, in certain embodiments of the invention, the engineered soluble α1-α2 domain fused to a heterologous polypeptide reversibly binds to a surface-specific target antigen on the target cell, resulting in a binding affinity of the engineered ULBP α1-α2 domain to NKG2D Sex will directly affect the stability of the artificial synapses formed between the NK cells and the target antigen expressing cells as already shown by the engineered soluble MIC protein (Example 2-4). To diversify the repertoire of non-natural α1-α2 domains engineered as NKG2D ligands, the ULBP protein was used as a starting point or substrate for phage display-based manipulation of its NKG2D-binding affinity. Conformational plasticity revealed by the cocrystal structure of NKG2D (2001), despite the observed structural homology between ULBP and MICA (Radaev, S., Rostro, B., Brooks, AG., Colonna, and its class I MHC-like ligand ULBP3. Immunity 15, 1039-49.), sequence homology is <50% for the ULBP α1-α2 domain compared to MICA (FIG. Therefore, the identity of codon positions in the ULBP alpha 1-alpha 2 domain, which improves NKG2D binding affinity, was explored.

ULBP2 and ULBP3 were selected for phage display, and selection of mutants with high affinity NKG2D binding, in order to manipulate the soluble, non-natural [alpha] 1- [alpha] 2 domain from the ULBP protein. Six amino acid positions (SEQ ID NO: 16) in the a1-a2 domain of ULBP2 and 36 amino acid positions (SEQ ID NO: 17) in the a1-a2 domain of ULBP3 were selected for extensive mutagenesis 18). In addition, conserved serine-serine mutations were generated in C103S (SEQ ID NO: 16) in ULBP2 and in C8S (SEQ ID NO: 17) in ULBP3, resulting in unpaired Free cysteine was removed. Individually synthesizing synthetic DNA libraries encoding these α1-α2 domains and containing NNK mutagenic codons at each of the selected amino acid positions; Cloned as a fusion to the pIII minor coat protein of M13 phage; Phage particles displaying mutated α1-α2 ULBP2 or ULBP3 variants were prepared in SS320 E. coli cells according to standard methodology (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, CF, 3rd. (2011) Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, Cold Spring Harbor protocols 2011 ). Using the human NKG2D-Fc as the target protein, the α1-α2 phage display library was classified for increased binding affinity for NKG2D and cyclically treated through intentionally extended binding, extended washing and repeated rounds of phage clone elution, A high affinity mutant enhanced with slow dissociation- or off-rate was selected. For ULBP2, specific amino acid mutations were found at high frequencies in positions R80, V151, V152 and A153 at a1-a2 and identified as preferred amino acid substitutions with enhanced NKG2D-binding affinity (Figure 19A and Table 6) .

Table 6 . A selected affinity mutation at the indicated four amino acid positions of the alpha 1-alpha 2 domain of ULBP2. The amino acids of SEQ ID NO: 16 in each of the four positions are shown in bold in the first row of the table. List the identified affinity mutations at a frequency that decreases from top to bottom. All amino acids are indicated by the single-letter IUPAC abbreviation.

For ULBP3, specific amino acid mutations were found at a higher frequency than at ULBP2 (Figure 18). Locations R162 and K165 in the alpha 1-alpha 2 domain of ULBP3 contained specific mutations identified as preferred amino acid substitutions with enhanced NKG2D-binding affinity (Figure 19b and Table 7). These modified non-natural [alpha] 1- [alpha] 2 domains derived from ULBP2 and ULBP3 can be used for NKG2D binding enhanced in multiple therapeutic formats as a fusion protein or a single protein to a heterologous peptide or polypeptide.

Table 7 . A selected affinity mutation at the indicated two amino acid positions of the alpha 1-alpha 2 domain of ULBP3. The amino acids of SEQ ID NO: 17 in each of the two positions are indicated in bold in the first row of the table. List the identified affinity mutations at a frequency that decreases from top to bottom. All amino acids are indicated by the single-letter IUPAC abbreviation.

Example 6 (Modified α1-α2 domain fused to an antibody peptide) . This is an embodiment in which an antibody polypeptide is attached to the modified NKG2DL so that the binding affinity for the human NKG2D receptor is significantly enhanced. The α1-α2 domain of the MIC protein is NKG2DL for the NKG2D receptor. Antibodies are highly stable glycoproteins made from two large heavy chains and two small light chains (Figure 1). A large amount of diversity that can be generated within the CDR regions of the variable domains allows specific antibodies to be generated for specific antigen targets (Hozumi N, Tonegawa S (1976). &Quot; Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant ..... regions "Proc Natl Acad Sci USA 73 (10):. 3628-3632 doi:.. 10.1073 / pnas.73.10.3628 PMC 431171. PMID 824647.) antibody significant therapeutic platform for drug development (Vidarsson, G., Dekkers, G., Rispens, T. (2014).) IgG subclasses and allotypes: from structure to effector functions. Frontiers in Immunology 5, 520.). Prior to the present invention, there was no IgG antibody format capable of directly activating immune cells using the non-natural α1-α2 domain, which binds NKG2D receptors more tightly than native NKG2DL. Previously, it was demonstrated that mouse NKG2D ligand, Rae1 beta, could be fused to anti-Her2 antibody for use as an antitumor agent in mice (Cho, HM., Rosenblatt, JD., Tolba, (NKG2D) ligand binding protein and its anti-Her2 antibody-NKG2D ligand fusion protein results in an enhanced innate and adaptive antitumor response Cancer Research 70 , 10121-30.). However, the mouse NKG2D ligand does not bind to human NKG2D, and there is no natural human NKG2D ligand with high affinity for human and mouse NKG2D. Fusions between the heavy or light chains of the engineered α1-α2 NKG2D ligands and IgG antibodies of the present invention (FIGS. 20a and 20b) overcome this limitation and allow flexibility of fusion of modified α1-α2 domains to heterologous proteins or peptides .

To generate mutant α1-α2 domain fusions for antibodies, DNA sequences encoding the α1-α2 domain for MIC WT, variants WED, 25 and 48 were synthesized and the heavy chain from the FGFR3-specific antibody (HC_WT, Terminal fusion to the light chain (LC_WT, LC_WED, LC_25, LC_48) sequences (Qing, J., Du, X., Chen, Y., Chan, P., Li , H., Wu, P., Marsters, S., Stawicki, S., Tien, J., Totpal, K., Ross, S., Stinson, S., Dornan, (2009) Antibody-based targeting of FGFR3 in bladder carcinoma and t (4; 14) -positive multiple myeloma in mice, The Journal of Clinical Pathology clinical investigation 119 , 1216-1229.) (SEQ ID NOS: 42-49, respectively). The resulting fusion was cloned into the mammalian expression vector pD2509 and expressed as a whole IgG antibody paired with the heavy or light chain fusion of the modified [alpha] l- [alpha] 2 domain (SEQ ID NO: 50-57, respectively). Transient expression was performed in HEK293 cells using the Expi293 expression system according to the manufacturer's protocol (Life Technologies) and purified using standard Protein A Affinity Chromatography. The ability of non-natural α1-α2-antibody fusions to re-induce NK cell-mediated dissolution of FGFR3-expressing target cells was demonstrated in vitro in calcein-emission assays. The human natural killer (NK) cell line, NKL, was co-cultured with callus-loaded P815 target cells expressing FGFR3 ectopically and titrated with the engineered antibody fusion protein. The results in Figures 20c and 20d showed that the killing activity of FGFR3-specific non-natural [alpha] 1- [alpha] 2-antibody fusions correlates with its engineered NKG2D affinity. Specifically, antibodies containing heavy chain or light chain fusions (HC_25, HC_48 and LC_25, LC_48) of non-natural variants 25 and 48 are more effective than FGFR3-expressing cells .

It is also possible that the variant 25 alpha 1-alpha 2 domain is replaced with the EGFR-specific antibody cetuximab (US Patent 6217866), the Her2-specific antibody trastuzumab (Carter, P., Presta, L., Gorman, CM, Ridgway, (1992) Proc Natl Acad Sci 15 , 4285-9), or an anti-PDL1 antibody &lt; RTI ID = 0.0 &gt; Terminal region of the heavy chain or light chain (SEQ ID NOs: 58-63, respectively) of the humanized antibody (US patent 20140341917). The resulting fusion was expressed as a light chain and heavy chain whole IgG antibody paired with a heavy chain or light chain fusion of the variant 25 [alpha] 1- alpha 2 domain. Transient expression was performed in HEK293 cells using the Expi293 expression system according to the manufacturer's protocol (Life Technologies) and purified using standard Protein A Affinity Chromatography. The ability of mutant 25 antibody fusions to re-induce NK cell-mediated dissolution of target-expressing cells was demonstrated in vitro in a Carlin-Emission assay. NK cells were co-cultured with callus-loaded A431 EGFR-expressing target cells, SKBR3 Her2-expressing target cells, or PDL1-expressing B16 cells, and each target-specific engineered antibody And then titrated with the fusion protein. The results in Figures 21a, 21b, and 21c show that the killing activity of the target-specific variant 25-antibody fusion is dramatically improved in all cases compared to the non-fused parent antibody and has a sub-nanomolar EC ₅₀ value It was very powerful. These data show that modified α1-α2 variant-antibody fusions are a universal platform that allows IgG antibodies to bind tightly to NKG2D and induce antigen-specific cell lysis.

Example 7 (Trastuzumab fusion to alpha 1-alpha2 mutant 25 binds to NK cells in vivo and leads to strong antigen presentation) . Fusion proteins containing alpha 1-alpha 2 domain variants that bind to NKG2D with high affinity bound to NK cells in vivo. Thus, an antigen-specific antibody containing a modified α1-α2 fusion binds tightly to NKG2D, thereby effectively arming the surface of the NK cell with the antibody in vivo to find a target cell expressing the specific antigen. These activities were similar to engineered CAR cells (Gill, S. and June, CH, 2015). Going viral: chimeric antigen receptor T-cell therapy for hematological malignancies. Immunol Rev 263 , 68-89.), NKG2D- No genetic modification of the expressed cell type was required.

To demonstrate that antibodies containing modified α1-α2 fusions bind to NK cells in vivo, the heavy and light chain fusion of Trastuzumab and the corresponding variant 25 was incubated with the pharmacodynamics (PD) of NK cell labeling and serum Pharmacokinetic (PK) profiles were analyzed in vivo. All three antibodies: Parent trastuzumab; Trastuzumab HC_25 fusions; And trastuzumab LC_25 fusions were conjugated to Alexa Flour 647 according to the manufacturer's protocol (Life Technologies). Groups of these C57BL / 6 mice were injected with a single dose of 100 μg of each antibody and blood samples were drawn at the indicated time points for flow cytometric analysis of plasma PK ELISA and peripheral NK cells. The PK profile of the parental Trastuzumab antibody showed a typical alpha phase distribution within 24 hours and the beta phase elimination was consistent with the one week half-life of the antibody in the mouse (Figure 22a). For both heavy and light chain fusions with variant 25, the initial alpha phase coincided with the NKG2D-synch, resulting in a much larger volume distribution relative to the parent antibody, while beta phase elimination also resulted in a typical antibody clearance ) (Fig. 22A). Using flow cytometry analysis of peripheral NK cells from mouse blood, the NK cell staining level with Alexa Fluor 647 showed a clear time-dependent increase in the percentage of NK cells labeled with antibody fusions, but not with parental antibodies (Fig. 22B). The increase in labeling by the fusion peak within 24 hours was consistent with the sync observed in the PK profile for the fusion and was stable for at least 3 days after injection. Combined PK and PD data demonstrate that a trastuzumab antibody containing a variant 25 [alpha] - [alpha] 2 fusion formed a stable complex with NKG2D in NK cells in vivo.

Plasma cells from PK / PD studies were evaluated for ADA using ELISA to assess the appearance of anti-drug antibodies (ADA) against human IgG Trastuzumab antibodies. In Figures 23a-c, ELISA for mouse IgG binding to wells coated with three each administered antibody revealed that only antibodies fused with variant 25 induced ADA within 7 days after a single dose of antibody. The parent trastuzumab antibody did not provide an ADA signal. To determine whether an antibody fusion elicited an immune (ADA) response to both the alpha 1-alpha 2 domain and the antibody (trastuzumab) component when the trastuzumab antibody itself did not elicit an immune response, antibody fusion ADA-positive plasma from water was tested separately for the parent antibody and variant 25 [alpha] 1- alpha 2 domains; Both moieties reacted with ADA from plasma (Figures 24a and 24b). These data demonstrate that the fusion of high affinity variant 25 to the parent antibody mediated NKG2D-dependent uptake and antigen presentation, leading to a potent and rapid immune response to the parent antibody (not immunogenic in mice alone) . Thus, the high-affinity mutant α1-α2 domain attached to an antigen or immunogen has provided powerful antigen presentation and epitope spreading, effectively acting as a powerful adjuvant for immunization.

The proven combinatorial effect of arming circulating NK cells and enhancing antigen presentation for induced target cell lysis is an important activity for antibody fusions against the modified [alpha] 1- [alpha] 2 domain that can provide therapeutic benefits.

Example 8 (Antibody heavy chain fusion to alpha 1-alpha2 mutant 25 showed anti-tumor activity in vivo) . In order to test the possibility that the antigen-specific antibody fused to the modified α1-α2 has anti-target cell activity, the anti-PD1 antibody heavy chain fusion to variant 25 α1-α2 was evaluated in the winter MC38 tumor model. MC38 tumors were subcutaneously transplanted into C57BL / 6 mice and tumors were grown to an average of 100 ㎣ before initiation of treatment. At the start of treatment, four cohorts of 10 mice per group on days 1, 4 and 7 were injected intravenously into vehicle, 100 μg ip, parent anti-PDL1 (300 ug iv) or anti-PDL1 HC_25 fusion 300 ug iv). In Figure 25, the tumor growth curve showed that anti-PD1 HC_25 mediates the most effective antitumor activity within the first two weeks of treatment. Tumor growth inhibition was significantly better over the first 12 days after initiation of treatment than established anti-CTLA4 treatment and parental anti-PDL1 antibody. By day 16, the anti-PDL1 HC_25 treatment began to lose efficacy consistent with the occurrence of the ADA response (Example 7), as observed for the Trastuzumab fusions. The significant antitumor activity observed for antibody heavy chain fusion to variant 25 compared to both parental antibody and standard anti-CTLA4 treatment was due to an impressive treatment of antibody fusions against the modified &lt; RTI ID = 0.0 &gt; ai-a2 &lt; / RTI & Proved its effectiveness.

Example 9 (Binding of modified α1-α2 domain of ULBP fused to antibody peptide and cell lysis) . The following example relates to attaching an antibody polypeptide to modified NKG2DL to significantly enhance its binding affinity for human and murine NKG2D receptors. The α1-α2 domain of the ULBP protein is a natural ligand for the NKG2D receptor, NKG2DL. Antibodies are highly stable glycoproteins made from two large heavy chains and two small light chains (Figure 1). The art does not have an IgG antibody format capable of directly activating immune cells using a non-natural ULBP alpha 1-alpha 2 domain that binds more tightly to the NKG2D receptor than the native ULBP domain. In addition, the ULBP α1-α2 domain provides an alternative NKG2DL such that an antibody fusion is constructed that may have differential in vivo characteristics over the MICA α1-α2 domain. For example, the in vivo anti-drug antibody response to the MICA alpha 1-alpha 2 domain in the antibody fusion reacts with or interferes with the modified ULBP alpha 1-alpha 2 domain due to the low sequence homology between the ULBP and the MICA alpha 1- (Fig. 18). This example demonstrates that fusion between engineered ULBP α1-α2 NKG2D ligands (Tables 6 and 7) and the heavy chain of IgG molecules (FIG. 20a) enhances NKG2D binding and target cell death relative to the natural ULBP α1-α2 NKG2D ligand . It also demonstrated the utility of a fusion of a modified α1-α2 domain to a heterologous protein or peptide.

(WT) ULBP2, mutants R80W and V151D (SEQ ID NOs: 16, 84 and 85, respectively), and natural (WT) ULBP3 and mutant R162G SEQ ID NOs: 17 and 86) were synthesized and cloned as a C-terminal fusion to the heavy chain sequence from the Her2-specific antibody used in Example 6 (Carter, (1992), Proc., Presta, L., Gorman, CM., Ridgway, JB., Henner, D., Wong, R., Rowland, A., Kotts, C, Carver, ME, Natl Acad Sci 15, 4285-9.). The resulting fusion was cloned into the mammalian expression vector pD2509 and expressed as a whole IgG antibody paired with the light chain of the parent antibody. Transient expression was performed in HEK293 cells using the Expi293 expression system according to the manufacturer's protocol (Life Technologies) and purified using standard Protein A Affinity Chromatography. Binding ELISAs performed on ULBP2 and ULBP3 alpha 1-alpha 2 antibody heavy chain fusion showed that modified ULBP2 fusions (HC_R80W and HC_V151D) and UBLP3 fusions (HC_R162G) were fused to their respective natural α1-α2 domains fused to the same heavy chain Lt; RTI ID = 0.0 &gt; NKG2D &lt; / RTI &gt; (Figs. 26A and 26B).

In order to analyze the target cell killing characteristics of the modified ULBP antibody fusion, the human natural killer (NK) cell line, NKL, was co-cultured with callus-loaded SKBR3 target cells expressing Her2 and titrated with the engineered antibody fusion protein Respectively. The results in Figures 27a and 27b show that enhanced cytolytic (killing) activity of Her2-specific non-natural ULBP2 and non-natural ULBP3 [alpha] 1- [alpha] 2-antibody fusion is associated with enhanced And reflects the affinity that has been achieved. Specifically, the ULBP2 mutant fusions HC_R80W and HC_V151D, and the ULBP3 mutant fusion product HC_R162G, more effectively killed SKBR3 cells than the antibody fusions containing the native α1-α2 domain. These data also show that modified α1-α2 variant-antibody fusions are a universal platform that allows IgG molecules to bind tightly to NKG2D and induce antigen-specific cell lysis.

SEQUENCE LISTING <110> AvidBiotics Corp. <120> INSERTABLE VARIABLE FRAGMENTS OF ANTIBODIES AND MODIFIED ALPHA1-ALPHA2 DOMAINS OF NKG2D LIGANDS <130> A227526 <150> US 62 / 088,456 <151> 2014-12-05 <150> US 14 / 959,745 <151> 2015-12-04 <160> 86 <170> PatentIn version 3.5 <210> 1 <211> 1126 <212> DNA <213> Artificial Sequence <220> Synthetic polynucleotide, encoding alpha3-iFv.1 <400> 1 ccccccatgg tgcaagttac ccgcagcgag gcctcaggag atcgcgtaac tatcacttgc 60 agagcttctc aggacgtgtc caccgcggtt gcttggtacc agcaaaagcc tggaaaggcg 120 ccgaagctgc tgatctactc cgcctcattc ttgtactcag gagtgcccag tcgatttagt 180 ggtagcggtt ctggtactga tttcaccctt accatcagca gtctccagcc cgaggatttc 240 gctacttatt actgccagca gtcatacacc actcctccca ctttcggcca aggtaccaag 300 gtcgagatta aaggcggaag ctctaggtcc tctagctccg gaggaggtgg ctctggcggc 360 ggcggagaag tccaactggt ggagagcgga ggcggactgg tgcagccagg cggatccttg 420 agacttagct gtgcggcttc gggttttacc tttacttcta ctggcatcag ttgggtcaga 480 caagcgcctg gcaagggact ggaatgggtt ggacgtatct accccactaa tggttcgacg 540 aactatgcgg atagtgtgaa aggtagattc acgatatctg ctgacacctc gaagaatacc 600 gcttaccttc aaatgaatag tttgcgtgcc gaagatactg ctgtctacta ttgcgccaga 660 acctatggaa tatacgacct ttatgtggac tacaccgagt acgtcatgga ttattggggc 720 cagggtacgt tggtgacagt gtcgagtggc ggaagctcta ggtcctctag ctccggagga 780 ggtggctctg gcggcggcgg agacattcag atgactcagt ctcccagttc tcttagtgcc 840 tctggccaaa ttaccgtcac gtgtcgtgct agcggcttct acccgtggaa tatcaccctg 900 agctggcgcc aagacggtgt tagcctgagc cacgacaccc aacaatgggg cgacgtgttg 960 ccagatggcc aaggtaccta ccagacgtgg gttgccaccc gtatttccca gggtgaagag 1020 cagcgtttta cctgctatat ggaacacagc ggccaacata gcacgcatcc ggtgccgagc 1080 ggtaaaggta gccaccatca tcaccaccat tagtaggaat tccgga <210> 2 <211> 1144 <212> DNA <213> Artificial Sequence <220> Synthetic polynucleotide, encoding alpha3-iFv.2 <400> 2 ccccccatgg tgcaagttac ccgcagcgag gcctcaggcg gaagcggaga tcgcgtaact 60 atcacttgca gagcttctca ggacgtgtcc accgcggttg cttggtacca gcaaaagcct 120 ggaaaggcgc cgaagctgct gatctactcc gcctcattct tgtactcagg agtgcccagt 180 cgatttagtg gtagcggttc tggtactgat ttcaccctta ccatcagcag tctccagccc 240 gaggatttcg ctacttatta ctgccagcag tcatacacca ctcctcccac tttcggccaa 300 ggtaccaagg tcgagattaa aggcggaagc tctaggtcct ctagctccgg aggaggtggc 360 tctggcggcg gcggagaagt ccaactggtg gagagcggag gcggactggt gcagccaggc 420 ggatccttga gacttagctg tgcggcttcg ggttttacct ttacttctac tggcatcagt 480 tgggtcagac aagcgcctgg caagggactg gaatgggttg gacgtatcta ccccactaat 540 ggttcgacga actatgcgga tagtgtgaaa ggtagattca cgatatctgc tgacacctcg 600 aagaataccg cttaccttca aatgaatagt ttgcgtgccg aagatactgc tgtctactat 660 tgcgccagaa cctatggaat atacgacctt tatgtggact acaccgagta cgtcatggat 720 tattggggcc agggtacgtt ggtgacagtg tcgagtggcg gaagctctag gtcctctagc 780 tccggaggag gtggctctgg cggcggcgga gacattcaga tgactcagtc tcccagttct 840 cttagtgcct ctggcggaag cggccaaatt accgtcacgt gtcgtgctag cggcttctac 900 ccgtggaata tcaccctgag ctggcgccaa gacggtgtta gcctgagcca cgacacccaa 960 caatggggcg acgtgttgcc agatggccaa ggtacctacc agacgtgggt tgccacccgt 1020 atttcccagg gtgaagagca gcgttttacc tgctatatgg aacacagcgg ccaacatagc 1080 acgcatccgg tgccgagcgg taaaggtagc caccatcatc accaccatta gtaggaattc 1140 cgga 1144 <210> 3 <211> 269 <212> PRT <213> Artificial Sequence <220> Synthetic peptide iFv (fgfr3) <400> 3 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr 15 10 15 Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 20 25 30 Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Arg Phe Ser 35 40 45 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 50 55 60 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro 65 70 75 80 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Ser Ser 85 90 95 Arg Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 100 105 110 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 115 120 125 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr Gly Ile 130 135 140 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg 145 150 155 160 Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 165 170 175 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 180 185 190 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 195 200 205 Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr Val Met 210 215 220 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 225 230 235 240 Ser Arg Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp 245 250 255 Ile Gln Met Thr Gln Ser Ser Ser Ser Leu Ser Ala Ser 260 265 <210> 4 <211> 807 <212> DNA <213> Artificial Sequence <220> Synthetic polynucleotide, encoding iFv (fgfr3) <400> 4 ggagatcgcg taactatcac ttgcagagct tctcaggacg tgtccaccgc ggttgcttgg 60 taccagcaaa agcctggaaa ggcgccgaag ctgctgatct actccgcctc attcttgtac 120 tcaggagtgc ccagtcgatt tagtggtagc ggttctggta ctgatttcac ccttaccatc 180 agcagtctcc agcccgagga tttcgctact tattactgcc agcagtcata caccactcct 240 cccactttcg gccaaggtac caaggtcgag attaaaggcg gaagctctag gtcctctagc 300 tccggaggag gtggctctgg cggcggcgga gaagtccaac tggtggagag cggaggcgga 360 ctggtgcagc caggcggatc cttgagactt agctgtgcgg cttcgggttt tacctttact 420 tctactggca tcagttgggt cagacaagcg cctggcaagg gactggaatg ggttggacgt 480 atctacccca ctaatggttc gacgaactat gcggatagtg tgaaaggtag attcacgata 540 tctgctgaca cctcgaagaa taccgcttac cttcaaatga atagtttgcg tgccgaagat 600 actgctgtct actattgcgc cagaacctat ggaatatacg acctttatgt ggactacacc 660 ggtacgtca tggattattg gggccagggt acgttggtga cagtgtcgag tggcggaagc 720 tctaggtcct ctagctccgg aggaggtggc tctggcggcg gcggagacat tcagatgact 780 cagtctccca gttctcttag tgcctct 807 <210> 5 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide linker region <400> 5 Gly Gly Ser Ser Arg Ser Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly 15 10 15 Gly Gly <210> 6 <211> 370 <212> PRT <213> Artificial Sequence <220> Synthetic peptide alpha3-iFv.1 <400> 6 Pro Pro Met Val Gln Val Thr Arg Ser Glu Ala Ser Gly Asp Arg Val 15 10 15 Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp 20 25 30 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 35 40 45 Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 50 55 60 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 65 70 75 80 Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro Thr Phe Gly 85 90 95 Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Ser Ser Ser Ser Ser Ser 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Tyr Pro Thr 165 170 175 Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile 210 215 220 Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr Val Met Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Ser Ser Ser Ser 245 250 255 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile Gln Met Thr 260 265 270 Gln Ser Ser Ser Leu Ser Ala Ser Gly Gln Ile Thr Val Thr Cys 275 280 285 Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln 290 295 300 Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu 305 310 315 320 Pro Asp Gly Gln Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser 325 330 335 Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln 340 345 350 His Ser Thr His Pro Val Ser Ser Gly Lys Gly Ser His His His 355 360 365 His His 370 <210> 7 <211> 376 <212> PRT <213> Artificial Sequence <220> Synthetic peptide alpha3-iFv.2 <400> 7 Pro Pro Met Val Gln Val Thr Arg Ser Glu Ala Ser Gly Gly Ser Gly 15 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser 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 Ser Ser Phe Ser Gly 50 55 60 Ser Gly 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 Ser Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Ser Ser Arg 100 105 110 Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Glu Val Gln 115 120 125 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser 145 150 155 160 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile 165 170 175 Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr 210 215 220 Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr Val Met Asp 225 230 235 240 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Ser 245 250 255 Arg Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile 260 265 270 Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Gly Gly Ser Ser 275 280 285 Gln Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile 290 295 300 Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr Gln 305 310 315 320 Gln Trp Gly Asp Val Leu Pro Asp Gly Gln Gly Thr Tyr Gln Thr Trp 325 330 335 Val Ala Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr 340 345 350 Met Glu His Ser Gly Gln His Ser Thr His Pro Val Ser Ser Gly Lys 355 360 365 Gly Ser His His His His His 370 375 <210> 8 <211> 368 <212> PRT <213> Artificial Sequence <220> Synthetic peptide alpha3-iFv.3 (CD20) <400> 8 Pro Pro Met Val Gln Val Thr Arg Ser Glu Ala Ser Gly Gly Ser Gly 15 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 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 Gly Gly Ser Ser Arg Ser 100 105 110 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly GInt GInt Leu 115 120 125 Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met 130 135 140 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp 145 150 155 160 Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly Ala Ile Tyr 165 170 175 Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala 180 185 190 Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser 195 200 205 Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Thr 210 215 220 Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr 225 230 235 240 Val Thr Val Ser Ala Gly Gly Ser Ser Ser Ser Ser Ser Ser Gly Gly 245 250 255 Gly Gly Gly Gly Gly Gly Gln Ile Val Leu Ser Gln Ser Pro Ala 260 265 270 Ile Leu Ser Ala Ser Gly Gly Ser Gln Ile Thr Val Thr Cys Arg Ala 275 280 285 Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly 290 295 300 Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp 305 310 315 320 Gly Gln Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly 325 330 335 Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser 340 345 350 Thr His Pro Val Ser Ser Gly Lys Gly Ser His His His His His 355 360 365 <210> 9 <211> 561 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding ULBP1 alpha1-alpha2 <400> 9 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> 10 <211> 567 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding ULBP2 alpha1-alpha2 <400> 10 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 ccatcggctg gtgccccccc catggtg 567 <210> 11 <211> 561 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding ULBP3 alpha1-alpha2 <400> 11 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> 12 <211> 561 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding ULBP4 alpha1-alpha2 <400> 12 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> 13 <211> 567 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding ILBP6 alpha1-alpha2 <400> 13 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> 14 <211> 483 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding OMCP alpha1-alpha2 <400> 14 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> 15 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide ULBP1 alpha1-alpha2 <400> 15 Ala Ala Glu Pro His Cys Leu Cys Tyr Asp Phe Ile Ile Thr Pro Lys 15 10 15 Ser Arg Pro Glu Pro Gln Trp Cys Glu Val Gln Gly Leu Val Asp Glu 20 25 30 Arg Pro Phe Leu His Tyr Asp Cys Val Asn His Lys Ala Lys Ala Phe 35 40 45 Ala Ser Leu Gly Lys Lys Val Asn Val Thr Lys Thr Trp Glu Glu Gln 50 55 60 Thr Glu Thr Leu Arg Asp Val Val Asp Phe Leu Lys Gly Gln Leu Leu 65 70 75 80 Asp Ile Gln Val Glu Asn Leu Ile Pro Ile Glu Pro Leu Thr Leu Gln 85 90 95 Ala Arg Met Ser Cys Glu His Glu Ala His Gly His Gly Arg Gly Ser 100 105 110 Trp Gln Phe Leu Phe Asn Gly Gln Lys Phe Leu Leu Phe Asp Ser Asn 115 120 125 Asn Arg Lys Trp Thr Ala Leu His Pro Gly Ala Lys Lys Met Thr Glu 130 135 140 Lys Trp Glu Lys Asn Arg Asp Val Thr Met Phe Phe Gln Lys Ile Ser 145 150 155 160 Leu Gly Asp Cys Lys Met Trp Leu Glu Glu Phe Leu Met Tyr Trp Glu 165 170 175 Gln Met Leu Asp Pro Thr Lys Pro Pro Met Val 180 185 <210> 16 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide ULBP2 alpha1-alpha2 <400> 16 Ala Ala Glu Pro His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys 15 10 15 Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu 20 25 30 Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val 35 40 45 Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln 50 55 60 Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Arg 65 70 75 80 Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln 85 90 95 Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser 100 105 110 Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu 115 120 125 Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu 130 135 140 Lys Trp Glu Asn Asp Lys Val Val Ala Met Ser Phe His Tyr Phe Ser 145 150 155 160 Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp 165 170 175 Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro Pro Met Val 180 185 <210> 17 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide ULBP3 alpha1-alpha2 <400> 17 Ala Ala Glu Pro His Ser Leu Trp Tyr Asn Phe Thr Ile Ile His Leu 15 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> 18 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide ULBP4 alpha1-alpha2 <400> 18 Ala Ala Glu Pro His Ser Leu Cys Phe Asn Phe Thr Ile Lys Ser Leu 15 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> 19 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide ULBP6 alpha1-alpha2 <400> 19 Ala Ala Glu Pro His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys 15 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> 20 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide OMCP alpha1-alpha2 <400> 20 Ala Ala Ala Glu Pro His Lys Leu Ala Phe Asn Phe Asn Leu Glu Ile 15 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> 21 <211> 580 <212> PRT <213> Artificial Sequence <220> Synthetic peptide ULBP1-alpha3-iFv.2 <400> 21 Met Gly Leu Gly Pro Val Phe Leu Leu Leu Ala Gly Ile Phe Pro Phe 15 10 15 Ala Pro Pro Gly Ala Ala Ala Glu Pro His Cys Leu Cys Tyr Asp Phe 20 25 30 Ile Ile Thr Pro Lys Ser Arg Pro Glu Pro Gln Trp Cys Glu Val Gln 35 40 45 Gly Leu Val Asp Glu Arg Pro Phe Leu His Tyr Asp Cys Val Asn His 50 55 60 Lys Ala Lys Ala Phe Ala Ser Leu Gly Lys Lys Val Asn Val Thr Lys 65 70 75 80 Thr Trp Glu Glu Gln Thr Glu Thr Leu Arg Asp Val Val Asp Phe Leu 85 90 95 Lys Gly Gln Leu Leu Asp Ile Gln Val Glu Asn Leu Ile Pro Ile Glu 100 105 110 Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu His Glu Ala His Gly 115 120 125 His Gly Arg Gly Ser Trp Gln Phe Leu Phe Asn Gly Gln Lys Phe Leu 130 135 140 Leu Phe Asp Ser Asn Asn Arg Lys Trp Thr Ala Leu His Pro Gly Ala 145 150 155 160 Lys Lys Met Thr Glu Lys Trp Glu Lys Asn Arg Asp Val Thr Met Phe 165 170 175 Phe Gln Lys Ile Ser Leu Gly Asp Cys Lys Met Trp Leu Glu Glu Phe 180 185 190 Leu Met Tyr Trp Glu Gln Met Leu Asp Pro Thr Lys Pro Pro Met Val 195 200 205 Gln Val Thr Arg Ser Glu Ala Ser Gly Gly Ser Gly Asp Arg Val Thr 210 215 220 Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr 225 230 235 240 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 245 250 255 Phe Leu Tyr Ser Gly Val Ser Ser Gly Ser Gly 260 265 270 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 275 280 285 Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln 290 295 300 Gly Thr Lys Val Glu Ile Lys Gly Ser Ser Ser Ser Ser Ser Ser 305 310 315 320 Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser 325 330 335 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 340 345 350 Ala Ser Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser Trp Val Arg Gln 355 360 365 Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn 370 375 380 Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 385 390 395 400 Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg 405 410 415 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr 420 425 430 Asp Leu Tyr Val Asp Tyr Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln 435 440 445 Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ser Ser Ser Ser Ser 450 455 460 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile Gln Met Thr Gln 465 470 475 480 Ser Pro Ser Ser Leu Ser Ala Ser Gly Gly Ser Gly Gln Ile Thr Val 485 490 495 Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp 500 505 510 Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp 515 520 525 Val Leu Pro Asp Gly Gln Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg 530 535 540 Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser 545 550 555 560 Gly Gln His Ser Thr His Pro Val Ser Ser Gly Lys Gly Ser His His 565 570 575 His His His His 580 <210> 22 <211> 582 <212> PRT <213> Artificial Sequence <220> Synthetic peptide ULBP2-alpha3-iFv.2 <400> 22 Met Gly Leu Gly Pro Val Phe Leu Leu Leu Ala Gly Ile Phe Pro Phe 15 10 15 Ala Pro Pro Gly Ala Ala Glu Pro His Ser Leu Cys Tyr Asp Ile 20 25 30 Thr Val Ile Pro Lys Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln 35 40 45 Gly Gln Val Asp Glu Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys 50 55 60 Thr Val Thr Pro Val Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr 65 70 75 80 Ala Trp Lys Ala Gln Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu 85 90 95 Thr Glu Gln Leu Arg Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu 100 105 110 Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly 115 120 125 His Ser Ser Gly Ser Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu 130 135 140 Leu Phe Asp Ser Glu Lys Arg Met Trp Thr Thr Val His Pro Gly Ala 145 150 155 160 Arg Lys Met Lys Glu Lys Trp Glu Asn Asp Lys Val Val Ala Met Ser 165 170 175 Phe His Tyr Phe Ser Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe 180 185 190 Leu Met Gly Met Asp Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro Pro 195 200 205 Met Val Gln Val Thr Arg Ser Glu Ala Ser Gly Gly Ser Gly Asp Arg 210 215 220 Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala 225 230 235 240 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser 245 250 255 Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly Ser Gly 260 265 270 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 275 280 285 Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro Thr Phe 290 295 300 Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Ser Ser Ser Ser Ser 305 310 315 320 Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Glu Val Gln Leu Val 325 330 335 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 340 345 350 Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser Trp Val 355 360 365 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Tyr Pro 370 375 380 Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 385 390 395 400 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 405 410 415 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Gly 420 425 430 Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr Val Met Asp Tyr Trp 435 440 445 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Ser Arg Ser 450 455 460 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile Gln Met 465 470 475 480 Thr Gln Ser Ser Ser Leu Ser Ala Ser Gly Gly 485 490 495 Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu 500 505 510 Ser Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp 515 520 525 Gly Asp Val Leu Pro Asp Gly Gln Gly Thr Tyr Gln Thr Trp Val Ala 530 535 540 Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu 545 550 555 560 His Ser Gly Gln His Ser Thr His Pro Val Ser Ser Gly Lys Gly Ser 565 570 575 His His His His His 580 <210> 23 <211> 580 <212> PRT <213> Artificial Sequence <220> Synthetic peptide ULBP3-alpha3-iFv.2 <400> 23 Met Gly Leu Gly Pro Val Phe Leu Leu Leu Ala Gly Ile Phe Pro Phe 15 10 15 Ala Pro Pro Gly Ala Ala Ala Glu Pro His Ser Leu Trp Tyr Asn Phe 20 25 30 Thr Ile Ile His Leu Pro Arg His Gly Gln Gln Trp Cys Glu Val Gln 35 40 45 Ser Gln Val Asp Gln Lys Asn Phe Leu Ser Tyr Asp Cys Gly Ser Asp 50 55 60 Lys Val Leu Ser Met Gly His Leu Glu Glu Gln Leu Tyr Ala Thr Asp 65 70 75 80 Ala Trp Gly Lys Gln Leu Glu Met Leu Arg Glu Val Gly Gln Arg Leu 85 90 95 Arg Leu Glu Leu Ala Asp Thr Glu Leu Glu Asp Phe Thr Pro Ser Gly 100 105 110 Pro Leu Thr Leu Gln Val Arg Met Ser Cys Glu Cys Glu Ala Asp Gly 115 120 125 Tyr Ile Arg Gly Ser Trp Gln Phe Ser Phe Asp Gly Arg Lys Phe Leu 130 135 140 Leu Phe Asp Ser Asn Asn Arg Lys Trp Thr Val Val His Ala Gly Ala 145 150 155 160 Arg Arg Met Lys Glu Lys Trp Glu Lys Asp Ser Gly Leu Thr Thr Phe 165 170 175 Phe Lys Met Val Ser Met Arg Asp Cys Lys Ser Trp Leu Arg Asp Phe 180 185 190 Leu Met His Arg Lys Lys Arg Leu Glu Pro Thr Ala Pro Pro Met Val 195 200 205 Gln Val Thr Arg Ser Glu Ala Ser Gly Gly Ser Gly Asp Arg Val Thr 210 215 220 Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr 225 230 235 240 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 245 250 255 Phe Leu Tyr Ser Gly Val Ser Ser Gly Ser Gly 260 265 270 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 275 280 285 Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln 290 295 300 Gly Thr Lys Val Glu Ile Lys Gly Ser Ser Ser Ser Ser Ser Ser 305 310 315 320 Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser 325 330 335 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 340 345 350 Ala Ser Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser Trp Val Arg Gln 355 360 365 Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn 370 375 380 Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 385 390 395 400 Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg 405 410 415 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr 420 425 430 Asp Leu Tyr Val Asp Tyr Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln 435 440 445 Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ser Ser Ser Ser Ser 450 455 460 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile Gln Met Thr Gln 465 470 475 480 Ser Pro Ser Ser Leu Ser Ala Ser Gly Gly Ser Gly Gln Ile Thr Val 485 490 495 Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp 500 505 510 Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp 515 520 525 Val Leu Pro Asp Gly Gln Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg 530 535 540 Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser 545 550 555 560 Gly Gln His Ser Thr His Pro Val Ser Ser Gly Lys Gly Ser His His 565 570 575 His His His His 580 <210> 24 <211> 580 <212> PRT <213> Artificial Sequence <220> Synthetic peptide ULBP4-alpha3-iFv.2 <400> 24 Met Gly Leu Gly Pro Val Phe Leu Leu Leu Ala Gly Ile Phe Pro Phe 15 10 15 Ala Pro Pro Gly Ala Ala Glu Pro His Ser Leu Cys Phe Asn Phe 20 25 30 Thr Ile Lys Ser Leu Ser Arg Pro Gly Gln Pro Trp Cys Glu Ala Gln 35 40 45 Val Phe Leu Asn Lys Asn Leu Phe Leu Gln Tyr Asn Ser Asp Asn Asn 50 55 60 Met Val Lys Pro Leu Gly Leu Leu Gly Lys Lys Val Tyr Ala Thr Ser 65 70 75 80 Thr Trp Gly Glu Leu Thr Gln Thr Leu Gly Glu Val Gly Arg Asp Leu 85 90 95 Arg Met Leu Leu Cys Asp Ile Lys Pro Gln Ile Lys Thr Ser Asp Pro 100 105 110 Ser Thr Leu Gln Val Glu Met Phe Cys Gln Arg Glu Ala Glu Arg Cys 115 120 125 Thr Gly Ala Ser Trp Gln Phe Ala Thr Asn Gly Glu Lys Ser Leu Leu 130 135 140 Phe Asp Ala Met Asn Met Thr Trp Thr Val Ile Asn His Glu Ala Ser 145 150 155 160 Lys Ile Lys Glu Thr Trp Lys Lys Asp Arg Gly Leu Glu Lys Tyr Phe 165 170 175 Arg Lys Leu Ser Lys Gly Asp Cys Asp His Trp Leu Arg Glu Phe Leu 180 185 190 Gly His Trp Glu Ala Met Pro Glu Pro Thr Val Ser Pro Pro Met Val 195 200 205 Gln Val Thr Arg Ser Glu Ala Ser Gly Gly Ser Gly Asp Arg Val Thr 210 215 220 Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr 225 230 235 240 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 245 250 255 Phe Leu Tyr Ser Gly Val Ser Ser Gly Ser Gly 260 265 270 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 275 280 285 Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln 290 295 300 Gly Thr Lys Val Glu Ile Lys Gly Ser Ser Ser Ser Ser Ser Ser 305 310 315 320 Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser 325 330 335 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 340 345 350 Ala Ser Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser Trp Val Arg Gln 355 360 365 Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn 370 375 380 Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 385 390 395 400 Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg 405 410 415 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr 420 425 430 Asp Leu Tyr Val Asp Tyr Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln 435 440 445 Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ser Ser Ser Ser Ser 450 455 460 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile Gln Met Thr Gln 465 470 475 480 Ser Pro Ser Ser Leu Ser Ala Ser Gly Gly Ser Gly Gln Ile Thr Val 485 490 495 Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp 500 505 510 Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp 515 520 525 Val Leu Pro Asp Gly Gln Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg 530 535 540 Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser 545 550 555 560 Gly Gln His Ser Thr His Pro Val Ser Ser Gly Lys Gly Ser His His 565 570 575 His His His His 580 <210> 25 <211> 582 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide ULBP6-alpha3-iFv.2 <400> 25 Met Gly Leu Gly Pro Val Phe Leu Leu Leu Ala Gly Ile Phe Pro Phe 15 10 15 Ala Pro Pro Gly Ala Ala Glu Pro His Ser Leu Cys Tyr Asp Ile 20 25 30 Thr Val Ile Pro Lys Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln 35 40 45 Gly Gln Val Asp Glu Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys 50 55 60 Thr Val Thr Pro Val Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr 65 70 75 80 Ala Trp Lys Ala Gln Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu 85 90 95 Thr Glu Gln Leu Leu Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu 100 105 110 Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly 115 120 125 His Ser Ser Gly Ser Trp Gln Phe Ser Ile Asp Gly Gln Thr Phe Leu 130 135 140 Leu Phe Asp Ser Glu Lys Arg Met Trp Thr Thr Val His Pro Gly Ala 145 150 155 160 Arg Lys Met Lys Glu Lys Trp Glu Asn Asp Lys Asp Val Ala Met Ser 165 170 175 Phe His Tyr Ile Ser Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe 180 185 190 Leu Met Gly Met Asp Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro Pro 195 200 205 Met Val Gln Val Thr Arg Ser Glu Ala Ser Gly Gly Ser Gly Asp Arg 210 215 220 Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala 225 230 235 240 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser 245 250 255 Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly Ser Gly 260 265 270 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 275 280 285 Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro Thr Phe 290 295 300 Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Ser Ser Ser Ser Ser 305 310 315 320 Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Glu Val Gln Leu Val 325 330 335 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 340 345 350 Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr Gly Ile Ser Trp Val 355 360 365 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Tyr Pro 370 375 380 Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 385 390 395 400 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 405 410 415 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Gly 420 425 430 Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr Val Met Asp Tyr Trp 435 440 445 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Ser Arg Ser 450 455 460 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile Gln Met 465 470 475 480 Thr Gln Ser Ser Ser Leu Ser Ala Ser Gly Gly 485 490 495 Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu 500 505 510 Ser Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp 515 520 525 Gly Asp Val Leu Pro Asp Gly Gln Gly Thr Tyr Gln Thr Trp Val Ala 530 535 540 Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu 545 550 555 560 His Ser Gly Gln His Ser Thr His Pro Val Ser Ser Gly Lys Gly Ser 565 570 575 His His His His His 580 <210> 26 <211> 553 <212> PRT <213> Artificial Sequence <220> Synthetic peptide OMCP-alpha3-iFv.2 <400> 26 Met Gly Leu Gly Pro Val Phe Leu Leu Leu Ala Gly Ile Phe Pro Phe 15 10 15 Ala Pro Pro Gly Ala Ala Ala Glu Pro His Lys Leu Ala Phe Asn Phe 20 25 30 Asn Leu Glu Ile Asn Gly Ser Asp Thr His Ser Thr Val Asp Val Tyr 35 40 45 Leu Asp Asp Ser Gln Ile Ile Thr Phe Asp Gly Lys Asp Ile Arg Pro 50 55 60 Thr Ile Pro Phe Met Ile Gly Asp Glu Ile Phe Leu Pro Phe Tyr Lys 65 70 75 80 Asn Val Phe Ser Glu Phe Phe Ser Leu Phe Arg Arg Val Thr Ser 85 90 95 Thr Pro Tyr Glu Asp Leu Thr Tyr Phe Tyr Glu Cys Asp Tyr Thr Asp 100 105 110 Asn Lys Ser Thr Phe Asp Gln Phe Tyr Leu Tyr Asn Gly Glu Glu Tyr 115 120 125 Thr Val Lys Thr Gln Glu Ala Thr Asn Lys Asn Met Trp Leu Thr Thr 130 135 140 Ser Glu Phe Arg Leu Lys Lys Trp Phe Asp Gly Glu Asp Cys Ile Met 145 150 155 160 His Leu Arg Ser Leu Val Arg Lys Met Glu Asp Ser Lys Arg Arg Thr 165 170 175 Val Pro Pro Met Val Gln Val Thr Arg Ser Glu Ala Ser Gly Gly Ser 180 185 190 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr 195 200 205 Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 210 215 220 Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Arg Phe Ser 225 230 235 240 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 245 250 255 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro 260 265 270 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Ser Ser 275 280 285 Arg Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val 290 295 300 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 305 310 315 320 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr Gly Ile 325 330 335 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg 340 345 350 Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly 355 360 365 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 370 375 380 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 385 390 395 400 Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr Val Met 405 410 415 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser 420 425 430 Ser Arg Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp 435 440 445 Ile Gln Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Gly Gly Ser 450 455 460 Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn 465 470 475 480 Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr 485 490 495 Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln Gly Thr Tyr Gln Thr 500 505 510 Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr Cys 515 520 525 Tyr Met Glu His Ser Gly Gln His Ser Thr His Pro Val Ser Ser Gly 530 535 540 Lys Gly Ser His His His His His His 545 550 <210> <211> <212> <213> 27 507 DNA Artificial Sequence <220> <223> Synthetic polynucleotide, encoding alpha1-alpha2 variant 15 <400> 27 gctgctgagc cacacagtct ccgctacaac cttacggtgt tgagctggga cggctctgtc 60 cagagtggct ttctgactga ggtacatctc gatggtcagc ccttcctccg atgcgacaga 120 caaaagtgca gggccaagcc acagggccaa tgggccgaag atgtacttgg caataagact 180 tgggacagag aaaccagaga tctgactggc tggggtaagg acttacgcat gactctcgca 240 cacattaaag accagaagga aggtcttcat tcgctccagg aaattagagt ctgtgaaatc 300 catgaagaca acagcacaag aagttcccaa catttctact acgacggcga gctgttctta 360 ccagaccctc gctatgaatg tgaggaattt ccttaaggag gacgctatgg aaactgacac ccactaccat 480 gcgatgcgcg ccgattgcct gcaggaa 507 <210> 28 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding alpha1-alpha2 variant 16 <400> 28 gctgctgagc cacacagtct ccgctacaac cttacggtgt tgagctggga cggctctgtc 60 cagagtggct ttctgactga ggtacatctc gatggtcagc ccttcctccg atgcgacaga 120 caaaagtgca gggccaagcc acagggccaa tgggccgaag atgtacttgg caataagact 180 tgggacagag aaaccagaga tctgactggc tggggtaagg acttacgcat gactctcgca 240 cacattaaag accagaagga aggtcttcat tcgctccagg aaattagagt ctgtgaaatc 300 catgaagaca acagcacaag aagttcccaa catttctact acgacggcga gctgttctta 360 ccagaccctc 420 gctatgaatg tgaggaattt ccttaaggag gacgctatgg aaactgacac ccactaccat 480 gcgatgcgcg ccgattgcct gcaggaa 507 <210> 29 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding alpha1-alpha2 variant 17 <400> 29 gctgctgagc cacacagtct ccgctacaac cttacggtgt tgagctggga cggctctgtc 60 cagagtggct ttctgactga ggtacatctc gatggtcagc ccttcctccg atgcgacaga 120 caaaagtgca gggccaagcc acagggccaa tgggccgaag atgtacttgg caataagact 180 tgggacagag aaaccagaga tctgactctc tggggtaagg acttacgcat gactctcgca 240 cacattaaag accagaagga aggtcttcat tcgctccagg aaattagagt ctgtgaaatc 300 catgaagaca acagcacaag aagttcccaa catttctact acgacggcga gctgttctta 360 ccagaccctc 420 gctatgaatg tgaggaattt ccttaaggag gacgctatgg aaactgacac ccactaccat 480 gcgatgcgcg ccgattgcct gcaggaa 507 <210> 30 <211> 507 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding alpha1-alpha2 variant 18 <400> 30 gctgctgagc cacacagtct ccgctacaac cttacggtgt tgagctggga cggctctgtc 60 cagcccggct ttctgactga ggtacatctc gatggtcagc ccttcctccg atgcgacaga 120 caaaagtgca gggccaagcc acagggccaa tgggccgaag atgtacttgg caataagact 180 tgggacagag aaaccagaga tctgactctc tggggtaagg acttacgcat gactctcgca 240 cacattaaag accagaagga aggtcttcat tcgctccagg aaattagagt ctgtgaaatc 300 catgaagaca acagcacaag aagttcccaa catttctact acgacggcga gctgttctta 360 ccagaccctc 420 gctatgaatg tgaggaattt ccttaaggag gacgctatgg aaactgacac ccactaccat 480 gcgatgcgcg ccgattgcct gcaggaa 507 <210> 31 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide MICA alpha1-alpha2 variant 15 <400> 31 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Asn Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met 145 150 155 160 Arg Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu 180 185 190 Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 195 200 205 Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 210 215 220 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 225 230 235 240 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 245 250 255 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 260 265 270 Ser Tyr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 275 280 285 Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 290 295 300 Gly Gly Gly Glu Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln 305 310 315 320 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe 325 330 335 Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 340 345 350 Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala 355 360 365 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 370 375 380 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 385 390 395 400 Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr 405 410 415 Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 420 425 430 Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 450 455 460 Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly 465 470 475 480 Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser 485 490 495 Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln 500 505 510 Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu 515 520 525 Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His 530 535 540 Pro Val Ser Ser Gly Lys Gly Ser His His His His His 545 550 555 <210> 32 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide MICA alpha1-alpha2 variant 16 <400> 32 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met 145 150 155 160 Arg Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu 180 185 190 Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 195 200 205 Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 210 215 220 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 225 230 235 240 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 245 250 255 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 260 265 270 Ser Tyr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 275 280 285 Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 290 295 300 Gly Gly Gly Glu Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln 305 310 315 320 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe 325 330 335 Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 340 345 350 Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala 355 360 365 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 370 375 380 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 385 390 395 400 Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr 405 410 415 Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 420 425 430 Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 450 455 460 Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly 465 470 475 480 Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser 485 490 495 Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln 500 505 510 Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu 515 520 525 Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His 530 535 540 Pro Val Ser Ser Gly Lys Gly Ser His His His His His 545 550 555 <210> 33 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide MICA alpha1-alpha2 variant 17 <400> 33 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Leu Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met 145 150 155 160 Arg Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu 180 185 190 Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 195 200 205 Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 210 215 220 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 225 230 235 240 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 245 250 255 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 260 265 270 Ser Tyr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 275 280 285 Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 290 295 300 Gly Gly Gly Glu Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln 305 310 315 320 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe 325 330 335 Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 340 345 350 Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala 355 360 365 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 370 375 380 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 385 390 395 400 Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr 405 410 415 Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 420 425 430 Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 450 455 460 Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly 465 470 475 480 Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser 485 490 495 Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln 500 505 510 Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu 515 520 525 Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His 530 535 540 Pro Val Ser Ser Gly Lys Gly Ser His His His His His 545 550 555 <210> 34 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide MICA alpha1-alpha2 variant 18 <400> 34 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Pro Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Leu Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met 145 150 155 160 Arg Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu 180 185 190 Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 195 200 205 Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 210 215 220 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 225 230 235 240 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 245 250 255 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 260 265 270 Ser Tyr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 275 280 285 Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 290 295 300 Gly Gly Gly Glu Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln 305 310 315 320 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe 325 330 335 Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 340 345 350 Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala 355 360 365 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 370 375 380 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 385 390 395 400 Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr 405 410 415 Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 420 425 430 Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 450 455 460 Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly 465 470 475 480 Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser 485 490 495 Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln 500 505 510 Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu 515 520 525 Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His 530 535 540 Pro Val Ser Ser Gly Lys Gly Ser His His His His His 545 550 555 <210> 35 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide MICA-WED <400> 35 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met 145 150 155 160 His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu 180 185 190 Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 195 200 205 Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 210 215 220 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 225 230 235 240 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 245 250 255 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 260 265 270 Ser Tyr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 275 280 285 Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 290 295 300 Gly Gly Gly Glu Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln 305 310 315 320 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe 325 330 335 Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 340 345 350 Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala 355 360 365 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 370 375 380 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 385 390 395 400 Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr 405 410 415 Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 420 425 430 Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 450 455 460 Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly 465 470 475 480 Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser 485 490 495 Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln 500 505 510 Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu 515 520 525 Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His 530 535 540 Pro Val Ser Ser Gly Lys Gly Ser His His His His His 545 550 555 <210> 36 <211> 1677 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding MICA alpha1-alpha2 variant 20 <400> 36 gagcctcaca gcctccggta taatttgact gtactctctt gggatggctc cgtgcagtcc 60 ggctttctga ctgaagttca tctcgacggt caacctttcc tgcgctgcga ccgacaaaaa 120 tgccgcgcca agccccaagg gcagtgggcc gaagatgtac tgggaaacaa gacctgggac 180 cgggagacac gagacctgac agcatgggga aaggacttgc gcatgacact cgcccatatc 240 aaggaccaga aggaaggatt gcactctttg caagagattc gcgtgtgtga aatccacgag 300 gacaattcaa cgaggagctc ccagcacttc tattacgatg gagaactctt cttgtcacag 360 aacttggaaa ccctggaatg gactatgcct cagagctccc gggcacagac tctcgctatg 420 aacgttagaa acttccttaa ggaggatgct atgcagaccg atactcacta ccgggccatg 480 cacgccgact gcctctttga actgcggaga tatctgaagt ccggcgtggt tttgagaaga 540 accgtgcccc ccatggtgca ggtgactcgc tctgaggcct ctggcggatc tggggaccgt 600 gtgacaatca cctgcagagc ctcccaggac gtctccactg ccgtggcgtg gtaccaacag 660 aagcccggga aggcacccaa actgctcatt tacagcgcat cctttctcta ctctggcgtg 720 ccgtctcgct ttagcgggtc cggcagcggt acagacttta ctctgaccat ctcctctctg 780 caaccggagg attttgcaac ctattattgc cagcaatcct acacaacccc ccccaccttt 840 ggccagggca ccaaggtgga gatcaaggga ggttctagcc gctccagcag ctctggaggt 900 ggaggctgg gcggaggagg cgaggtgcaa ctggtggagt ctgggggcgg cctggtccag 960 cccggcggaa gcttgcgcct gagctgtgcc gcctccggtt ttaccttcac cagcactgga 1020 atctcctggg tgcgccaagc tcccggcaaa gggctcgaat gggtgggccg tatctacccc 1080 accaacggaa gcaccaacta tgcagacagc gtgaaggggc gcttcactat ctccgccgac 1140 accagcaaaa acaccgcgta cctgcagatg aattctttga gggcagagga tactgccgtg 1200 tactactgcg cgaggacata cggcatttac gatctgtatg tggattacac cgaatacgtg 1260 atggactatt ggggccaggg cactctggtc acagtgtcta gcggtggcag ctcccgcagc 1320 tccagcagcg gtggtggcgg tagcggaggc ggaggcgata tccagatgac tcagagtccc 1380 tcttctctga gtgcttctgg cggaagtggg cagatcaccg tcacatgtcg cgcaagcggc 1440 ttttatcctt ggaacatcac cctgagctgg cggcaggacg gcgtcagcct gtcccatgat 1500 acccaacagt ggggagatgt gctcccggac ggtcagggaa cttaccagac ctgggttgca 1560 actcgcatct cccaggggga ggagcagcgt ttcacatgtt atatggagca ctctggccag 1620 cacagcactc atccggtgcc gtccggaaag ggatctcatc accatcacca ccactag 1677 <210> 37 <211> 1677 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding MICA alpha1-alpha2 variant 25 <400> 37 gagcctcaca gcctccggta taatttgact gtactctctt gggatggctc cgtgcagtcc 60 ggctttctga ctgaagttca tctcgacggt caacctttcc tgcgctgcga ccgacaaaaa 120 tgccgcgcca agccccaagg gcagtgggcc gaagatgtac tgggaaacaa gacctgggac 180 cgggagacac gagacctgac aggctggggc aaggacttgc gcatgacact cgcccatatc 240 aaggaccaga aggaaggatt gcactctttg caagagattc gcgtgtgtga aatccacgag 300 gacaattcaa cgaggagctc ccagcacttc tattacgatg gagaactctt cttgtcacag 360 cctcgaatg aacgttagaa acttccttaa ggaggatgct atggagaccg atactcacta ccacgccatg 480 cgcgccgact gcctctctga actgcggaga tatctgaagt ccggcgtggt tttgagaaga 540 accgtgcccc ccatggtgca ggtgactcgc tctgaggcct ctggcggatc tggggaccgt 600 gtgacaatca cctgcagagc ctcccaggac gtctccactg ccgtggcgtg gtaccaacag 660 aagcccggga aggcacccaa actgctcatt tacagcgcat cctttctcta ctctggcgtg 720 ccgtctcgct ttagcgggtc cggcagcggt acagacttta ctctgaccat ctcctctctg 780 caaccggagg attttgcaac ctattattgc cagcaatcct acacaacccc ccccaccttt 840 ggccagggca ccaaggtgga gatcaaggga ggttctagcc gctccagcag ctctggaggt 900 ggaggctgg gcggaggagg cgaggtgcaa ctggtggagt ctgggggcgg cctggtccag 960 cccggcggaa gcttgcgcct gagctgtgcc gcctccggtt ttaccttcac cagcactgga 1020 atctcctggg tgcgccaagc tcccggcaaa gggctcgaat gggtgggccg tatctacccc 1080 accaacggaa gcaccaacta tgcagacagc gtgaaggggc gcttcactat ctccgccgac 1140 accagcaaaa acaccgcgta cctgcagatg aattctttga gggcagagga tactgccgtg 1200 tactactgcg cgaggacata cggcatttac gatctgtatg tggattacac cgaatacgtg 1260 atggactatt ggggccaggg cactctggtc acagtgtcta gcggtggcag ctcccgcagc 1320 tccagcagcg gtggtggcgg tagcggaggc ggaggcgata tccagatgac tcagagtccc 1380 tcttctctga gtgcttctgg cggaagtggg cagatcaccg tcacatgtcg cgcaagcggc 1440 ttttatcctt ggaacatcac cctgagctgg cggcaggacg gcgtcagcct gtcccatgat 1500 acccaacagt ggggagatgt gctcccggac ggtcagggaa cttaccagac ctgggttgca 1560 actcgcatct cccaggggga ggagcagcgt ttcacatgtt atatggagca ctctggccag 1620 cacagcactc atccggtgcc gtccggaaag ggatctcatc accatcacca ccactag 1677 <210> 38 <211> 1677 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding MICA alpha1-alpha2 variant 48 <400> 38 gagccccaca gtcttcgtta taacctcacg gtgctgtcct gggacggatc tgttgtttca 60 gggtttctca ctgaggtaca tctggatggt cagcccttcc tgcgctgtga caggcagaaa 120 tgcagggcaa agccccaggg acagtgggca gaagatgtcc tgggaaataa gacatgggac 180 agagagacca gagacttgga cgggtgggga aaggacctca ggatgaccct ggctcatatt 240 aaggaccaga aagaaggctt gcattccctc caggagatta gggtctgtga gatccatgaa 300 gacaaguca ccaggagctc ccagcatttc tactacgatg gggagctgtt cctctcccaa 360 aacctggaga ctctggagtg gacaatgccc cagtcctcca gagctcagac cttggccatg 420 aacgtcagga acttcttgaa agaggacgcc atggcgaccg acacacacta cattgcaatg 480 cgggcagact gcctggctga actacggcga tatctgaaga gcggcgtagt cctgaggaga 540 acagtgcccc ccatggtgca ggtgactcgc tctgaggcct ctggcggatc tggggaccgt 600 gtgacaatca cctgcagagc ctcccaggac gtctccactg ccgtggcgtg gtaccaacag 660 aagcccggga aggcacccaa actgctcatt tacagcgcat cctttctcta ctctggcgtg 720 ccgtctcgct ttagcgggtc cggcagcggt acagacttta ctctgaccat ctcctctctg 780 caaccggagg attttgcaac ctattattgc cagcaatcct acacaacccc ccccaccttt 840 ggccagggca ccaaggtgga gatcaaggga ggttctagcc gctccagcag ctctggaggt 900 ggaggctgg gcggaggagg cgaggtgcaa ctggtggagt ctgggggcgg cctggtccag 960 cccggcggaa gcttgcgcct gagctgtgcc gcctccggtt ttaccttcac cagcactgga 1020 atctcctggg tgcgccaagc tcccggcaaa gggctcgaat gggtgggccg tatctacccc 1080 accaacggaa gcaccaacta tgcagacagc gtgaaggggc gcttcactat ctccgccgac 1140 accagcaaaa acaccgcgta cctgcagatg aattctttga gggcagagga tactgccgtg 1200 tactactgcg cgaggacata cggcatttac gatctgtatg tggattacac cgaatacgtg 1260 atggactatt ggggccaggg cactctggtc acagtgtcta gcggtggcag ctcccgcagc 1320 tccagcagcg gtggtggcgg tagcggaggc ggaggcgata tccagatgac tcagagtccc 1380 tcttctctga gtgcttctgg cggaagtggg cagatcaccg tcacatgtcg cgcaagcggc 1440 ttttatcctt ggaacatcac cctgagctgg cggcaggacg gcgtcagcct gtcccatgat 1500 acccaacagt ggggagatgt gctcccggac ggtcagggaa cttaccagac ctgggttgca 1560 actcgcatct cccaggggga ggagcagcgt ttcacatgtt atatggagca ctctggccag 1620 cacagcactc atccggtgcc gtccggaaag ggatctcatc accatcacca ccactag 1677 <210> <211> <212> <213> 39 558 PRT Artificial Sequence <220> <223> Synthetic peptide MICA alpha1-alpha2 variant 20 <400> 39 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 1 5 10 15 Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Ala Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Gln Thr Asp Thr His Tyr Arg Ala Met 145 150 155 160 His Ala Asp Cys Leu Phe Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu 180 185 190 Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 195 200 205 Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 210 215 220 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 225 230 235 240 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 245 250 255 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 260 265 270 Ser Tyr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 275 280 285 Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 290 295 300 Gly Gly Gly Glu Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln 305 310 315 320 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe 325 330 335 Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 340 345 350 Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala 355 360 365 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 370 375 380 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 385 390 395 400 Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr 405 410 415 Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 420 425 430 Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 450 455 460 Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly 465 470 475 480 Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser 485 490 495 Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln 500 505 510 Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu 515 520 525 Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His 530 535 540 Pro Val Ser Ser Gly Lys Gly Ser His His His His His 545 550 555 <210> 40 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide MICA alpha1-alpha2 variant 25 <400> 40 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met 145 150 155 160 Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu 180 185 190 Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 195 200 205 Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 210 215 220 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 225 230 235 240 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 245 250 255 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 260 265 270 Ser Tyr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 275 280 285 Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 290 295 300 Gly Gly Gly Glu Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln 305 310 315 320 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe 325 330 335 Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 340 345 350 Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala 355 360 365 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 370 375 380 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 385 390 395 400 Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr 405 410 415 Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 420 425 430 Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 450 455 460 Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly 465 470 475 480 Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser 485 490 495 Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln 500 505 510 Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu 515 520 525 Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His 530 535 540 Pro Val Ser Ser Gly Lys Gly Ser His His His His His 545 550 555 <210> 41 <211> 558 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide MICA alpha1-alpha2 variant 48 <400> 41 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Ala Thr Asp Thr His Tyr Ile Ala Met 145 150 155 160 Arg Ala Asp Cys Leu Ala Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu 180 185 190 Ala Ser Gly Gly Ser Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 195 200 205 Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 210 215 220 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 225 230 235 240 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 245 250 255 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 260 265 270 Ser Tyr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 275 280 285 Lys Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 290 295 300 Gly Gly Gly Glu Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln 305 310 315 320 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe 325 330 335 Thr Ser Thr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 340 345 350 Glu Trp Val Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala 355 360 365 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 370 375 380 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 385 390 395 400 Tyr Tyr Cys Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr 405 410 415 Thr Glu Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 420 425 430 Ser Ser Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 450 455 460 Ala Ser Gly Gly Ser Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly 465 470 475 480 Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser 485 490 495 Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gln 500 505 510 Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu 515 520 525 Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His 530 535 540 Pro Val Ser Ser Gly Lys Gly Ser His His His His His 545 550 555 <210> 42 <211> 1974 <212> DNA <213> Artificial Sequence <220> Synthetic polynucleotide, encoding HC_WT <400> 42 ggtgcaactg 60 gtggagtctg ggggcggcct ggtccagccc ggcggaagct tgcgcctgag ctgtgccgcc 120 tccggtttta ccttcaccag cactggaatc tcctgggtgc gccaagctcc cggcaaaggg 180 ctcgaatggg tgggccgtat ctaccccacc aacggaagca ccaactatgc agacagcgtg 240 aaggggcgct tcactatctc cgccgacacc agcaaaaaca ccgcgtacct gcagatgaac 300 tttttgaggg cagaggatac tgccgtgtac tactgcgcga ggacatacgg catttacgat 360 ctgtatgtgg attacaccga atacgtgatg gactattggg gccagggcac tctggtcaca 420 gtgtctagcg cgtcgaccaa gggcccgtca gtgttcccgc tggccccgtc atccaagtcc 480 acgtctgggg gcacagcagc cctgggatgc ttggtcaagg actacttccc cgagcccgtg 540 actgtgtcct ggaactccgg agcactgacc tccggagtgc acacctttcc cgcggtgctg 600 cagtcctccg gactgtactc cctgtcgtcg gtcgtgaccg tgccgagctc ctcgctcgga 660 acccagacct acatctgcaa cgtgaaccac aagccctcga acaccaaagt ggacaagaag 720 gtcgagccca aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 780 ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 840 cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 900 ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 960 cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 1020 aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 1080 accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 1140 cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 1200 tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 1260 ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 1320 tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 1380 cactacaccc agaagtcact gagcctctcc cccggaggag gtggcagcga gcctcacagc 1440 ctccggtata atttgactgt actctcttgg gatggctccg tgcagtccgg ctttctgact 1500 gaagttcatc tcgacggtca acctttcctg cgctgcgacc gacaaaaatg ccgcgccaag 1560 ccccaagggc agtgggccga agatgtactg ggaaacaaga cctgggaccg ggagacacga 1620 gacctgacag gcaacggcaa ggacttgcgc atgacactcg cccatatcaa ggaccagaag 1680 gaaggattgc actctttgca agagattcgc gtgtgtgaaa tccacgagga caattcaacg 1740 aggagctccc agcacttcta ttacgatgga gaactcttct tgtcacagaa cttggaaacc 1800 aaggaatgga ctatgcctca gagctctcgg gcacagactc tcgctatgaa cgttagaaac 1860 ttccttaagg aggatgctat gaagaccaaa actcactacc acgccatgca cgccgactgc 1920 ctccaggaac tgcggagata tctgaagtcc ggcgtggttt tgagaagaac ctag 1974 <210> 43 <211> 1974 <212> DNA <213> Artificial Sequence <220> Synthetic polynucleotide, encoding HC_WED <400> 43 ggtgcaactg 60 gtggagtctg ggggcggcct ggtccagccc ggcggaagct tgcgcctgag ctgtgccgcc 120 tccggtttta ccttcaccag cactggaatc tcctgggtgc gccaagctcc cggcaaaggg 180 ctcgaatggg tgggccgtat ctaccccacc aacggaagca ccaactatgc agacagcgtg 240 aaggggcgct tcactatctc cgccgacacc agcaaaaaca ccgcgtacct gcagatgaac 300 tttttgaggg cagaggatac tgccgtgtac tactgcgcga ggacatacgg catttacgat 360 ctgtatgtgg attacaccga atacgtgatg gactattggg gccagggcac tctggtcaca 420 gtgtctagcg cgtcgaccaa gggcccgtca gtgttcccgc tggccccgtc atccaagtcc 480 acgtctgggg gcacagcagc cctgggatgc ttggtcaagg actacttccc cgagcccgtg 540 actgtgtcct ggaactccgg agcactgacc tccggagtgc acacctttcc cgcggtgctg 600 cagtcctccg gactgtactc cctgtcgtcg gtcgtgaccg tgccgagctc ctcgctcgga 660 acccagacct acatctgcaa cgtgaaccac aagccctcga acaccaaagt ggacaagaag 720 gtcgagccca aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 780 ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 840 cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 900 ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 960 cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 1020 aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 1080 accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 1140 cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 1200 tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 1260 ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 1320 tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 1380 cactacaccc agaagtcact gagcctctcc cccggaggag gtggcagcga gcctcacagc 1440 ctccggtata atttgactgt actctcttgg gatggctccg tgcagtccgg ctttctgact 1500 gaagttcatc tcgacggtca acctttcctg cgctgcgacc gacaaaaatg ccgcgccaag 1560 ccccaagggc agtgggccga agatgtactg ggaaacaaga cctgggaccg ggagacacga 1620 gacctgacag gctggggcaa ggacttgcgc atgacactcg cccatatcaa ggaccagaag 1680 gaaggattgc actctttgca agagattcgc gtgtgtgaaa tccacgagga caattcaacg 1740 aggagctccc agcacttcta ttacgatgga gaactcttct tgtcacagaa cttggaaacc 1800 aaggaatgga ctatgcctca gagctctcgg gcacagactc tcgctatgaa cgttagaaac 1860 ttccttaagg aggatgctat ggagaccgat actcactacc acgccatgca cgccgactgc 1920 ctccaggaac tgcggagata tctgaagtcc ggcgtggttt tgagaagaac ctag 1974 <210> 44 <211> 1974 <212> DNA <213> Artificial Sequence <220> Synthetic polynucleotide, encoding HC_25 <400> 44 ggtgcaactg 60 gtggagtctg ggggcggcct ggtccagccc ggcggaagct tgcgcctgag ctgtgccgcc 120 tccggtttta ccttcaccag cactggaatc tcctgggtgc gccaagctcc cggcaaaggg 180 ctcgaatggg tgggccgtat ctaccccacc aacggaagca ccaactatgc agacagcgtg 240 aaggggcgct tcactatctc cgccgacacc agcaaaaaca ccgcgtacct gcagatgaac 300 tttttgaggg cagaggatac tgccgtgtac tactgcgcga ggacatacgg catttacgat 360 ctgtatgtgg attacaccga atacgtgatg gactattggg gccagggcac tctggtcaca 420 gtgtctagcg cgtcgaccaa gggcccgtca gtgttcccgc tggccccgtc atccaagtcc 480 acgtctgggg gcacagcagc cctgggatgc ttggtcaagg actacttccc cgagcccgtg 540 actgtgtcct ggaactccgg agcactgacc tccggagtgc acacctttcc cgcggtgctg 600 cagtcctccg gactgtactc cctgtcgtcg gtcgtgaccg tgccgagctc ctcgctcgga 660 acccagacct acatctgcaa cgtgaaccac aagccctcga acaccaaagt ggacaagaag 720 gtcgagccca aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 780 ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 840 cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 900 ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 960 cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 1020 aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 1080 accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 1140 cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 1200 tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 1260 ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 1320 tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 1380 cactacaccc agaagtcact gagcctctcc cccggaggag gtggcagcga gcctcacagc 1440 ctccggtata atttgactgt actctcttgg gatggctccg tgcagtccgg ctttctgact 1500 gaagttcatc tcgacggtca acctttcctg cgctgcgacc gacaaaaatg ccgcgccaag 1560 ccccaagggc agtgggccga agatgtactg ggaaacaaga cctgggaccg ggagacacga 1620 gacctgacag gctggggcaa ggacttgcgc atgacactcg cccatatcaa ggaccagaag 1680 gaaggattgc actctttgca agagattcgc gtgtgtgaaa tccacgagga caattcaacg 1740 aggagctccc agcacttcta ttacgatgga gaactcttct tgtcacagaa cttggaaacc 1800 ctcgaatgga ctatgcctca gagctctcgg gcacagactc tcgctatgaa cgttagaaac 1860 ttccttaagg aggatgctat ggagaccgat actcactacc acgccatgcg cgccgactgc 1920 ctctctgaac tgcggagata tctgaagtcc ggcgtggttt tgagaagaac ctag 1974 <210> 45 <211> 1974 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding HC_48 <400> 45 ggtgcaactg 60 gtggagtctg ggggcggcct ggtccagccc ggcggaagct tgcgcctgag ctgtgccgcc 120 tccggtttta ccttcaccag cactggaatc tcctgggtgc gccaagctcc cggcaaaggg 180 ctcgaatggg tgggccgtat ctaccccacc aacggaagca ccaactatgc agacagcgtg 240 aaggggcgct tcactatctc cgccgacacc agcaaaaaca ccgcgtacct gcagatgaac 300 tttttgaggg cagaggatac tgccgtgtac tactgcgcga ggacatacgg catttacgat 360 ctgtatgtgg attacaccga atacgtgatg gactattggg gccagggcac tctggtcaca 420 gtgtctagcg cgtcgaccaa gggcccgtca gtgttcccgc tggccccgtc atccaagtcc 480 acgtctgggg gcacagcagc cctgggatgc ttggtcaagg actacttccc cgagcccgtg 540 actgtgtcct ggaactccgg agcactgacc tccggagtgc acacctttcc cgcggtgctg 600 cagtcctccg gactgtactc cctgtcgtcg gtcgtgaccg tgccgagctc ctcgctcgga 660 acccagacct acatctgcaa cgtgaaccac aagccctcga acaccaaagt ggacaagaag 720 gtcgagccca aaagctgcga caagactcac acttgtccgc cgtgccccgc ccccgaactg 780 ctgggtggcc cctccgtgtt cctgttcccg cctaagccta aggacaccct tatgatcagc 840 cgcacccctg aagtgacctg tgtcgtcgtg gatgtgtcac acgaggaccc ggaggtcaag 900 ttcaattggt acgtggacgg cgtggaagtg cataacgcaa agaccaagcc tcgggaggaa 960 cagtacaact cgacctaccg cgtggtgtca gtcctgactg tgctgcacca ggactggctg 1020 aacgggaagg agtacaagtg caaagtgtcg aacaaggccc tgccggctcc aattgaaaag 1080 accatcagca aggccaaggg ccagccaagg gaaccacagg tgtacaccct ccctccttcc 1140 cgggacgagc tgaccaaaaa ccaagtgtcc ctgacttgcc ttgtgaaggg gttctaccct 1200 tctgacattg ccgtcgaatg ggaatcgaac ggacagcctg aaaacaacta taagactacc 1260 ccgcccgtgc tggattccga cggaagcttc ttcctgtact ccaagctgac cgtggacaag 1320 tcgagatggc agcagggaaa tgtgttcagc tgctccgtga tgcatgaggc gctgcacaac 1380 cactacaccc agaagtcact gagcctctcc cccggaggag gtggcagcga gcctcacagc 1440 ctccggtata atttgactgt actctcttgg gatggctccg tgcagtccgg ctttctgact 1500 gaagttcatc tcgacggtca acctttcctg cgctgcgacc gacaaaaatg ccgcgccaag 1560 ccccaagggc agtgggccga agatgtactg ggaaacaaga cctgggaccg ggagacacga 1620 gacctgacag gctggggcaa ggacttgcgc atgacactcg cccatatcaa ggaccagaag 1680 gaaggattgc actctttgca agagattcgc gtgtgtgaaa tccacgagga caattcaacg 1740 aggagctccc agcacttcta ttacgatgga gaactcttct tgtcacagaa cttggaaacc 1800 ctcgaatgga ctatgcctca gagctctcgg gcacagactc tcgctatgaa cgttagaaac 1860 ttccttaagg aggatgctat ggctaccgat actcactaca tcgccatgcg cgccgactgc 1920 ctcgctgaac tgcggagata tctgaagtcc ggcgtggttt tgagaagaac ctag 1974 <210> 46 <211> 1269 <212> DNA <213> Artificial Sequence <220> Synthetic polynucleotide, encoding LC_WT <400> 46 atgagaccta tcgttcttgt actccttttc gctacctccg ccctcgccga cattcagatg 60 gt gcttctcagg acgtgtccac cgcggttgct tggtaccagc aaaagcctgg aaaggcgccg 180 aagctgctga tctactccgc ctcattcttg tactcaggag tgcccagtcg atttagtggt 240 agcggttctg gtactgattt cacccttacc atcagcagtc tccagcccga ggatttcgct 300 acttattact gccagcagtc atacaccact cctcccactt tcggccaagg taccaaggtc 360 gagattaaac ggaccgtggc cgccccgagc gtgttcattt tccctccctc cgacgagcag 420 ttgaaatcgg gcaccgctag cgtggtctgc cttctcaaca atttctatcc acgggaagcc 480 aaagtgcagt ggaaggtcga caacgcgctc caatccggga actcacagga atccgtgact 540 gagcaggatt ccaaggactc gacctactcc ctgtcatcca cgctgaccct gagcaaggca 600 gactacgaga agcacaaggt ctacgcctgc gaagtgacac accagggact gtccagcccc 660 gtgaccaaga gcttcaacag aggagaatgc gcacctacct caagctctgg aggaggtggc 720 agcgagcctc acagcctccg gtataatttg actgtactct cttgggatgg ctccgtgcag 780 tccggctttc tgactgaagt tcatctcgac ggtcaacctt tcctgcgctg cgaccgacaa 840 aaatgccgcg ccaagcccca agggcagtgg gccgaagatg tactgggaaa caagacctgg 900 gaccgggaga cacgagacct gacaggcaac ggcaaggact tgcgcatgac actcgcccat 960 atcaaggacc agaaggaagg attgcactct ttgcaagaga ttcgcgtgtg tgaaatccac 1020 gaggacaatt caacgaggag ctcccagcac ttctattacg atggagaact cttcttgtca 1080 cagaacttgg aaaccaagga atggactatg cctcagagct ctcgggcaca gactctcgct 1140 atgaacgtta gaaacttcct taaggaggat gctatgaaga ccaaaactc ctaccacgcc 1200 atgcacgccg actgcctcca ggaactgcgg agatatctga agtccggcgt ggttttgaga 1260 agaacctaa 1269 <210> 47 <211> 1269 <212> DNA <213> Artificial Sequence <220> Synthetic polynucleotide, encoding LC_WED <400> 47 atgagaccta tcgttcttgt actccttttc gctacctccg ccctcgccga cattcagatg 60 gt gcttctcagg acgtgtccac cgcggttgct tggtaccagc aaaagcctgg aaaggcgccg 180 aagctgctga tctactccgc ctcattcttg tactcaggag tgcccagtcg atttagtggt 240 agcggttctg gtactgattt cacccttacc atcagcagtc tccagcccga ggatttcgct 300 acttattact gccagcagtc atacaccact cctcccactt tcggccaagg taccaaggtc 360 gagattaaac ggaccgtggc cgccccgagc gtgttcattt tccctccctc cgacgagcag 420 ttgaaatcgg gcaccgctag cgtggtctgc cttctcaaca atttctatcc acgggaagcc 480 aaagtgcagt ggaaggtcga caacgcgctc caatccggga actcacagga atccgtgact 540 gagcaggatt ccaaggactc gacctactcc ctgtcatcca cgctgaccct gagcaaggca 600 gactacgaga agcacaaggt ctacgcctgc gaagtgacac accagggact gtccagcccc 660 gtgaccaaga gcttcaacag aggagaatgc gcacctacct caagctctgg aggaggtggc 720 agcgagcctc acagcctccg gtataatttg actgtactct cttgggatgg ctccgtgcag 780 tccggctttc tgactgaagt tcatctcgac ggtcaacctt tcctgcgctg cgaccgacaa 840 aaatgccgcg ccaagcccca agggcagtgg gccgaagatg tactgggaaa caagacctgg 900 gaccgggaga cacgagacct gacaggctgg ggcaaggact tgcgcatgac actcgcccat 960 atcaaggacc agaaggaagg attgcactct ttgcaagaga ttcgcgtgtg tgaaatccac 1020 gaggacaatt caacgaggag ctcccagcac ttctattacg atggagaact cttcttgtca 1080 cagaacttgg aaaccaagga atggactatg cctcagagct ctcgggcaca gactctcgct 1140 atgaacgtta gaaacttcct taaggaggat gctatggaga ccgatactca ctaccacgcc 1200 atgcacgccg actgcctcca ggaactgcgg agatatctga agtccggcgt ggttttgaga 1260 agaacctaa 1269 <210> 48 <211> 1269 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding LC_25 <400> 48 atgagaccta tcgttcttgt actccttttc gctacctccg ccctcgccga cattcagatg 60 gt gcttctcagg acgtgtccac cgcggttgct tggtaccagc aaaagcctgg aaaggcgccg 180 aagctgctga tctactccgc ctcattcttg tactcaggag tgcccagtcg atttagtggt 240 agcggttctg gtactgattt cacccttacc atcagcagtc tccagcccga ggatttcgct 300 acttattact gccagcagtc atacaccact cctcccactt tcggccaagg taccaaggtc 360 gagattaaac ggaccgtggc cgccccgagc gtgttcattt tccctccctc cgacgagcag 420 ttgaaatcgg gcaccgctag cgtggtctgc cttctcaaca atttctatcc acgggaagcc 480 aaagtgcagt ggaaggtcga caacgcgctc caatccggga actcacagga atccgtgact 540 gagcaggatt ccaaggactc gacctactcc ctgtcatcca cgctgaccct gagcaaggca 600 gactacgaga agcacaaggt ctacgcctgc gaagtgacac accagggact gtccagcccc 660 gtgaccaaga gcttcaacag aggagaatgc gcacctacct caagctctgg aggaggtggc 720 agcgagcctc acagcctccg gtataatttg actgtactct cttgggatgg ctccgtgcag 780 tccggctttc tgactgaagt tcatctcgac ggtcaacctt tcctgcgctg cgaccgacaa 840 aaatgccgcg ccaagcccca agggcagtgg gccgaagatg tactgggaaa caagacctgg 900 gaccgggaga cacgagacct gacaggctgg ggcaaggact tgcgcatgac actcgcccat 960 atcaaggacc agaaggaagg attgcactct ttgcaagaga ttcgcgtgtg tgaaatccac 1020 gaggacaatt caacgaggag ctcccagcac ttctattacg atggagaact cttcttgtca 1080 cagaacttgg aaaccctcga atggactatg cctcagagct ctcgggcaca gactctcgct 1140 atgaacgtta gaaacttcct taaggaggat gctatggaaa ccgatactca ctaccatgcc 1200 atgagagccg actgcctctc tgaactgcgg agatatctga agtccggagt ggttttgaga 1260 agaacttaa 1269 <210> 49 <211> 1269 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide, encoding LC_48 <400> 49 atgagaccta tcgttcttgt actccttttc gctacctccg ccctcgccga cattcagatg 60 gt gcttctcagg acgtgtccac cgcggttgct tggtaccagc aaaagcctgg aaaggcgccg 180 aagctgctga tctactccgc ctcattcttg tactcaggag tgcccagtcg atttagtggt 240 agcggttctg gtactgattt cacccttacc atcagcagtc tccagcccga ggatttcgct 300 acttattact gccagcagtc atacaccact cctcccactt tcggccaagg taccaaggtc 360 gagattaaac ggaccgtggc cgccccgagc gtgttcattt tccctccctc cgacgagcag 420 ttgaaatcgg gcaccgctag cgtggtctgc cttctcaaca atttctatcc acgggaagcc 480 aaagtgcagt ggaaggtcga caacgcgctc caatccggga actcacagga atccgtgact 540 gagcaggatt ccaaggactc gacctactcc ctgtcatcca cgctgaccct gagcaaggca 600 gactacgaga agcacaaggt ctacgcctgc gaagtgacac accagggact gtccagcccc 660 gtgaccaaga gcttcaacag aggagaatgc gcacctacct caagctctgg aggaggtggc 720 agcgagcctc acagcctccg gtataatttg actgtactct cttgggatgg ctccgtgcag 780 tccggctttc tgactgaagt tcatctcgac ggtcaacctt tcctgcgctg cgaccgacaa 840 aaatgccgcg ccaagcccca agggcagtgg gccgaagatg tactgggaaa caagacctgg 900 gaccgggaga cacgagacct gacaggctgg ggcaaggact tgcgcatgac actcgcccat 960 atcaaggacc agaaggaagg attgcactct ttgcaagaga ttcgcgtgtg tgaaatccac 1020 gaggacaatt caacgaggag ctcccagcac ttctattacg atggagaact cttcttgtca 1080 cagaacttgg aaaccctcga atggactatg cctcagagct ctcgggcaca gactctcgct 1140 atgaacgtta gaaacttcct taaggaggat gctatggcta ccgatactca ctacatcgcc 1200 atgcgcgccg actgcctcgc tgaactgcgg agatatctga agtccggcgt ggttttgaga 1260 agaacctaa 1269 <210> 50 <211> 657 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide HC_WT <400> 50 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 20 25 30 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr 35 40 45 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 50 55 60 Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val 65 70 75 80 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 85 90 95 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 100 105 110 Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 115 120 125 Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 130 135 140 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Ser Ser Ser 145 150 155 160 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 165 170 175 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 180 185 190 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 195 200 205 Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr 210 215 220 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 225 230 235 240 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 245 250 255 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 260 265 270 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 275 280 285 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 290 295 300 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 305 310 315 320 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 325 330 335 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 340 345 350 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 355 360 365 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 370 375 380 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 385 390 395 400 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 405 410 415 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 420 425 430 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 435 440 445 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 450 455 460 Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Glu Pro His Ser 465 470 475 480 Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly Ser Val Gln Ser 485 490 495 Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro Phe Leu Arg Cys 500 505 510 Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln Trp Ala Glu Asp 515 520 525 Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg Asp Leu Thr Gly 530 535 540 Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile Lys Asp Gln Lys 545 550 555 560 Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys Glu Ile His Glu 565 570 575 Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr Asp Gly Glu Leu 580 585 590 Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr Met Pro Gln Ser 595 600 605 Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn Phe Leu Lys Glu 610 615 620 Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met His Ala Asp Cys 625 630 635 640 Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val Val Leu Arg Arg 645 650 655 Thr <210> 51 <211> 657 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide HC_WED <400> 51 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 20 25 30 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr 35 40 45 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 50 55 60 Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val 65 70 75 80 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 85 90 95 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 100 105 110 Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 115 120 125 Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 130 135 140 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Ser Ser Ser 145 150 155 160 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 165 170 175 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 180 185 190 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 195 200 205 Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr 210 215 220 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 225 230 235 240 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 245 250 255 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 260 265 270 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 275 280 285 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 290 295 300 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 305 310 315 320 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 325 330 335 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 340 345 350 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 355 360 365 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 370 375 380 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 385 390 395 400 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 405 410 415 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 420 425 430 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 435 440 445 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 450 455 460 Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Glu Pro His Ser 465 470 475 480 Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly Ser Val Gln Ser 485 490 495 Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro Phe Leu Arg Cys 500 505 510 Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln Trp Ala Glu Asp 515 520 525 Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg Asp Leu Thr Gly 530 535 540 Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile Lys Asp Gln Lys 545 550 555 560 Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys Glu Ile His Glu 565 570 575 Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr Asp Gly Glu Leu 580 585 590 Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr Met Pro Gln Ser 595 600 605 Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn Phe Leu Lys Glu 610 615 620 Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met His Ala Asp Cys 625 630 635 640 Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val Val Leu Arg Arg 645 650 655 Thr <210> 52 <211> 657 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide HC_25 <400> 52 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 20 25 30 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr 35 40 45 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 50 55 60 Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val 65 70 75 80 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 85 90 95 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 100 105 110 Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 115 120 125 Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 130 135 140 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Ser Ser Ser 145 150 155 160 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 165 170 175 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 180 185 190 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 195 200 205 Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr 210 215 220 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 225 230 235 240 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 245 250 255 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 260 265 270 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 275 280 285 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 290 295 300 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 305 310 315 320 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 325 330 335 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 340 345 350 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 355 360 365 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 370 375 380 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 385 390 395 400 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 405 410 415 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 420 425 430 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 435 440 445 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 450 455 460 Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Glu Pro His Ser 465 470 475 480 Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly Ser Val Gln Ser 485 490 495 Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro Phe Leu Arg Cys 500 505 510 Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln Trp Ala Glu Asp 515 520 525 Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg Asp Leu Thr Gly 530 535 540 Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile Lys Asp Gln Lys 545 550 555 560 Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys Glu Ile His Glu 565 570 575 Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr Asp Gly Glu Leu 580 585 590 Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr Met Pro Gln Ser 595 600 605 Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn Phe Leu Lys Glu 610 615 620 Asp Ala Met Glu Thr Asp Thr His Tyr His Ala Met Arg Ala Asp Cys 625 630 635 640 Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val Val Leu Arg Arg 645 650 655 Thr <210> 53 <211> 657 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide HC_48 <400> 53 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 20 25 30 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Thr 35 40 45 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 50 55 60 Gly Arg Ile Tyr Pro Thr Asn Gly Ser Thr Asn Tyr Ala Asp Ser Val 65 70 75 80 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 85 90 95 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 100 105 110 Ala Arg Thr Tyr Gly Ile Tyr Asp Leu Tyr Val Asp Tyr Thr Glu Tyr 115 120 125 Val Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 130 135 140 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Ser Ser Ser 145 150 155 160 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 165 170 175 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 180 185 190 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 195 200 205 Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr 210 215 220 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 225 230 235 240 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 245 250 255 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 260 265 270 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 275 280 285 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 290 295 300 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 305 310 315 320 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 325 330 335 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 340 345 350 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 355 360 365 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 370 375 380 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 385 390 395 400 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 405 410 415 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 420 425 430 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 435 440 445 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 450 455 460 Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Glu Pro His Ser 465 470 475 480 Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly Ser Val Gln Ser 485 490 495 Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro Phe Leu Arg Cys 500 505 510 Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln Trp Ala Glu Asp 515 520 525 Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg Asp Leu Thr Gly 530 535 540 Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile Lys Asp Gln Lys 545 550 555 560 Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys Glu Ile His Glu 565 570 575 Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr Asp Gly Glu Leu 580 585 590 Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp Thr Met Pro Gln Ser 595 600 605 Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn Phe Leu Lys Glu 610 615 620 Asp Met Ala Thr Asp Thr His Tyr Ile Ala Met Arg Ala Asp Cys 625 630 635 640 Leu Ala Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val Val Leu Arg Arg 645 650 655 Thr <210> 54 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide LC_WT <400> 54 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 20 25 30 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 35 40 45 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 50 55 60 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly 65 70 75 80 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 85 90 95 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro 100 105 110 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 115 120 125 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 130 135 140 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 145 150 155 160 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 165 170 175 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 180 185 190 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 195 200 205 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 210 215 220 Phe Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Gly Gly Gly Gly 225 230 235 240 Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp 245 250 255 Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln 260 265 270 Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly 275 280 285 Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr 290 295 300 Arg Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His 305 310 315 320 Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val 325 330 335 Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr 340 345 350 Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp 355 360 365 Thr Met Pro Gln Ser Ser Ala Gln Thr Leu Ala Met Asn Val Arg 370 375 380 Asn Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala 385 390 395 400 Met His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly 405 410 415 Val Val Leu Arg Arg Thr 420 <210> 55 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide LC_WED <400> 55 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 20 25 30 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 35 40 45 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 50 55 60 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly 65 70 75 80 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 85 90 95 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro 100 105 110 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 115 120 125 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 130 135 140 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 145 150 155 160 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 165 170 175 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 180 185 190 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 195 200 205 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 210 215 220 Phe Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Gly Gly Gly Gly 225 230 235 240 Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp 245 250 255 Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln 260 265 270 Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly 275 280 285 Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr 290 295 300 Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His 305 310 315 320 Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val 325 330 335 Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr 340 345 350 Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp 355 360 365 Thr Met Pro Gln Ser Ser Ala Gln Thr Leu Ala Met Asn Val Arg 370 375 380 Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala 385 390 395 400 Met His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly 405 410 415 Val Val Leu Arg Arg Thr 420 <210> 56 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide LC_25 <400> 56 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 20 25 30 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 35 40 45 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 50 55 60 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly 65 70 75 80 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 85 90 95 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro 100 105 110 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 115 120 125 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 130 135 140 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 145 150 155 160 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 165 170 175 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 180 185 190 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 195 200 205 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 210 215 220 Phe Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Gly Gly Gly Gly 225 230 235 240 Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp 245 250 255 Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln 260 265 270 Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly 275 280 285 Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr 290 295 300 Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His 305 310 315 320 Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val 325 330 335 Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr 340 345 350 Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp 355 360 365 Thr Met Pro Gln Ser Ser Ala Gln Thr Leu Ala Met Asn Val Arg 370 375 380 Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala 385 390 395 400 Met Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly 405 410 415 Val Val Leu Arg Arg Thr 420 <210> 57 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide LC_48 <400> 57 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 20 25 30 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 35 40 45 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 50 55 60 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly 65 70 75 80 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 85 90 95 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro 100 105 110 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 115 120 125 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 130 135 140 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 145 150 155 160 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 165 170 175 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 180 185 190 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 195 200 205 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 210 215 220 Phe Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Gly Gly Gly Gly 225 230 235 240 Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp 245 250 255 Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln 260 265 270 Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly 275 280 285 Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr 290 295 300 Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His 305 310 315 320 Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val 325 330 335 Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr 340 345 350 Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp 355 360 365 Thr Met Pro Gln Ser Ser Ala Gln Thr Leu Ala Met Asn Val Arg 370 375 380 Asn Phe Leu Lys Glu Asp Ala Met Ala Thr Asp Thr His Tyr Ile Ala 385 390 395 400 Met Arg Ala Asp Cys Leu Ala Glu Leu Arg Arg Tyr Leu Lys Ser Gly 405 410 415 Val Val Leu Arg Arg Thr 420 <210> 58 <211> 649 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide cetuximab HC_25 <400> 58 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 20 25 30 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 35 40 45 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 50 55 60 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 65 70 75 80 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Ser Ser Ser Gln Val Phe Phe 85 90 95 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 100 105 110 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 130 135 140 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 145 150 155 160 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 165 170 175 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 180 185 190 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser 195 200 205 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 210 215 220 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 225 230 235 240 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp 275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 450 455 460 Gly Gly Gly Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu 465 470 475 480 Ser Trp Asp Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu 485 490 495 Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys 500 505 510 Pro Gln Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp 515 520 525 Arg Glu Thr Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr 530 535 540 Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu 545 550 555 560 Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln 565 570 575 His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr 580 585 590 Leu Glu Trp Thr Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met 595 600 605 Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His 610 615 620 Tyr His Ala Met Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu 625 630 635 640 Lys Ser Gly Val Val Leu Arg Arg Thr 645 <210> 59 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide cetuximab LC_25 <400> 59 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 20 25 30 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 35 40 45 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 50 55 60 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 65 70 75 80 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 85 90 95 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 100 105 110 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 115 120 125 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 130 135 140 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 145 150 155 160 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 165 170 175 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 180 185 190 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 195 200 205 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 210 215 220 Phe Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Gly Gly Gly Gly 225 230 235 240 Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp 245 250 255 Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln 260 265 270 Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly 275 280 285 Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr 290 295 300 Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His 305 310 315 320 Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val 325 330 335 Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr 340 345 350 Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp 355 360 365 Thr Met Pro Gln Ser Ser Ala Gln Thr Leu Ala Met Asn Val Arg 370 375 380 Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala 385 390 395 400 Met Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly 405 410 415 Val Val Leu Arg Arg Thr 420 <210> 60 <211> 650 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide trastuzumab HC_25 <400> 60 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 20 25 30 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 35 40 45 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 50 55 60 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 65 70 75 80 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 85 90 95 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 100 105 110 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Gly Gly Gly Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val 465 470 475 480 Leu Ser Trp Asp Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His 485 490 495 Leu Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala 500 505 510 Lys Pro Gln Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp 515 520 525 Asp Arg Glu Thr Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met 530 535 540 Thr Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln 545 550 555 560 Glu Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser 565 570 575 Gln His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu 580 585 590 Thr Leu Glu Trp Thr Met Pro Gln Ser Ser Ala Gln Thr Leu Ala 595 600 605 Met Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr 610 615 620 His Tyr His Ala Met Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr 625 630 635 640 Leu Lys Ser Gly Val Val Leu Arg Arg Thr 645 650 <210> 61 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide trastuzumab LC_25 <400> 61 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 20 25 30 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 35 40 45 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 50 55 60 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly 65 70 75 80 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 85 90 95 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 100 105 110 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 115 120 125 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 130 135 140 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 145 150 155 160 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 165 170 175 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 180 185 190 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 195 200 205 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 210 215 220 Phe Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Gly Gly Gly Gly 225 230 235 240 Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp 245 250 255 Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln 260 265 270 Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly 275 280 285 Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr 290 295 300 Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr Leu Ala His 305 310 315 320 Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val 325 330 335 Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr 340 345 350 Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Leu Glu Trp 355 360 365 Thr Met Pro Gln Ser Ser Ala Gln Thr Leu Ala Met Asn Val Arg 370 375 380 Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His Tyr His Ala 385 390 395 400 Met Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu Lys Ser Gly 405 410 415 Val Val Leu Arg Arg Thr 420 <210> 62 <211> 650 <212> PRT <213> Artificial Sequence <220> Synthetic peptide anti-PDL1 HC_25 <400> 62 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 20 25 30 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 35 40 45 Met Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Val Trp 50 55 60 Ser Ser Ile Tyr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser. 65 70 75 80 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 85 90 95 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 100 105 110 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Ser Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Gly Gly Gly Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val 465 470 475 480 Leu Ser Trp Asp Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His 485 490 495 Leu Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala 500 505 510 Lys Pro Gln Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp 515 520 525 Asp Arg Glu Thr Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met 530 535 540 Thr Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln 545 550 555 560 Glu Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser 565 570 575 Gln His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu 580 585 590 Thr Leu Glu Trp Thr Met Pro Gln Ser Ser Ala Gln Thr Leu Ala 595 600 605 Met Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr 610 615 620 His Tyr His Ala Met Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr 625 630 635 640 Leu Lys Ser Gly Val Val Leu Arg Arg Thr 645 650 <210> 63 <211> 425 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide anti-PDL1 LC_25 <400> 63 Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu Ala 15 10 15 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 20 25 30 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr 35 40 45 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 50 55 60 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 65 70 75 80 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 85 90 95 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 100 105 110 Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys Ala Pro Thr Ser Ser Ser Gly 225 230 235 240 Gly Gly Gly Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu 245 250 255 Ser Trp Asp Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu 260 265 270 Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys 275 280 285 Pro Gln Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp 290 295 300 Arg Glu Thr Arg Asp Leu Thr Gly Trp Gly Lys Asp Leu Arg Met Thr 305 310 315 320 Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu 325 330 335 Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln 340 345 350 His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr 355 360 365 Leu Glu Trp Thr Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met 370 375 380 Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Glu Thr Asp Thr His 385 390 395 400 Tyr His Ala Met Arg Ala Asp Cys Leu Ser Glu Leu Arg Arg Tyr Leu 405 410 415 Lys Ser Gly Val Val Leu Arg Arg Thr 420 425 <210> 64 <211> 274 <212> PRT <213> Homo sapiens <400> 64 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr Leu Tyr His Ala Met 145 150 155 160 His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu 180 185 190 Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr 195 200 205 Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val 260 265 270 Pro Ser <210> 65 <211> 274 <212> PRT <213> Homo sapiens <400> 65 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Ile Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met 145 150 155 160 His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu 180 185 190 Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr 195 200 205 Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val 260 265 270 Pro Ser <210> 66 <211> 274 <212> PRT <213> Homo sapiens <400> 66 Glu Pro His Ser Leu Pro Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Tyr Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 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 Glu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met 145 150 155 160 His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu 180 185 190 Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr 195 200 205 Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val 260 265 270 Pro Ser <210> 67 <211> 274 <212> PRT <213> Homo sapiens <400> 67 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Tyr Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 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 Glu Glu Trp Thr 115 120 125 Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met 145 150 155 160 His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Glu Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu 180 185 190 Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr 195 200 205 Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val 260 265 270 Pro Ser <210> 68 <211> 274 <212> PRT <213> Homo sapiens <400> 68 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met 145 150 155 160 His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu 180 185 190 Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr 195 200 205 Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val 260 265 270 Pro Ser <210> 69 <211> 274 <212> PRT <213> Homo sapiens <400> 69 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met 145 150 155 160 His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu 180 185 190 Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr 195 200 205 Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val 260 265 270 Pro Ser <210> 70 <211> 276 <212> PRT <213> Homo sapiens <400> 70 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met 145 150 155 160 His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu 180 185 190 Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr 195 200 205 Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val 260 265 270 Pro Ser Gly Lys 275 <210> 71 <211> 306 <212> PRT <213> Homo sapiens <400> 71 Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Ala Lys Thr Trp Asp Thr Glu Thr Glu 50 55 60 Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile 65 70 75 80 Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr 115 120 125 Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn 130 135 140 Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met 145 150 155 160 Gln Ala Asp Cys Leu Gln Lys Leu Gln Leu Pro Pro Met Val Asn Val 165 170 175 Ile Cys Ser Glu Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala 180 185 190 Ser Ser Phe Tyr Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly 195 200 205 Val Ser Leu Ser His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp 210 215 220 Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly 225 230 235 240 Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly 245 250 255 Thr His Pro Val Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg 260 265 270 Thr Asp Phe Pro Tyr Val Ser Ala Ala Met Pro Cys Phe Val Ile Ile 275 280 285 Ile Ile Leu Cys Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu 290 295 300 Gly Pro 305 <210> 72 <211> 318 <212> PRT <213> Homo sapiens <400> 72 Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Ala Glu Thr Trp Asp Thr Glu Thr Glu 50 55 60 Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile 65 70 75 80 Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Met His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr 100 105 110 Asn Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr 115 120 125 Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn 130 135 140 Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met 145 150 155 160 Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val 165 170 175 Ala Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Cys Ser Glu 180 185 190 Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr 195 200 205 Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val 260 265 270 Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro 275 280 285 Tyr Val Ser Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys 290 295 300 Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro 305 310 315 <210> 73 <211> 318 <212> PRT <213> Homo sapiens <400> 73 Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Ala Lys Thr Trp Asp Thr Glu Thr Glu 50 55 60 Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile 65 70 75 80 Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr 115 120 125 Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn 130 135 140 Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met 145 150 155 160 Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val 165 170 175 Ala Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Ile Cys Ser Glu 180 185 190 Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr 195 200 205 Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val 260 265 270 Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro 275 280 285 Tyr Val Ser Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys 290 295 300 Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro 305 310 315 <210> 74 <211> 318 <212> PRT <213> Homo sapiens <400> 74 Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asn Val Leu Gly Ala Lys Thr Trp Asp Thr Glu Thr Glu 50 55 60 Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile 65 70 75 80 Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr 115 120 125 Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn 130 135 140 Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met 145 150 155 160 Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val 165 170 175 Ala Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Cys Ser Glu 180 185 190 Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr 195 200 205 Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val 260 265 270 Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro 275 280 285 Tyr Val Ser Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys 290 295 300 Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro 305 310 315 <210> 75 <211> 318 <212> PRT <213> Homo sapiens <400> 75 Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Ala Glu Thr Trp Asp Thr Glu Thr Glu 50 55 60 Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile 65 70 75 80 Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr 100 105 110 Asn Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr 115 120 125 Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn 130 135 140 Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met 145 150 155 160 Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val 165 170 175 Ala Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Cys Ser Glu 180 185 190 Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr 195 200 205 Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Lys 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val 260 265 270 Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro 275 280 285 Tyr Val Ser Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys 290 295 300 Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro 305 310 315 <210> 76 <211> 318 <212> PRT <213> Homo sapiens <400> 76 Glu Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly 15 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Ala Glu Thr Trp Asp Thr Glu Thr Glu 50 55 60 Asp Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile 65 70 75 80 Lys Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr 100 105 110 Asn Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr 115 120 125 Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn 130 135 140 Phe Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met 145 150 155 160 Gln Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val 165 170 175 Ala Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Cys Ser Glu 180 185 190 Val Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr 195 200 205 Pro Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val 260 265 270 Pro Ser Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro 275 280 285 Tyr Val Ser Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys 290 295 300 Val Pro Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro 305 310 315 <210> 77 <211> 171 <212> PRT <213> Homo sapiens <400> 77 His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Gly Asp Gly Ser Val 15 10 15 Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln Pro Phe Leu 20 25 30 Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln Trp Ala 35 40 45 Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg Asp Leu 50 55 60 Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile Lys Asp 65 70 75 80 Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys Glu Ile 85 90 95 His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr Asp Gly 100 105 110 Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr Met Pro 115 120 125 Gln Ser Ser Ala Gln Thr Leu Ala Met Asn Val Arg Asn Phe Leu 130 135 140 Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met Met His Ala 145 150 155 160 Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys 165 170 <210> 78 <211> 169 <212> PRT <213> Homo sapiens <400> 78 His Cys Leu Cys Tyr Asp Phe Ile Ile Thr Pro Lys Ser Arg Pro Glu 15 10 15 Pro Gln Trp Cys Glu Val Gln Gly Leu Val Asp Glu Arg Pro Phe Leu 20 25 30 His Tyr Asp Cys Val Asn His Lys Ala Lys Ala Phe Ala Ser Leu Gly 35 40 45 Lys Lys Val Asn Val Thr Lys Thr Trp Glu Glu Gln Thr Glu Thr Leu 50 55 60 Arg Asp Val Val Asp Phe Leu Lys Gly Gln Leu Leu Asp Ile Gln Val 65 70 75 80 Glu Asn Leu Ile Pro Ile Glu Pro Leu Thr Leu Gln Ala Arg Met Ser 85 90 95 Cys Glu His Glu Ala His Gly His Gly Arg Gly Ser Trp Gln Phe Leu 100 105 110 Phe Asn Gly Gln Lys Phe Leu Leu Phe Asp Ser Asn Asn Arg Lys Trp 115 120 125 Thr Ala Leu His Pro Gly Ala Lys Lys Met Thr Glu Lys Trp Glu Lys 130 135 140 Asn Arg Asp Val Thr Met Phe Phe Gln Lys Ile Ser Leu Gly Asp Cys 145 150 155 160 Lys Met Trp Leu Glu Glu Phe Leu Met 165 <210> 79 <211> 169 <212> PRT <213> Homo sapiens <400> 79 His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg Pro Gly 15 10 15 Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu 20 25 30 His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro Leu Gly 35 40 45 Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro Val Leu 50 55 60 Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Arg Asp Ile Gln Leu 65 70 75 80 Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser 85 90 95 Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln Phe Ser 100 105 110 Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Lys Arg Met Trp 115 120 125 Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn 130 135 140 Asp Lys Val Val Ala Met Ser Phe His Tyr Phe Ser Met Gly Asp Cys 145 150 155 160 Ile Gly Trp Leu Glu Asp Phe Leu Met 165 <210> 80 <211> 169 <212> PRT <213> Homo sapiens <400> 80 His Ser Leu Trp Tyr Asn Phe Thr Ile Ile His Leu Pro Arg His Gly 15 10 15 Gln Gln Trp Cys Glu Val Gln Ser Gln Val Asp Gln Lys Asn Phe Leu 20 25 30 Ser Tyr Asp Cys Gly Ser Asp Lys Val Leu Ser Met Gly His Leu Glu 35 40 45 Glu Gln Leu Tyr Ala Thr Asp Ala Trp Gly Lys Gln Leu Glu Met Leu 50 55 60 Arg Glu Val Gly Gln Arg Leu Arg Leu Glu Leu Ala Asp Thr Glu Leu 65 70 75 80 Glu Asp Phe Thr Pro Ser Gly Pro Leu Thr Leu Gln Val Arg Met Ser 85 90 95 Cys Glu Cys Glu Ala Asp Gly Tyr Ile Arg Gly Ser Trp Gln Phe Ser 100 105 110 Phe Asp Gly Arg Lys Phe Leu Leu Phe Asp Ser Asn Asn Arg Lys Trp 115 120 125 Thr Val Val His His Ala Gly Ala Arg Arg Met Lys Glu Lys Trp Glu Lys 130 135 140 Asp Ser Gly Leu Thr Thr Phe Phe Lys Met Val Ser Met Arg Asp Cys 145 150 155 160 Lys Ser Trp Leu Arg Asp Phe Leu Met 165 <210> 81 <211> 168 <212> PRT <213> Homo sapiens <400> 81 His Ser Leu Cys Phe Asn Phe Thr Ile Lys Ser Leu Ser Arg Pro Gly 15 10 15 Gln Pro Trp Cys Glu Ala Gln Val Phe Leu Asn Lys Asn Leu Phe Leu 20 25 30 Gln Tyr Asn Ser Asp Asn Asn Met Val Lys Pro Leu Gly Leu Leu Gly 35 40 45 Lys Lys Val Tyr Ala Thr Ser Thr Trp Gly Glu Leu Thr Gln Thr Leu 50 55 60 Gly Glu Val Gly Arg Asp Leu Arg Met Leu Leu Cys Asp Ile Lys Pro 65 70 75 80 Gln Ile Lys Thr Ser Asp Pro Ser Thr Leu Gln Val Glu Met Phe Cys 85 90 95 Gln Arg Glu Ala Glu Arg Cys Thr Gly Ala Ser Trp Gln Phe Ala Thr 100 105 110 Asn Gly Glu Lys Ser Leu Leu Phe Asp Ala Met Asn Met Thr Trp Thr 115 120 125 Val Ile Asn His Glu Ala Ser Lys Ile Lys Glu Thr Trp Lys Lys Asp 130 135 140 Arg Gly Leu Glu Lys Tyr Phe Arg Lys Leu Ser Lys Gly Asp Cys Asp 145 150 155 160 His Trp Leu Arg Glu Phe Leu Gly 165 <210> 82 <211> 169 <212> PRT <213> Homo sapiens <400> 82 His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg Pro Gly 15 10 15 Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu 20 25 30 His Tyr Asp Cys Gly Ser Lys Thr Val Thr Pro Val Ser Pro Leu Gly 35 40 45 Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro Val Leu 50 55 60 Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Leu Asp Ile Gln Leu 65 70 75 80 Glu Asn Tyr Ile Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser 85 90 95 Cys Glu Gln Lys Ala Glu Gly His Gly Ser Gly Ser Trp Gln Leu Ser 100 105 110 Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu Asn Arg Met Trp 115 120 125 Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn 130 135 140 Asp Lys Asp Met Thr Met Ser Phe His Tyr Ile Ser Met Gly Asp Cys 145 150 155 160 Thr Gly Trp Leu Glu Asp Phe Leu Met 165 <210> 83 <211> 169 <212> PRT <213> Homo sapiens <400> 83 His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg Pro Gly 15 10 15 Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu 20 25 30 His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro Leu Gly 35 40 45 Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln Asn Pro Val Leu 50 55 60 Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Leu Asp Ile Gln Leu 65 70 75 80 Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser 85 90 95 Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln Phe Ser 100 105 110 Ile Asp Gly Gln Thr Phe Leu Leu Phe Asp Ser Glu Lys Arg Met Trp 115 120 125 Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn 130 135 140 Asp Lys Asp Val Ala Met Ser Phe His Tyr Ile Ser Met Gly Asp Cys 145 150 155 160 Ile Gly Trp Leu Glu Asp Phe Leu Met 165 <210> 84 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide ULBP2 alpha1-alpha2 R80W <400> 84 Ala Ala Glu Pro His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys 15 10 15 Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu 20 25 30 Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val 35 40 45 Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln 50 55 60 Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Trp 65 70 75 80 Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln 85 90 95 Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser 100 105 110 Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu 115 120 125 Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu 130 135 140 Lys Trp Glu Asn Asp Lys Val Val Ala Met Ser Phe His Tyr Phe Ser 145 150 155 160 Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp 165 170 175 Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro Pro Met Val 180 185 <210> 85 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide ULBP2 alpha1-alpha2 V151D <400> 85 Ala Ala Glu Pro His Ser Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys 15 10 15 Phe Arg Pro Gly Pro Arg Trp Cys Ala Val Gln Gly Gln Val Asp Glu 20 25 30 Lys Thr Phe Leu His Tyr Asp Cys Gly Asn Lys Thr Val Thr Pro Val 35 40 45 Ser Pro Leu Gly Lys Lys Leu Asn Val Thr Thr Ala Trp Lys Ala Gln 50 55 60 Asn Pro Val Leu Arg Glu Val Val Asp Ile Leu Thr Glu Gln Leu Arg 65 70 75 80 Asp Ile Gln Leu Glu Asn Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln 85 90 95 Ala Arg Met Ser Cys Glu Gln Lys Ala Glu Gly His Ser Ser Gly Ser 100 105 110 Trp Gln Phe Ser Phe Asp Gly Gln Ile Phe Leu Leu Phe Asp Ser Glu 115 120 125 Lys Arg Met Trp Thr Thr Val His Pro Gly Ala Arg Lys Met Lys Glu 130 135 140 Lys Trp Glu Asn Asp Lys Asp Val Ala Met Ser Phe His Tyr Phe Ser 145 150 155 160 Met Gly Asp Cys Ile Gly Trp Leu Glu Asp Phe Leu Met Gly Met Asp 165 170 175 Ser Thr Leu Glu Pro Ser Ala Gly Ala Pro Pro Met Val 180 185 <210> 86 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide ULBP3 alpha1-alpha2 R162G <400> 86 Ala Ala Glu Pro His Ser Leu Trp Tyr Asn Phe Thr Ile Ile His Leu 15 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 Gly Asp Cys Lys Ser Trp Leu Arg Asp Phe Leu Met His Arg Lys 165 170 175 Lys Arg Leu Glu Pro Thr Ala Pro Pro Met Val 180 185

Claims (62)

A non-natural, modified α1-α2 domain molecule from a mammalian-born MHC class I chain-associated (MIC) molecule, comprising an amino acid sequence having at least 80% identity to the conformational α1-α2 domain of the MIC molecule, Naturally occurring, modified α1-α2 domain molecule having two or more of its congenital amino acids whose molecules are replaced at positions selected from the group consisting of 20, 68, 125, 152, 161 and 166. The non-natural, modified? 1-alpha 2 domain molecule of claim 1, wherein the congenital MIC molecule is selected from the group consisting of SEQ ID NOs: 64-76. The non-natural, modified α1-α2 domain molecule of claim 1, wherein the domain molecule is modified to alter its binding affinity for human NKG2D as compared to the congenital α1-α2 domain. 4. The non-natural, modified α1-α2 domain molecule of claim 3, wherein its altered binding affinity for NKG2D is affected by an altered off-rate. 4. The non-natural, modified α1-α2 domain molecule of claim 3, wherein its altered binding affinity for NKG2D is affected by a slower off-rate. The non-natural, modified α1-α2 domain molecule as set forth in claim 1:
The amino acid at position 20 is P, T, D, A, L or N;
The amino acid at position 68 is L, F, S, A, Y, I, E, T or W;
The amino acid at position 125 is L, R, F, T, A, N, V, Y, I or S;
Wherein the amino acid at position 152 is E, T, V, G, F, Y, A, Q, D, I, N, S, H, M or P;
Amino acid at position 161 is R, S, A, K, G, L, F or Y; or
The amino acid at position 166 is F, S, H, Y, W, V, L or M;
Or a combination of these position changes. 7. A non-natural, modified α1-α2 domain molecule according to claim 6 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 32, 33, 34, 39, 40 and 41. The non-natural, modified? 1-alpha 2 domain molecule of claim 1, which exhibits greater binding affinity for NKG2D compared to the congenital alpha-alpha 2 domain. The non-natural, modified α1-α2 domain molecule according to claim 8, which exhibits enhanced activation of cells expressing NKG2D resulting in cells having a greater target cell killing effect. The non-natural, modified α1-α2 domain molecule of claim 1, further comprising an attached heterologous peptide. The non-natural, modified α1-α2 domain molecule of claim 10, wherein the attached heterologous peptide is an antibody, antibody light chain, antibody heavy chain or fragment thereof. 11. A non-natural, modified? 1-alpha 2 domain molecule according to claim 10, wherein the peptide is delivered to NKG2D. 11. The non-natural, modified &lt; RTI ID = 0.0 &gt; ai-a2 &lt; / RTI &gt; domain molecule of claim 10 wherein the heterologous peptide induces binding of the domain molecule to the target molecule on the target cell, thereby delivering the domain molecule to the target cell. 14. The non-natural, modified? 1-α2 domain molecule of claim 13, exhibiting greater target cell killing efficacy compared to the native α1-α2 domain attached to the antibody, antibody light chain, antibody heavy chain or fragment thereof. 15. The non-natural, modified α1-α2 domain molecule of claim 14, which exhibits greater in vivo target cell killing efficacy. A composition comprising a non-natural, modified α1-α2 domain molecule according to claim 1, and a carrier and an excipient. 17. The composition of claim 16, further comprising a therapeutic or diagnostic agent. 18. The composition of claim 17, wherein the therapeutic or diagnostic agent is attached to a non-natural, modified α1-α2 domain molecule. A nucleic acid molecule encoding a non-natural, modified? 1-a2 domain molecule according to claim 1. 19. An expression cassette comprising a nucleic acid molecule according to claim 19. A pharmaceutical composition for treating a mammal suspected of having a malignant tumor or viral infection comprising an effective amount of a non-natural, modified α1-α2 domain molecule according to claim 12, wherein the target cell is a malignant cell or a virus- Lt; / RTI &gt; A pharmaceutical composition for treating a mammal suspected of having a malignant tumor or viral infection, comprising an effective amount of a non-natural, modified α1-α2 domain molecule according to claim 10. As an insertable Fv (iFv) polypeptide exhibiting specific antigen-binding properties comprising a variable fragment (Fv) of an antibody, wherein the specific antigen-binding domain of Fv is one variable domain is from a light chain (VL) The variable domain comprises two antibody variable domains (each having 1-3 complementarity determining regions (CDRs), each having a C-terminus and an N-terminus) from the heavy chain (VH); Wherein the VL and VH domains are joined by two linker moieties joining both the canonical ends of the VL domains to both the canonical ends of the VH domains. 24. The iFv polypeptide of claim 23, wherein a non-natural pair of N- and C-termini are generated in the VL or VH domain to provide an attachment site for attachment to the recipient molecule or insertion into the recipient molecule. 26. The iFv polypeptide of claim 24, wherein the non-natural N- and C-termini are spatially adjacent to each other. 25. The iFv polypeptide of claim 24, further comprising a recipient polypeptide or protein comprising at least one solvent-exposed loop, wherein the iFv polypeptide is inserted into at least one of the loops. 26. An iFv polypeptide according to claim 24, further comprising a recipient polypeptide or protein comprising at least one solvent-exposed loop, wherein the iFv is inserted into at least one of the loops to generate non-natural N- and C- An iFv polypeptide wherein the ends are spatially adjacent to one another. 24. The method of claim 23, wherein the C-terminus of the VL domain is linked to the N-terminus of the VH domain and the N-terminus of the VL domain is connected to the C-terminus of the VH domain; An iFv polypeptide wherein the N-terminus of the VL domain is linked to the C-terminus of the VH domain and the C-terminus of the VL domain is linked to the N-terminus of the VH domain. 28. The iFv polypeptide of claim 27, wherein the distance between the non-natural N- and C-termini ranges from about 0.2 to about 2.0 nm. 30. The iFv polypeptide of claim 29, wherein the distance between the ends is from about 0.5 nm to about 1.5 nm. 24. The iFv polypeptide of claim 23, wherein the two linker sites comprise the same or non-identical peptides of about 10 to 25 amino acids. 28. The method of claim 27, wherein the recipient polypeptide or protein is selected from the group consisting of an antibody, an Ig fold, an Ig domain, a globulin, an albumin, a fibronectin, a fibronectin domain, an integrin, a fluorescent protein, an enzyme, an external membrane protein, , A viral antigen, a viral capsid, a viral ligand for a cell receptor, a high molecular weight bacteriocin, a histone, a hormone, a knottin, a cyclic peptide, a ULB protein, a lectin, or a lectin. 24. The iFv polypeptide of claim 23, wherein the iFv further comprises a non-protein molecule into which is inserted or attached. 34. The method of claim 33, wherein the non-protein molecule is selected from the group consisting of a polysaccharide, a dendrimer, a polyglycol, a peptidoglycan, a chemotherapeutic agent, a toxin, a fluorescent moiety, a chromophore, a pharmacophore, an antibiotic or a polyketide, Polypeptide. 27. The iFv polypeptide of claim 24, further comprising a non-protein molecule or atom conjugated to one or both of the non-natural N-terminal and non-natural C-termini. 28. The iFv polypeptide of claim 27, wherein all or part of one or more solvent-exposed loops of the recipient molecule are deleted and replaced by an iFv. A non-natural, modified α1-α2 domain molecule from a human-derived NKG2D ligand molecule comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 16 and having a domain molecule consisting of 80, 151, 152 and 153 Naturally occurring, modified &lt; RTI ID = 0.0 &gt; ai-a2 &lt; / RTI &gt; domain molecule having at least one of its native amino acids substituted at a position selected from the group. 38. The non-natural, modified? 1-? 2 domain molecule of claim 37, wherein the congenital ligand molecule is selected from the group consisting of SEQ ID NOs: 15, 16, 39. The non-natural, modified? 1-α2 domain molecule of claim 38, wherein the domain molecule is modified to alter its binding affinity for human NKG2D compared to its native α1-α2 domain. 40. The method of claim 39, wherein the non-natural, modified? 1-? 2 domain molecule:
The amino acid at position 80 is L, W, V, F, I, S, A, E, P or T;
The amino acid at position 151 is D, E, Q, K, N, R or T;
The amino acid at position 152 is L or W;
The amino acid at position 153 is E, K, G or P;
Or a combination of these position changes. A naturally occurring, modified α1-α2 domain molecule from a human-derived NKG2D ligand molecule comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 17, wherein the domain molecule is selected from the group consisting of 162 and 165 Non-natural, modified &lt; RTI ID = 0.0 &gt; ai-a2 &lt; / RTI &gt; domain molecule having at least one of its native amino acids substituted in position. 42. The non-natural, modified? 1-? 2 domain molecule of claim 41, wherein the congenital ligand molecule is selected from the group consisting of SEQ ID NOs: 17 and 18. 43. The non-natural, modified? 1-? 2 domain molecule of claim 42, wherein the domain molecule is modified to alter its binding affinity for human NKG2D compared to its native? 44. The non-natural, modified α1-α2 domain molecule of claim 43,
The amino acid at position 162 is G, A, or Y; And / or
The amino acid at position 165 is S, P, A, T, H, N, Q or G; 9. A non-natural, modified α1-α2 domain molecule attached to an immunogen, wherein the α1-α2 domain has an adjuvant activity to accelerate and / or enhance the efficacy of the recipient animal's immune response to the immunogen Lt; RTI ID = 0.0 &gt; a1-a2 &lt; / RTI &gt; domain molecule. 41. The non-natural, modified? 1-alpha 2 domain molecule of claim 40, which exhibits greater binding affinity for NKG2D compared to its native alpha 1-alpha 2 domain. 47. The non-natural, modified α1-α2 domain molecule of claim 46, further comprising an attached heterologous peptide. 48. The non-natural, modified α1-α2 domain molecule of claim 47, which exhibits enhanced activation of cells expressing NKG2D resulting in cells having greater target cell killing efficacy. 47. The non-natural, modified? 1-? 2 domain molecule of claim 47, wherein the attached heterologous peptide is an antibody, an antibody light chain, an antibody heavy chain or a fragment thereof. 49. The non-natural, modified α1-α2 domain molecule of claim 48, wherein the peptide is delivered to NKG2D. 49. The non-natural, modified α1-α2 domain molecule of claim 48, wherein the heterologous peptide induces binding of the domain molecule to the target molecule on the target cell, thereby transferring the domain molecule to the target cell. 47. The non-natural, modified? 1-? 2 domain molecule of claim 47, exhibiting greater target cell killing efficacy compared to its native α1-α2 domain attached to the peptide. 47. The non-natural, modified α1-α2 domain molecule of claim 46 comprising the amino acid sequence of SEQ ID NO: 84. 47. The non-natural, modified α1-α2 domain molecule of claim 46 comprising the amino acid sequence of SEQ ID NO: 85. 44. The non-natural, modified α1-α2 domain molecule of claim 44, which exhibits greater binding affinity for NKG2D compared to its native α1-α2 domain. 56. The non-natural, modified? 1-? 2 domain molecule of claim 55, further comprising an attached heterologous peptide. 56. The non-natural, modified? 1-? 2 domain molecule of claim 55, which exhibits enhanced activation of cells expressing NKG2D resulting in cells having greater target cell killing efficiency. 57. The non-natural, modified? 1-? 2 domain molecule of claim 56, wherein the attached heterologous peptide is an antibody, an antibody light chain, an antibody heavy chain or a fragment thereof. 57. The non-natural, modified? 1-? 2 domain molecule of claim 56, wherein the peptide is delivered to NKG2D. 56. The non-natural, modified &lt; RTI ID = 0.0 &gt; ai-a2 &lt; / RTI &gt; domain molecule of claim 56 wherein the heterologous peptide induces binding of the domain molecule to the target molecule on the target cell, thereby delivering the domain molecule to the target cell. 60. The non-natural, modified α1-α2 domain molecule of claim 60, which exhibits greater target cell killing efficacy compared to its native α1-α2 domain attached to the peptide. 61. The non-natural, modified? 1-? 2 domain molecule of claim 60 comprising the amino acid sequence of SEQ ID NO: 86.

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