Dap10 / dap12 fusion polypeptides

Abstract

The present invention relates to a fusion polypeptide comprising a DNAX-activating protein 10 (DAP10) polypeptide and a DNAX-activating protein 12 (DAP12) polypeptide. The present invention also relates to a cell comprising said fusion protein and its use in the treatment of cancer.

Description

Technical Field

[0001] This invention relates to a fusion polypeptide comprising a DNAX-activated protein 10 (DAP10) polypeptide and a DNAX-activated protein 12 (DAP12) polypeptide. The invention also relates to cells comprising said fusion polypeptide and their use in the treatment of cancer. Background Technology

[0002] Immunotherapy using chimeric antigen receptor (CAR) engineered T cells has proven transformative in the treatment of B-cell malignancies and multiple myeloma. However, the application of this technology in solid tumor immunotherapy is hampered by the lack of tumor-selective targets. Most tumor antigens are intracellular, making them difficult for CAR-T cells to recognize. Consequently, most of the targets involved in currently developed targeted CARs for solid tumors are upregulated in tumor cells but present at lower levels in normal tissues.

[0003] NKG2D ligands are among the few target groups exhibiting high tumor selectivity. In humans, these ligands comprise a group of eight stress-inducible proteins (MICA, MICB, ULBP1-6), which are aberrantly expressed in almost all tumor cell types. Furthermore, NKG2D ligands are also present on tumor-associated stromal elements such as endothelial cells, regulatory T cells, and myeloid-derived suppressor cells (Parihar, R., et al., 2019, Cancer Immunol. Res. 7(3):363-375; Schmiedel & Mandelboim, 2018, Front. Immunol. (9) 2040). Mice genetically lacking NKG2D exhibit impaired immune surveillance against both epithelial and lymphoid malignancies. The absence of NKG2D ligands in healthy tissues suggests that NKG2D ligands are a safe therapeutic target. Furthermore, no significant safety issues have been identified in ongoing clinical trials involving NKG2D-targeted CARs, even when combined with fludarabine / cyclophosphamide lymphoablation chemotherapy (see https: / / www.celyad.com / en / news / celyad-presents-update-on-autologous-allogeneic-nkg2d-based-car-t-therapies-in-solid-tumors).

[0004] The NKG2D receptor is naturally expressed by natural killer (NK) cells and some T cell populations. Each NKG2D homodimer binds to two DAP10 adaptor molecules via complementary charged amino acids within the plasma membrane. This interaction is essential for the cell surface expression and function of NKG2D. DAP10 provides co-stimulation via phosphatidylinositol 3-kinase in a manner similar to CD28, but importantly, it lacks the p56lck binding motif to promote recruitment of regulatory T cells that is not desired (Kofler, et al., 2011, Mol. Ther. 19:760-767). The potency of DAP10 co-stimulation is enhanced by its ability to continue signaling after internalization. However, due to the lack of an immune receptor tyrosine-based activation motif (ITAM) in DAP10, the involvement of NKG2D does not lead to complete T cell activation.

[0005] Several CARs have been developed that utilize different approaches to provide co-stimulation (also known as signal 2) in addition to ITAM-dependent signal 1, as both signal types are essential for the complete activation of T cells. The first NKG2D-targeting CAR was developed by Sentman et al., which consists of a fusion of NKG2D and CD3ζ (Zhang et al., 2005, Blood 106:1544-1551). Although nominally a first-generation CAR, it is associated with endogenous DAP10 in T cells, meaning it provides both signals 1 and 2 simultaneously. This CAR is currently being clinically developed by Celyad SA as Cyad-01. More recently, Chang et al. (2013, Cancer Res. 73:1777-1786) engineered NK cells that co-expressed the same CAR in addition to exogenous DAP10. Two other NKG2D CARs were also described as combining 4-1BB (Song et al., 2013, Hum. Gene Ther. 24:295-305) or CD28 (Lehner et al., 2012, PLoS One 7:e31210) to provide alternative forms of co-stimulation, rather than that provided by DAP10. All of these CARs enabled T cell-mediated killing of tumor cells, accompanied by cytokine production, while the CAR described by Chang et al. also showed transient in vivo antitumor activity. Invention Overview

[0007] An isoform of mouse NKG2D found in NK cells can trans-bind to DAP10 and DAP12 (which contain a single ITAM), potentially providing signal 1 and signal 2 for T cell activation. Based on this observation, we designed a fusion peptide comprising DAP10 and DAP12. When co-expressed with NKG2D, the fusion peptide and NKG2D bind together on the plasma membrane via complementary charged amino acid residues, forming a small CAR based on a tight adaptor that drives full T cell activation.

[0008] Therefore, the fusion polypeptide provided by the present invention comprises (i) a DNAX-activated protein 10 (DAP10) polypeptide, or a functional variant thereof, and (ii) a DNAX-activated protein 12 (DAP12) polypeptide, or a functional variant thereof. The DAP10 polypeptide (or its functional variant thereof) and the DAP12 polypeptide (or its functional variant thereof) may be directly fused together or linked by a linker. The fusion polypeptide may further include an N-terminal or C-terminal amino acid sequence, for example, to assist in detection or purification, to improve expression, or to increase half-life.

[0009] In one embodiment, the present invention provides a fusion polypeptide having the following general formula, from N-terminus to C-terminus: ABCDE.

[0010] in,

[0011] A = an arbitrary N-terminal sequence;

[0012] B = A DAP10 polypeptide or a functional variant thereof;

[0013] C = an optional concatenation sequence;

[0014] D = a DAP12 polypeptide or a functional variant thereof;

[0015] E = an optional C-terminal sequence.

[0016] In one embodiment, the fusion polypeptide does not include or is not composed of SEQ ID NO:1 of WO 2019 / 182425 (hereinafter referred to as SEQ ID NO:84). In some embodiments, the fusion polypeptide does not include an anti-EpCAM peptide. In some embodiments, the fusion polypeptide does not include an EpCAM-specific antigen receptor. In some embodiments, the fusion polypeptide does not include or is not composed of SEQ ID NO:9 of WO 2019 / 182425 (hereinafter referred to as SEQ ID NO:85). In some embodiments, the DAP10 polypeptide or a functional variant thereof does not include or is not composed of SEQ ID NO:85. In some embodiments, the fusion polypeptide does not include or is not composed of SEQ ID NO:11 of WO 2019 / 182425 (hereinafter referred to as SEQ ID NO:86). In some embodiments, the DAP12 polypeptide or a functional variant thereof does not include or is not composed of SEQ ID NO:86. In some embodiments, the fusion polypeptide does not include or is not composed of both SEQ ID NO:85 and SEQ ID NO:86.

[0017] In other respects, the present invention relates to a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding the fusion polypeptide described herein, comprising DNA and RNA molecules. Such a nucleic acid molecule may also optionally encode an NKG2D polypeptide or a functional variant thereof.

[0018] The present invention also discloses vectors, particularly expression vectors, containing the nucleic acid molecules of the present invention.

[0019] The present invention also provides host cells comprising nucleic acids and / or vectors encoding the fusion polypeptide described herein. The present invention further provides host cells comprising the fusion polypeptide of the present invention.

[0020] The present invention also provides a method for preparing the fusion polypeptide of the present invention, which includes maintaining the host cell of the present invention under conditions suitable for expressing the nucleic acid, thereby expressing the recombinant nucleic acid and generating the fusion polypeptide.

[0021] The present invention also provides a pharmaceutical composition comprising the fusion polypeptide, nucleic acid molecule, vector or host cell of the present invention, optionally further comprising a pharmaceutically or physiologically acceptable vector.

[0022] The present invention also provides a method for treating patients with pathological diseases, comprising administering a fusion polypeptide comprising the fusion polypeptide of the present invention, a nucleic acid molecule, a vector, or a host cell to the patient in need.

[0023] The present invention also provides fusion peptides, nucleic acid molecules, vectors, host cells or pharmaceutical compositions according to the present invention for (i) treating and (ii) preparing medicaments for treating diseases or conditions of the present invention. Attached Figure Description

[0024] Figure 1 A schematic diagram of each constructed structure is shown. N1012 comprises a complex consisting of exogenous human NKG2D protein and a fusion exogenous DAP10 / 12 homodimer according to the invention. N1012 comprises the sequence shown in SEQ ID NO:64, described in Table 2. The NKG2D complex comprises exogenous human NKG2D protein that interacts with endogenous DAP10 already present in the cells and is provided for comparative purposes only.

[0025] Figure 2 The results showed that, compared with untransfected (UT) 293T cells, cells transfected with a retroviral plasmid expressing N1012 (e.g., Figure 1 (As shown) or NKG2D expression on the surface and inside the cells of 293T cells three days later (ICS).

[0026] Figure 3 The percentage of NKG2D expression on the cell surface was shown after transduction into activated undifferentiated human T cells using a retroviral vector encoding either NKG2D or N1012 alone. Figure 3 (A) and median fluorescence intensity (MFI) Figure 3 (B). Expression in UTT cells and MFI are provided as a comparison.

[0027] Figure 4 The image shows CD4+ of activated undifferentiated T cells transduced to express N1012, NKG2D, or a control. + and CD8 + Representative expression patterns of NKG2D on the cell surface in the subsets. NKG2D expression in UTT cells is provided for comparison.

[0028] Figure 5 Eleven different tumor cell lines were shown compared with N1012. + NKG2D + Survival of CAR T cells co-cultured with different CAR T cell:target cell ratios. After 72 hours, tumor cell survival was assessed using the MTT assay and expressed as a percentage observed in the absence of T cell co-culture.

[0029] Figure 6 shows the cytokines (IFN-γ) secreted when N1012, NKG2D, and UT T cells were co-cultured with eight different tumor cell lines at a 1:1 CAR T cell:target cell ratio. Figure 6A ) and IL-2 ( Figure 6B The levels of cytokines were assessed. After 72 hours, the co-culture supernatant was removed, and the presence of cytokines was evaluated using enzyme-linked immunosorbent assay (ELISA).

[0030] Figure 7 Showing N1012 + NKG2D + Survival rates of tumor and pancreatic stellate cells PS1 after co-culturing CAR T cells with different CAR T cell:target cell ratios. Figure 7 A shows the relationship with N1012 + NKG2D + The survival rate of pancreatic stellate cells PS1 after co-culturing CAR T cells with different CAR T cell:target cell ratios. When CAR T cells are added at a 1:1 CAR T cell:target cell ratio, N1012+ T cells can also effectively lyse monolayers of PS1 cells and BxPC3 cells grown together at a 1:1 ratio. Figure 7 (B). This is related to the addition of NKG2D. + This contrasts with the lack of potency observed after UT T cells. In both cases, target cell survival was assessed by an MTT assay after 48 hours and expressed as a percentage observed without T cell co-culture. Figure 7 C(BxPC3_LT+PS1) and Figure 7 The D(PaTU+PS1) value shows the relationship with N1012. + NKG2D + The survival rate of tumor cells and stromal cells in monolayers after co-culturing CAR T cells at a 1:1 ratio of CAR T cells to target cells. Although compared with N1012 + Effective lysis of tumor and stromal cells was observed during T cell culture, but this was not observed when using NKG2D or UT T cells ( Figure 7 When co-cultured with CD, a slight decrease in the survival rate of target cells was observed. Figure 7 E and Figure 7 F shows the relationship with N1012 + A2028z + NKG2D + Or, after co-culturing CAR T cells at a 1:1 ratio of CAR T cells to target cells, the survival rate of tumor cells and stromal cell monolayers (after up to 5 repeated stimulations). Figure 7 G and H indicate N1012 + A2028z+ NKG2D + The levels of IFN-γ secreted by UTT cells during the above co-culture period were compared. The level of IFN-γ secreted solely from the tumor served as a negative control. Figure 7 I and J show the relationship with N1012 + NKG2D + Survival rate of tumor spheroids after co-culture with UTT cells. Figure 7 K shows N1012 during co-culture with tumor spheroids + The levels of IFN-γ secreted by UTT cells and tumors alone.

[0031] Figure 8 illustrates the ability of N1012-expressing T cells to undergo multiple rounds of repeated stimulation (“restimulation”) compared to UT T cells or T cells expressing NKG2D alone. N1012-expressing T cells are able to undergo multiple rounds of repeated stimulation (…). Figure 8B ) mediates mesothelioma (Ju77) cells and head and neck (HN3_LUC) cells ( Figure 8A The cleavage of ) during which they exhibited a large number of proliferations ( Figure 8C ) and T cell fold expansion ( Figure 8D ). Figure 8B and 8D It was also shown that T cells expressing N1012 could undergo repeated cyclic stimulation and exhibited significant proliferation when cultured with Ren or BxP3_LT cells. In contrast, UT T cells or those T cells expressing NKG2D alone exhibited minimal target cell lysis or proliferation.

[0032] Figure 9 shows the evaluation of retroviral transduction of CAR T cells in tumor-bearing mice. Figure 9A The CD4 transduced using N1012 or NKG2D is shown. + and CD8 + NKG2D expression on the surface of T cells. Comparisons were made with T cells expressing a pan-ErbB-targeting CAR and T4 cells from the same donor (e.g., Davies et al., 2012, Mol. Med. 18: 565-576). A second pan-ErbB-targeting CAR, named TMY, was also used, lacking the 4αβ domain found in T4 and possessing a slightly altered CD28 hinge (where the MYPPPY sequence was replaced by a 10-amino acid linear myc tag sequence). Figure 9B The in vitro cytotoxic function of the residual T cells against the tumor cell line was demonstrated after transfection into mice.

[0033] Figure 10 shows the growth of ffLUC-labeled BxPC3 cells in NSG mice, determined by bioluminescence imaging (BLI). Figure 10AThe summary of BLI emission for each group of mice is shown. Figure 10B The continuous bioluminescence emission of individual mice was shown. Figure 10C This shows the weight of the mouse. Figure 10D The continuous bioluminescence emission of individual mice was shown after tumor re-excitation on day 88. Figure 10E Survival curves for experiments that ended after 145 days.

[0034] Figure 11 shows the results of another experiment, demonstrating the growth of ffLUC-labeled BxPC3 cells in NSG mice, as determined by bioluminescence imaging (BLI). Figure 11A The average total flux (photons / second) of BLI is shown for each treatment group. Figure 11B The total BLI flux (photons / second) for each mouse is shown. On day 41 (29 days after T-cell infusion), tumor-free mice were re-injected intravenously with ffLUC-labeled BxPC3 cells (shown as dashed lines).

[0035] Figure 12 shows the use of N1012 + The growth of ffLUC-labeled H226 malignant mesothelioma cells in NSG mice after T-cell therapy was determined by bioluminescence imaging (BLI). Figure 12A The average total flux (photons / second) of BLI is shown for each treatment group. Figure 12B The total BLI flux (photons / second) for each mouse is shown. To confirm the persistence and retention of T cells, all tumor-free mice were re-inoculated with 1 × 10⁻⁶ cells 91 days after the initial tumor inoculation. 6 H226 cells labeled with ffLUC, re-excited, are shown as dashed lines in the figure.

[0036] Figure 13 The NKG2D, N1012, and CYAD-01 were shown on CD4. + Surface expression in T cells.

[0037] Figure 14 Comparison of N1012 + Restimulation of CYAD-01T cells and NKG2D T cells when T cells are co-cultured with BxPC3-LT, Ren, or Ju77 cells Figure 14 A) and proliferation potential ( Figure 14 (B).

[0038] Figure 15A Showing N1012 + Survival rate of tumor spheroids after co-culture with CYAD-01 or UTT cells. Figure 15B The proliferation level of T cells during co-culture with tumor spheroids was shown. Invention Details

[0040] DAP10 peptide and its functional variants

[0041] The DAP10 peptide used in the fusion peptides described herein can be from mammals, such as humans. Wild-type human DAP10 is encoded by the amino acid sequence of UniProt accession number Q9UBK5 (SEQ ID NO:1). It is a 93-amino acid peptide. The first 18 amino acids are considered to be the signal / guide sequence, amino acids 19-48 are the extracellular domain, amino acids 49-69 are the transmembrane domain, and amino acids 70-93 are the cytoplasmic / intracellular domain.

[0042] In one embodiment, the DAP10 peptide used in the fusion peptide of the present invention comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the DAP10 peptide shown in SEQ ID NO:1. In some embodiments, the DAP10 peptide used in the fusion peptide of the present invention comprises an amino acid sequence shown in SEQ ID NO:1.

[0043] In another embodiment, the functional variant DAP10 peptide used in the fusion peptide of the present invention may include one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete or substitute any amino acid in the DAP10 amino acid (e.g., the amino acid of wild-type human DAP10 (SEQ ID NO: 1)).

[0044] Truncated variants of the DAP10 peptide can also be used in the fusion peptide of the present invention. For example, a truncated variant of DAP10 comprising only 19-93 amino acids as shown in SEQ ID NO:1 (i.e., lacking 1-18 amino acids, the signal / guide sequence) can also be used in the fusion peptide of the present invention. This sequence is referred to herein as SEQ ID NO:2. Other truncated variants may comprise 19-69 amino acids of SEQ ID NO:1, such a sequence comprising only the extracellular and transmembrane domains of DAP10, referred to herein as SEQ ID NO:3. Further truncated variants of DAP10 used in the present invention may comprise 1-71 amino acids of SEQ ID NO:1 (i.e., the signal / guide sequence, the extracellular domain, the transmembrane domain, and 2 amino acids from the cytoplasmic / intracellular domain), referred to herein as SEQ ID NO:4. Further truncated variants of DAP10 used in the present invention may comprise 19-71 amino acids of SEQ ID NO:1 (i.e., the extracellular domain, the transmembrane domain, and 2 amino acids from the cytoplasmic / intracellular domain), referred to herein as SEQ ID NO:5. Further truncated variants of DAP10 used in this invention may include 70-93 amino acids of SEQ ID NO:1 (i.e., the intracellular domain), referred to herein as SEQ ID NO:6. Further truncated variants of DAP10 used in this invention may include 49-93 amino acids of SEQ ID NO:1 (i.e., the transmembrane and cytoplasmic / intracellular domains), referred to herein as SEQ ID NO:7. Further truncated variants of DAP10 used in this invention may include 49-69 amino acids of SEQ ID NO:1 (i.e., the transmembrane domain), referred to herein as SEQ ID NO:8.

[0045] Other mutant versions or truncated variants of the DAP10 peptide are also suitable for use in the present invention. In some embodiments, such mutant versions or truncated variants used as functional variants of the DAP10 peptide of the present invention retain the activity of the wild-type peptide as shown in SEQ ID NO:1.

[0046] In one embodiment, the functional variant of the DAP10 peptide of the present invention retains at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the activity of the wild-type peptide as shown in SEQ ID NO:1. In one embodiment, the activity can be measured by evaluating tyrosine phosphorylation of DAP10 and / or recruitment and activation of the p85 subunit of phosphatidylinositol 3-kinase and the downstream anti-apoptotic kinase AKT.

[0047] DAP12 peptide and its functional variants

[0048] The DAP12 peptide used in the fusion peptide described in this article can be from mammals, such as humans. Wild-type human DAP12 is encoded by the amino acid sequence of UniProt accession number: O43914 (SEQ ID NO:9). The first 21 amino acids are considered to be the signal / guide sequence, amino acids 22-40 are the extracellular domain, amino acids 41-61 are the transmembrane domain, and amino acids 62-113 are the cytoplasmic / intracellular domain.

[0049] In one embodiment, the DAP12 peptide used in the fusion peptide of the present invention comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the DAP12 peptide shown in SEQ ID NO:9. In some embodiments, the DAP12 peptide used in the fusion peptide of the present invention comprises an amino acid sequence shown in SEQ ID NO:9.

[0050] In another embodiment, the functional variant DAP12 peptide used in the fusion peptide of the present invention may include one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete or substitute any amino acid in the DAP12 amino acid (such as the amino acid of wild-type human DAP12 (SEQ ID NO: 9)).

[0051] Truncated variants of the DAP12 peptide can also be used in the fusion peptide of the present invention. For example, a truncated variant of DAP1 comprising only amino acids 22-113 of SEQ ID NO:9 (i.e., lacking 1-21 amino acids, the signal / guide sequence) can be used in the fusion peptide of the present invention. Such a sequence is referred to herein as SEQ ID NO:10. Other truncated variants may comprise 62-113 amino acids of SEQ ID NO:9, such a sequence comprising only the cytoplasmic / intracellular domain of DAP12, and is referred to herein as SEQ ID NO:11. Other truncated variants may comprise 41-61 amino acids of SEQ ID NO:9 (i.e., the transmembrane domain), and is referred to herein as SEQ ID NO:12. Another truncated variant may comprise 22-61 amino acids of SEQ ID NO:9 (i.e., the extracellular and transmembrane domains), and is referred to herein as SEQ ID NO:13.

[0052] Other mutants or truncated variants of the DAP12 peptide are also suitable for use in the present invention. Mutants or truncated variants used as functional variants of the DAP12 peptide in the present invention can retain the activity of the wild-type peptide as shown in SEQ ID NO:9.

[0053] In one embodiment, the functional variant of the DAP12 peptide of the present invention retains at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the activity of the wild-type peptide as shown in SEQ ID NO:9. In one embodiment, the activity can be determined using functional assays, such as MTT and by measuring cytokine secretion via ELISA.

[0054] Polypeptides that bind to the fusion polypeptide of the present invention

[0055] The fusion peptide of this invention can bind to other peptides. This binding may be due to electrostatic interactions, such as those provided by complementary charged amino acids.

[0056] An example of a peptide that can bind to the fusion peptide of the present invention is the NKG2D peptide (Wu et al., 2000, J. Exp. Med., 192(7): 1059-1067 and Rosen et al., 2004, J. Immunol., 173(4): 2470-2478).

[0057] In one embodiment, this polypeptide can be genetically encoded as part of a continuous chimeric construct encoding the gene for the fusion polypeptide of the present invention. The fusion polypeptide and other polypeptides can then be separated during translation (e.g., using ribosomal jumping peptides) or by post-translational cleavage (e.g., using a furin cleavage site). Therefore, the fusion polypeptide and other polypeptides can be linked by an optional linker. Such a linker may include a cleavage site to facilitate cleavage.

[0058] NKG2D peptides and their functional variants

[0059] The NKG2D peptide used in the chimeric peptides described herein can be from mammals, such as humans. Wild-type human NKG2D is encoded by the amino acid sequence of UniProt accession number: P26718 (SEQ ID NO:14). This peptide is believed to include a cytoplasmic domain (amino acids 1-51), a transmembrane domain (amino acids 52-72), and an extracellular domain (amino acids 73-216).

[0060] In one embodiment, the NKG2D polypeptide used in this invention comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the NKG2D polypeptide shown in SEQ ID NO:14. In some embodiments, the NKG2D polypeptide used in this invention comprises the amino acid sequence of SEQ ID NO:14.

[0061] In another embodiment, the functional variant NKG2D polypeptide used in this invention may include one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete or substitute any amino acid in NKG2D amino acids (such as the amino acids in wild-type human NKG2D (SEQ ID NO: 14)).

[0062] Truncated variants of the NKG2D peptide can also be used in the peptides of the present invention. For example, a truncated variant of NKG2D comprising only amino acids 73-216 of SEQ ID NO:14 (i.e., the extracellular domain) can be used in the present invention. Such a sequence is referred to herein as SEQ ID NO:15. Other truncated variants may comprise amino acids 82-216 of SEQ ID NO:14, such a sequence comprising a portion of the extracellular domain of NKG2D, and is referred to herein as SEQ ID NO:16. Another truncated variant comprises amino acids 52-216 of SEQ ID NO:14 (i.e., the transmembrane and extracellular domains), and is referred to herein as SEQ ID NO:17.

[0063] Other mutants or truncated variants of the NKG2D peptide are also suitable for use in the present invention. Of course, any such mutants or truncated variants used in the present invention as functional variants of the NKG2D peptide preferably retain the activity of the wild-type peptide as shown in SEQ ID NO:14.

[0064] In one embodiment, the functional variant of the NKG2D peptide disclosed herein retains at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the activity of the wild-type peptide as shown in SEQ ID NO:14. In one embodiment, the activity can be measured using flow cytometry to confirm sustained binding to the NKG2D ligand and by various cell culture assays (e.g., MTT and ELISA) to confirm target cell lysis, cytokine secretion, and co-stimulation.

[0065] connector

[0066] The DAP10 and DAP12 molecules described in this statement can bind directly to each other in a continuous polypeptide chain or indirectly to each other via suitable linkers. The linkers can be peptide linkers. Peptide linkers are commonly used for fusion polypeptides, and methods for selecting or designing linkers are well known (e.g., see Chen X et al., 2013, Adv. Drug Deliv. Rev. 65(10):135701369 and Wriggers W et al., 2005, Biopolymers 80:736-746). As mentioned above, linkers can also be used to link the fusion polypeptide of this invention to another polypeptide (such as the NKG2D polypeptide) in a chimeric construct.

[0067] Peptide linkers are generally classified into i) flexible linkers, ii) helical linkers, and iii) cleavable linkers, examples of each type being known in the art. In one example, the fusion peptide described herein includes a flexible linker. Flexible linkers may contain mostly non-sterically hindered amino acids, such as glycine and alanine. The hydrophilic amino acid Ser is also conventionally used in flexible linkers. Examples of flexible linkers include, but are not limited to: polyglycine (e.g., (Gly)4 and (Gly)5), polyalanine poly(Gly-Ala), and poly(Gly-Ser) (e.g., (Gly... n -Ser n ) n or (Ser) n -Gly n ) n , where each n is an independent integer equal to or greater than 1.

[0068] Peptide linkers can have suitable lengths. The peptide linker sequence can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more amino acid residues long. For example, the length of a peptide linker can be about 5 to about 50 amino acids; about 10 to about 40 amino acids; about 15 to about 30 amino acids; or about 15 to about 20 amino acids. Variations in peptide linker length can preserve or enhance activity, producing superior efficacy in activity studies. The peptide linker sequence can consist of natural or non-natural amino acids, or a mixture of natural and non-natural amino acids.

[0069] In some respects, the amino acids glycine and serine include amino acids within the linker sequence. In some respects, the linker region includes a glycine repeat sequence group (GSG3). n(SEQ ID NO:18), where n is a positive integer equal to or greater than 1 (e.g., 1 to approximately 20). More specifically, the linker sequence may be GSGGG (SEQ ID NO:19). The linker sequence may be GSGG (SEQ ID NO:20). In some other respects, the orientation of the linker region includes a glycine repeat sequence group (SerGly3). n , where n is a positive integer equal to or greater than 1 (e.g., 1 to about 20) (SEQ ID NO:21).

[0070] In other embodiments, the linker may contain glycine (G) and serine (S) randomly or repeatedly. For example, the linker may be (GGGGS). n (SEQ ID NO:22), where n is an integer from 1 to 20, for example, 1 to 4. In one particular embodiment, n is 4, and the connector is GGGGSGGGGSGGGGS (SEQ ID NO:23). In another particular embodiment, n is 3, and the connector is GGGGSGGGGSGGGGS (SEQ ID NO:24).

[0071] In other embodiments, the linker may contain glycine (G), serine (S), and proline (P) randomly or repeatedly. For example, the linker may be (GPGS). n , where n is an integer from 1 to 20, for example 1 to 4. In a particular embodiment, n is 1, and the connector is GPGS (SEQ ID NO:25).

[0072] Generally, linkers are not immunogenic when administered to patients (such as humans). Therefore, linkers can be selected because they have low immunogenicity or are considered to have low immunogenicity.

[0073] The linkers described herein are exemplary, and may include other amino acids, such as Glu and Lys, if desired. If desired, peptide linkers may include multiple repeats of, for example, (G3S)(SEQ ID NO:26), (G4S)(SEQ ID NO:27), (GYS)(SEQ ID NO:28), and / or (GlySer)(SEQ ID NO:29). In some aspects, peptide linkers may include multiple repeats of, for example, (SG4)(SEQ ID NO:30), (SG3)(SEQ ID NO:31), (SG2)(SEQ ID NO:32), (SG)2(SEQ ID NO:33), or (SerGly)(SEQ ID NO:34).

[0074] In other aspects, the peptide linker may comprise combinations and multiples of repeating amino acid sequence units, such as (G3S)+(G4S)+(GlySer)(SEQ ID NO:26+SEQ ID NO:27+SEQ ID NO:29). In other aspects, Ser may be replaced by Ala, for example, (G4A)(SEQ ID NO:35) or (G3A)(SEQ ID NO:36). In other aspects, the linker comprises (EAAK)n, where n is a positive integer equal to or greater than 1, for example, 1 to about 20 (SEQ ID NO:37). In some aspects, the peptide linker may also comprise a cleavable linker.

[0075] The linker may further include domains and / or features such as furanase cleavage site (RRKR) (SEQ ID NO:38), P2A ribosomal jumping peptide (ATNFSLLKQAGDVEENPGP) (SEQ ID NO:39) and / or T2A ribosomal jumping peptide (EGRGSLLTCGDVEENPGP) (SEQ ID NO:40). Examples of linkers including these domains include SGSG+P2A ribosomal jumping peptide (SGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO:41), SGSG+T2A ribosomal jumping peptide (SGSGEGRGSLLTCGDVEENPGP) (SEQ ID NO:42), and versions including furin cleavage sites, namely furin cleavage site+SGSG+P2A ribosomal jumping peptide (RRKRSGSGAT NFSLLKQAGDVEENPGP) (SEQ ID NO:43) and furin cleavage site+SGSG+T2A ribosomal jumping peptide (RRKRSGSGEGRGSLLTCGDVEENPGP) (SEQ ID NO:44). Alternative ribosomal jumping peptides that can be used in this invention include F2A (VKQTLNFDLLKLAGDVESNPGP) (SEQ ID NO:45) and E2A (QCTNYALLKLAGDVESNPGP) (SEQ ID NO:46).

[0076] N-terminal sequence and C-terminal sequence

[0077] Various sequences can be attached to the N-terminus or C-terminus of the fusion peptide of this disclosure, or to the NKG2D peptide of this invention. These sequences can be functional, such as signal peptides, purified tags / sequences, or half-life extension molecules, or may simply include spacer sequences. Additionally, they may include a function, such as T-cell stimulation.

[0078] Purification Labels and Markers

[0079] Various tags or markers can be attached to the N-terminus or C-terminus of the fusion peptide of the present invention to assist in purification. Any affinity tag can bind to the fusion peptide of the present invention to aid in purification. Examples of such affinity tags include His-tags, FLAG-tags, Arg-tags, T7-tags, Strep-tags, S-tags, aptamer-tags, V5 tags, and AviTags. TM The affinity tag is a myc epitope tag or any combination of these tags. In one embodiment, the affinity tag is a His-tag (typically comprising 5-10 histidine residues), such as a 6His tag (i.e., HHHHH) (SEQ ID NO:47). In another embodiment, the affinity tag is a FLAG tag (i.e., DYKDDDDK) (SEQ ID NO:48). In yet another embodiment, the affinity tag is an AviTag. TM (i.e. GLNDIFEAQKIEWHE) (SEQ ID NO: 49).

[0080] In another embodiment, the affinity tag is a V5 tag (GKPIPNPLLGLDST) (SEQ ID NO:50) or (IPNPLLGLD) (SEQ ID NO:51). In another embodiment, the affinity tag is a myc epitope tag (EQKLISEEDL) recognized by the 9e10 antibody (SEQ ID NO:52). Various other tags used in this invention are well known in the art.

[0081] This combination of affinity tags can also be used, including one or more tags at the N-end, one or more tags at the C-end, or one or more tags at both the N-end and C-end. Examples of this combination include a His tag (H) combined with an Avi tag (A), or a His tag (H) combined with an Avi tag (A) and a FLAG tag (F). Tags can be positioned in any orientation; therefore, the Avi / His tag can have an N-AH-C or N-HA-C orientation, while the Avi / His / FLAG tag can have an N-AHF-C, N-FHA-C, and so on.

[0082] In one embodiment, the fusion polypeptide according to the invention comprises an "AHF" tag (SEQ ID NO: 53) having the sequence "GLNDIFEAQKIEWHEGGHHHHDYKDDK". In another embodiment, the fusion polypeptide according to the invention comprises an "F HA" tag (SEQ ID NO: 54) having the sequence "DYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE".

[0083] The CD8α guide sequence (amino acids 1-21 of UniProt. P01732 or a shortened derivative comprising 1-18 amino acids) is a commonly used T-cell sequence, referred to herein as SEQ ID NO:55. P01732 or a shortened derivative comprising amino acids 1-18 is a commonly used T-cell sequence, referred to herein as SEQ ID NO:55.

[0084] Co-stimulatory sequences

[0085] Various T cell co-stimulatory active sequences are known from previous work on CAR-T cells. These sequences can also be added to the fusion peptide of this invention.

[0086] The 4-1BB intracellular domain (amino acids 214-255 of UniProt:Q07011) can also be used as an N-terminal or C-terminal sequence. The 4-1BB intracellular domain is referred to herein as SEQ ID NO:56. The 4-1BB intracellular domain can also serve as a co-stimulatory domain.

[0087] The CD27 intracellular domain (amino acids 213-260 of UniProt: P26842) can also be used as an N-terminal or C-terminal sequence. The CD27 intracellular domain is referred to herein as SEQ ID NO: 57. The CD27 intracellular domain can also serve as a co-stimulatory domain.

[0088] The human IgG1 hinge (amino acids 218-229 of UniProt:P0DOX5) can also be used as an N- or C-terminal sequence. The human IgG1 hinge is designated SEQ ID NO:58.

[0089] The truncated CD8α hinge (amino acids 138-182 of Uniprot: P01732) can also be used as an N-terminal or C-terminal sequence. The truncated CD8α hinge is designated as SEQ ID NO: 59.

[0090] Exemplary builder

[0091] The present invention provides the following exemplary fusion peptide constructs in Table 1.

[0092] Table 1: Exemplary DAP10 / DAP12 fusion peptide constructs

[0093]

[0094] Furthermore, as described above, the fusion peptide of the present invention can be expressed as a single chimeric construct in conjunction with the NKG2D peptide, undergoing translation-related or post-translational cleavage. In such a construct, after expression, the post-translational peptide cleaves to form independent peptides, which then self-bind to form a CAR. In one embodiment, the fusion peptide of the present invention is derived from the cleavage of the NKG2D peptide. Examples of such constructs are shown in Table 2.

[0095] Table 2: Exemplary chimeric constructs

[0096]

[0097]

[0098]

[0099] Nucleic acid molecules encoding the fusion polypeptide of the present invention

[0100] Another aspect of this invention relates to nucleic acid molecules encoding the fusion polypeptides or chimeric constructs of this invention. The nucleic acid molecule may be DNA or RNA. Unless specifically defined herein, the term includes nucleic acids containing known analogs of natural nucleotides, which have properties similar to a reference nucleic acid and are metabolized in a similar manner to the natural nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, phosphoramides, methylphosphonates, chiral methylphosphates, 2-O-methylribonucleic acid, and peptide-nucleic acids (PNAs). Unless otherwise stated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be achieved through synthetic sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batze et al., 1991, Nucleic Acid Res. 19: 5081; Ohtsuka et al., 1985, J. Biol. Chem. 260: 2605-2608; and Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98).

[0101] Therefore, the present invention also provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide sequence of any one or more of SEQ ID NO:60-69.

[0102] The present invention further provides a nucleic acid comprising a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity with any nucleic acid encoding SEQ ID NO: 60-69. Sequence identity is typically determined by the full length of a reference sequence.

[0103] The present invention further provides a nucleic acid comprising a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity with any one of SEQ ID NO:70-79.

[0104] The present invention also provides a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NO:70-79. The present invention further provides a nucleic acid composed of a nucleotide sequence of any one of SEQ ID NO:70-79.

[0105] Polynucleotide sequences can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences (e.g., the sequences described in the examples below). Direct chemical synthesis of nucleic acids can be performed using methods known in the art, such as the phosphate ester method in Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method in Brown et al., 1979, Meth. Enzymol. 68:109; the diethylphosphoramide method in Beaucage et al., 1981, Tetra. Lett., 22:1859; and the solid-phase support method in US Pat. No. 4,458,066. Introducing mutations into polynucleotide sequences via PCR can be performed as described, for example, in the following books: Principles and Applications for DNA Amplification, HAErlich (Ed.), Freeman Press, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., 1991, Nucleic Acids Res. 19:967; and Eckert et al., 1991, PCR Methods and Applications 1:17.

[0106] carrier

[0107] The present invention also provides a vector comprising one or more nucleic acid molecules of the present invention.

[0108] For expression in host cells, the nucleic acid encoding the fusion polypeptide can be present in a suitable vector, which, upon introduction into a suitable host, can express the sequence to produce the encoded fusion polypeptide according to standard cloning and expression techniques known in the art (e.g., Sambrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). The present invention also relates to such vectors comprising the nucleic acid sequences of the present invention.

[0109] Various expression vectors can be used to express polynucleotides encoding the fusion peptides of the present invention. Both viral and non-viral expression vectors can be used to generate the fusion peptides in host cells (such as mammalian host cells). Non-viral vectors and systems include plasmids, free vectors (typically carrying expression cassettes for expressing proteins or RNA), and human artificial chromosomes (see, e.g., Harrington et al., 1997, Nat Genet. 15:345). For example, non-viral vectors useful for expressing polynucleotides and peptides of the fusion peptides of the present invention in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1 / His, pEBVHis A, B, and C (Invitrogen, San Diego, Calif.), MPS V vectors, and many other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, SV40, papillomaviruses, HBP Epstein Barr virus, vaccine viral vectors, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, 1995, Annu. Rev. Microbiol. 49:807 and Rosenfeld et al., 1992, Cell 68:143. Specifically, retroviral, lentiviral, adenoviral, or adeno-associated virus vectors are typically expressed on T cells. Examples of such vectors include the SFG retroviral expression vector (see Riviere et al., 1995, Proc. Natl. Acad. Sci. (USA) 92:6733-6737). In one embodiment, lentiviral vectors are used, including self-inactivating lentiviral vectors (referred to as SIN vectors).

[0110] The choice of expression vector depends on the intended host cell for which the vector will be expressed. Expression vectors for mammalian host cells may include expression control sequences such as origin of replication, promoters, and enhancers (see, for example, Queen, et al., 1986, Immunol. Rev. 89:49-68), as well as necessary information processing sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription termination sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or modulotropic or tunable. Useful promoters include, but are not limited to: metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-induced MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPS V promoters, tetracycline-induced CMV promoters (such as human immediate early CMV promoters), constitutive CMV promoters, EF1 alpha promoters, phosphoglycerate kinase (PGK) promoters, and combinations of promoter enhancers known in the art.

[0111] Transformed organisms can be cultured and expanded under non-inducible conditions without bias towards populations whose expression products are better tolerated by host cells. In addition to promoters, other regulatory elements may be required or desired for efficient expression of the antibodies or fragments thereof of the present invention. These elements typically include the ATG start codon and adjacent ribosome binding sites or other sequences. Furthermore, expression efficiency can be improved by adding enhancers suitable for the cellular system (e.g., see Scharf et al., 1994, Results Probl. Cell Differ. 20:125; and Bittner et al., 1987, Meth. Enzymol. 153:516). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0112] This invention provides a cloning or expression vector comprising a nucleic acid having a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity with any nucleic acid encoding SEQ ID NO:60-69. Furthermore, this invention provides a cloning or expression vector comprising a nucleic acid encoding one or more of SEQ ID NO:60-69. This invention provides a cloning or expression vector comprising a nucleic acid sequence of any one of SEQ ID NO:70-79.

[0113] host cells

[0114] The host cells provided by this invention comprise the polypeptides, nucleic acids, vectors, or a combination of one or two thereof. Such cells are typically used to express the fusion polypeptides of this invention.

[0115] The nucleic acid or vector can be transfected into host cells using standard techniques.

[0116] The term “transfection” is intended to encompass a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc.

[0117] Furthermore, the nucleic acid or vector can be transduced into host cells. For example, viral vectors as disclosed above can be used to deliver the nucleic acid or vector.

[0118] It is possible to express the fusion peptide of the present invention in prokaryotic or eukaryotic host cells. Representative host cells include various strains of *Escherichia coli*; mammalian cell lines such as CHO, CHO-K1, and HEK293; insect cells such as Sf9 cells; and yeast cells such as *S. cerevisiae* and *P. pastoris*. In one embodiment, the host cell is an immune-response cell, such as NK cells (primary NK cells, or NK cell lines) or T cells (primary T cells or T cell lines). Other types of host cells include macrophages, induced pluripotent stem cells (iPSCs), neutrophils, and invariant NKT (iNKT) cells. The T cells may be CD4+. + or CD8 + T cells. In one embodiment, the host cell is a human cell. In another embodiment, the host cell is a human T cell. In yet another embodiment, the host cell is a primary human T cell. Cell lines that may be used include the NK cell line NK-92.

[0119] Mammalian host cells used to express the fusion polypeptides of the present invention include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, using DH FR selective markers, for example, as described in RJ Kaufman and PA Sharp, 1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. In one embodiment, the host cell is CHO K1PD cells. In another embodiment, the host cell is NSO1 cells. In particular, another expression system for using NSO myeloma cells is the GS gene expression system shown in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding a fusion peptide is introduced into mammalian host cells, the fusion peptide can be produced either by culturing the host cells for a period sufficient to express the fusion peptide within the host cells or by secreting the fusion peptide into the culture medium in which the host cells are growing. The fusion peptide can be recovered from the culture medium using standard protein purification methods.

[0120] Preparation method

[0121] The present invention also provides a method for producing immune-response cells comprising the fusion polypeptide of the present invention. This method may include transducing cells with a nucleic acid or vector encoding the fusion polypeptide of the present invention. The method may further include culturing cells to express the fusion polypeptide and bind it to the NKG2D polypeptide to form a CAR.

[0122] In one embodiment, the present invention provides a method for preparing immune response cells, comprising the steps of: (i) transferring a nucleic acid or vector encoding the fusion polypeptide of the present invention into the immune response cells, and (ii) culturing the immune response cells to express the fusion polypeptide and bind it to the NKG2D polypeptide to form a CAR.

[0123] In another embodiment, the present invention provides a method comprising: (i) obtaining T cells and / or NK cells from a patient; (ii) transferring a nucleic acid or vector encoding the fusion polypeptide of the present invention into the T cells and / or NK cells; and (iii) culturing the T cells and / or NK cells to express the fusion polypeptide and bind it to the NKG2D polypeptide to form a CAR.

[0124] Various methods for culturing immune-response cells are well known in the art. For example, see Parente-Pereira AC et al. 2014, J. Biol. Methods 1(2):e7, Ghassemi S et al. 2018, Cancer Immunol Res 6(9):1100-110, and Denman CJ et al. 2012, PLoS One 7(1):e30264.

[0125] Composition

[0126] The present invention also provides pharmaceutical compositions comprising the fusion peptides, nucleic acids, carriers, or host cells described herein. Such pharmaceutical compositions may include pharmaceutically or physiologically acceptable diluents and / or carriers. The carriers are typically selected to suit the intended route of administration and may include agents for altering, maintaining, or preserving, for example, the composition's pH, osmotic pressure, viscosity, transparency, color, isotonicity, taste, sterility, stability, solubility or release rate, adsorption, or permeability. Typically, these carriers comprise aqueous solutions or alcohol / aqueous solutions; emulsions or suspensions, including saline and / or buffer media.

[0127] Pharmaceutical agents suitable for use in pharmaceutical compositions include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antibacterial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), leavening agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as free serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, and hydrophilic polymers (such as polyethylene glycol). The following substances are used: pyrrolidone, low molecular weight peptides, counterions that form salts (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl benzoate, propyl benzoate, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as polymers; PEG; sorbitol esters; polysorbates, such as polysorbate 20 or polysorbate 80; Triton; tryptophan; lecithin; cholesterol or tyloxapal), stability enhancers (such as sucrose or sorbitol), elasticity enhancers (such as alkali metal halides, such as sodium chloride or potassium chloride, or mannitol sorbitol), delivery carriers, diluents, excipients, and / or drug adjuvants.

[0128] Parenteral carriers include sodium chloride solution, Ringer's glucose, glucose and sodium chloride, and lactated Ringer's. Suitable physiologically acceptable thickeners may be included, such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin, and alginate. Intravenous formulations include fluid and nutrient supplements and electrolyte supplements, such as those based on Ringer's glucose. In some cases, the carrier may include agents that adjust the elasticity of the composition, such as sugars; polyols such as mannitol and sorbitol; or sodium chloride in the pharmaceutical composition. For example, in many cases, it is desirable that the composition be substantially isotonic. The composition may also contain preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases. The precise formulation is determined by the route of administration. The principles, methods, and ingredients of other relevant pharmaceutical formulations are well known (e.g., see Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22). nd Edition).

[0129] The pharmaceutical compositions of the present invention can be administered via one or more routes of administration, using one or more of the various methods known in the art. As is well known to those skilled in the art, the route and / or manner of administration will vary depending on the desired outcome. Routes of administration for the pharmaceutical compositions of the present invention include intravenous injection, intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, spinal injection, or other parenteral administration routes, such as injection or infusion. As used herein, “parenteral administration” refers to a method of administration other than enteral and local administration, typically performed by injection, including but not limited to intravenous, intramuscular, intra-articular, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrathoracic injections and infusions. In one embodiment, the pharmaceutical composition is for intratumoral administration. When considering parenteral administration, the pharmaceutical composition is generally presented in a sterile, pyrogen-free, parenteral-acceptable form. Carriers particularly suitable for parenteral injection are sterile, isotonic, and appropriately preserved solutions. The pharmaceutical composition may be in the form of a lyophilized product, such as a lyophilized cake.

[0130] In addition, the pharmaceutical compositions described herein can be administered via non-enteric routes, such as local, epidermal, or mucosal routes, for example, intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0131] In some embodiments, the pharmaceutical composition is intended for subcutaneous administration. Suitable formulations and methods for subcutaneous administration of peptide therapeutics (such as antibodies, fusion peptides, etc.) are known in the art, for example, see US2011 / 0044977, US8465739, and US8476239. Typically, pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (such as amino acids, such as methionine, and / or sugars; such as sucrose), buffers, and reinforcing agents.

[0132] Typically, in cell therapy, a composition containing host cells is administered to the patient via intravenous injection.

[0133] Uses and methods

[0134] The fusion peptides, nucleic acids, carriers, host cells, or pharmaceutical compositions of the present invention can be administered to subjects and can be used for the treatment, prevention, and / or delay of disease symptoms.

[0135] Therefore, the present invention provides the use of the fusion peptide, nucleic acid, carrier, host cell, or pharmaceutical composition of the present invention in treatment or as a medicament. The present invention further provides the use of the fusion peptide, nucleic acid, carrier, host cell, or pharmaceutical composition of the present invention in the treatment of pathological diseases. The present invention also provides the use of the fusion peptide, nucleic acid, carrier, host cell, or pharmaceutical composition of the present invention in the preparation of a medicament for treating pathological diseases. The present invention further provides a method of treating a patient suffering from a pathological disease, comprising administering to the patient a therapeutically effective amount of the fusion peptide, nucleic acid, carrier, host cell, or pharmaceutical composition of the present invention.

[0136] As used herein, the term "pathological disease" includes cancer, including but not limited to solid tumor cancer; soft tissue tumors; metastatic lesions and blood cancers; for example, said cancers may be liver cancer, lung cancer, breast cancer, prostate cancer, lymphoma, colon cancer, kidney cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, cancers of the endocrine system, cancers of the thyroid gland, cancers of the parathyroid gland, cancers of the adrenal gland, soft tissue sarcomas, urethral cancer, penile cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and chronic lymphocytic leukemia. Cellular leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary central nervous system lymphoma, angiogenic tumors, spinal axonoma, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal cancer, squamous cell carcinoma, T-cell lymphoma, myelodysplastic syndrome (MDS), chronic myeloid leukemia-chronic phase (CMLCP), diffuse large B-cell lymphoma (DLBCL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), hepatocellular carcinoma (HCC), gastrointestinal stromal tumor (GIST), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN); environmentally induced cancers, including cancers induced by asbestos; and combinations of the above cancers. In particular, the cancers can be breast cancer, such as estrogen receptor-positive (ERpos) breast cancer and / or metastatic forms of breast cancer.

[0137] In one embodiment, the cancer is a solid tumor. In another embodiment, treatment of the pathological disease involves targeting non-tumor cells, such as tumor-associated stromal cells. Examples of such tumor-associated stromal cells include pancreatic stromal cells. Other non-tumor cell types that can be targeted include macrophages, regulatory T cells, and myeloid-derived suppressor cells.

[0138] In one implementation plan, the patient has already undergone pretreatment with chemotherapy drugs.

[0139] In one implementation, administering host cells to a patient can reduce tumor size by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% compared to untreated tumors.

[0140] The number of host cells granted a patent should take into account the route of administration, the cancer being treated, the patient's weight, and / or the patient's age. Generally, it is approximately 1 × 10⁻⁶.6 To approximately 1×10 11 Cells are administered to the patient. In one implementation, approximately 1 × 10⁻⁶ cells are used. 7 To approximately 1×10 10 Cells, or about 1 × 10 8 To approximately 1×10 9 Cells are administered to patients.

[0141] conventional

[0142] Sequence identity can be determined using standard methods typically used to compare the similarity of amino acid positions of two peptides. Computer programs such as BLAST, FASTA, or Clustal Omega are used to align two peptides to achieve optimal amino acid matching (along the full length of one or both sequences or along predetermined portions of one or both sequences). These programs provide a default gap penalty and a default interstitial penalty, along with a scoring matrix such as PAM 250 [a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol.5, supp.3 (1978)] that can be used with the computer program. For example, the percentage of identity can be calculated as: the total number of identical matches multiplied by 100, then divided by the sum of the length of the longer sequence within the span of the match and the number of gaps introduced to align the two sequences.

[0143] Throughout the description and claims of this specification, the words “comprising” and “including”, and variations of these words, such as “comprising” meaning “including but not limited to”, do not exclude other components, integers, or steps. Furthermore, unless the context otherwise requires, the singular includes the plural: particularly in the case of the use of the indefinite article, unless the context otherwise requires, this specification should be understood to take into account both the plural and the majority.

[0144] The term "approximately" in relation to the numerical value x means, for example, x + 5%.

[0145] Features of each aspect of the invention may be described in relation to any other aspect. Within the scope of this application, it is expressly indicated that the aspects, embodiments, examples, and alternatives listed in the preceding paragraphs, claims, and / or the following description and drawings, particularly their individual features, may be combined independently or in any manner. That is, all embodiments and / or features of any embodiment may be combined and / or combined in any manner, unless such features are incompatible. Detailed Implementation Plan

[0146] conventional methods

[0147] T cell isolation and retroviral transduction

[0148] Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of healthy volunteers using density-mediated centrifugation. T cells were activated for 48 hours using anti-CD3 and anti-CD28 coated paramagnetic beads at a T cell:bead ratio of 1:2. 1×10⁻⁶ cells were then used. 6 T cells were seeded onto reverse transcriptase-binding protein-coated plates pretreated with 3 mL of retroviral supernatant. Each well was then treated with 3 mL of fresh viral supernatant and 100 IU / mL IL-2. Retroviral transduction was performed using viral particles generated from stable GALV pseudotype 93TVec packaging cells. Subsequently, T cells were cultured at 100 IU / mL in RPMI 1640 medium supplemented with 5% normal human AB serum, with fresh medium and IL-2 (100 IU / mL) provided three times weekly.

[0149] Flow cytometry

[0150] T cell transduction and transfection of 293T cells were evaluated by flow cytometry and compared with appropriate isotype controls. To assess the expression of NKG2D-based constructs, cells were stained with mouse anti-human CD4-FITC, mouse anti-human NKG2D-PE, and mouse anti-human CD8-APC, with appropriate compensation. Because endogenous NKG2D is present in CD8... + High-level expression of NKG2D in T cells was used, thus comparing transduction efficiency with that in untransduced CD4+ T cells. PanErbB-specific T4 and TMY CAR expression were assessed using biotinylated goat anti-human EGF and PE-bound streptavidin. Transduction efficiency was calculated by comparison with N1012+ T cells stained with the same reagents. Transduction efficiency between constructs was normalized prior to use by increasing the necessary proportion of untransformed T cells. This ensured that all conditions regarding the total number of CAR+ T cells and total T cell concentration were identical.

[0151] For intracellular staining, transfected 293T cells were fixed in 4% formaldehyde for 10 minutes at room temperature, and then washed twice in osmotic solution (PBS + 0.5% BSA + 0.1% saponins). Subsequently, cells were stained with 500 ng PE combined with anti-human NKG2D or a suitable isotype control in the presence of 100 μL osmotic solution. Cells were further washed twice in osmotic solution before flow cytometry analysis.

[0152] Dose-response analysis

[0153] Add 1×10 to each well (100 μL) 4Tumor cells were seeded in 96-well plates and cultured overnight at 37°C and 5% CO2. After 24 hours, T cells were added at a logarithmic CAR T cell:tumor cell ratio of 1:1 to 1:64. After 72 hours, T cells were removed and 100 μL of MTT solution (500 μg / mL) was added, followed by incubation at 37°C and 5% CO2 for approximately 1 hour. After removing the MTT solution, the resulting formalin crystals were dissolved in DMSO (100 μL / well), and absorbance was measured at 560 nm. Tumor cell viability was calculated as follows: (Absorbance of T cell monolayer / Absorbance of non-T cell monolayer) * 100.

[0154] Restimulation test

[0155] 1×10 5 Tumor cells were placed in three wells of a 24-well plate and cultured at 37°C and 5% CO2 for 24 hours. After 24 hours, 1 mL of a solution containing 1×10⁻⁶ cells was added to each well. 5 CAR + T cells. After 72 hours, the T cells were gently removed, and the wells were washed with 1 mL of PBS. After removing the PBS, 1 mL of MTT (final concentration 500 μg / mL) was added to each well, and the plates were incubated at 37°C and 5% CO2 for approximately 1 hour. The absorbance in the appropriate wells was measured at 560 nm, and tumor cell viability was calculated according to the detailed instructions in the "Dose-Response" section. Restimulation was considered successful if the tumor cell viability was less than 50%.

[0156] T cells removed from the culture dish were centrifuged at 400×g for 5 minutes, and the supernatant was removed. The precipitate was resuspended in 3.2 mL of R5 medium, and 1 mL was added in triplicate to each well of a fresh tumor monolayer (1×10⁶ cells per well for a 24-well plate). 5 The total number of T cells was assessed by performing trypan blue exclusion on a small fraction of the remaining 200 μL of the tumor cells.

[0157] ELISA

[0158] The secretion of IFN-γ and IL-2 by T cells was assessed using the Duo-set and Ready-Staddy-Go ELISA kits, respectively, in the supernatant removed 24 hours after the start of co-culture.

[0159] Tumor spheroid formation

[0160] To generate tumor spheroids, 1×10⁻⁶ ppm was added to each well of an ultra-low affinity 96-well plate. 3 Tumor cells and 1×10 3PS1 stellate cells were cultured at 37°C and 5% CO2 for 72 hours. The formation of spheroids was confirmed by observation using a standard optical microscope.

[0161] in vivo

[0162] 1×10 5 Firefly luciferase (ffLUC)-labeled B×PC3 cells were injected intraperitoneally into NSG mice. Twelve days after tumor inoculation, mice (n=5 per group) were intraperitoneally injected with PBS (4×10⁻⁶ cells / mL). 6 (N1012 + (N1012(lo)), T4 + Or TMY + CAR T cells) or 1×10 7 (N1012(hi) or NKG2D)CAR + T cells. Alternatively, NSG mice can be intraperitoneally inoculated with 1×10⁻⁶ T cells. 6 H226 malignant mesothelioma cells labeled with ffLUC. Eight days after tumor inoculation, mice were given PBS or 4×10⁻⁶ malignant mesothelioma cells. 6 N1012+ T cell therapy. As a control, a group of mice were treated alone with 4×10⁴ N1012+ T cells expressing NKG2D. 6 T-cell therapy.

[0163] BLI was used to monitor tumor growth, and all data were expressed as total flow rate (photons / second) or average total flow rate (photons / second) per treatment. Mice were closely monitored and weighed three times a week to identify signs of poor health.

[0164] Example 1: Expression of NKG2D and DAP10 / 12 fusion protein in 293T cells

[0165] 2293T cells were transfected with an SFG retroviral plasmid backbone containing the DAP10 / 12 fusion protein N1012 or the NKG2D expression cassette. N1012 (SEQ ID NO:64) comprises a complex containing the exogenous human NKG2D protein according to the invention and a fusion exogenous DAP10 / 12 homodimer. The N1012 plasmid contains SEQ ID NO:74, encoding the sequence shown in SEQ ID NO:64. NKG2D surface expression was detected by flow cytometry after 72 hours. Although NKG2D expression was readily detected on the surface of 293T cells transfected with the N1012 plasmid, no NKG2D expression was detected on the surface of cells transfected with a plasmid encoding the control NKG2D. Figure 2(Top column). Given that NKG2D surface expression depends on DAP10 expression, the lack of NKG2D surface expression could be explained by the absence of DAP10 co-expression in the NKG2D plasmid. To confirm that the lack of NKG2D expression on the surface of NKG2D-transfected 293T cells was not due to poor transfection, intracellular staining was performed to verify the presence of NKG2D. Crucially, intracellular expression of NKG2D was observed in 293T cells transfected with either N1012 or NKG2D plasmids. Figure 2 (See the bottom column). This demonstrates the successful expression of NKG2D from both structures and confirms the necessity of DAP10 co-expression for achieving NKG2D surface expression.

[0166] Example 2: Expression of N1012 and NKG2D in primary human T cells

[0167] Primary human T cells were activated using paramagnetic beads coated with anti-human CD3 and anti-human CD28 antibodies. After 48 hours of activation, the T cells were engineered via retroviral transduction to express N1012 or NKG2D. NKG2D surface expression was detected by flow cytometry and combined CD4 and CD8 staining. The percentage of NKG2D expression was calculated. Figure 3 (A) and median fluorescence intensity (MFI, Figure 3 B) was compared with untransformed T cells. Because NKG2D is present in CD8... + Endogenous expression in T cells, data in CD4 + Gating was performed on T cells. As shown in the figure, NKG2D and N1012 constructs were repeatedly expressed at high levels on the surface of primary human T cells compared to UT T cells or T cells expressing control CAR. Figure 4 ).

[0168] Example 3: Evaluation of N1012 T cell destruction and recognition of target cells

[0169] To assess cell lysis capacity, N1012 + T cells were co-cultured with 11 different human tumor cell lines representing five different tumor types (mesothelioma, ovarian cancer, head and neck squamous cell carcinoma, pancreatic cancer, and breast cancer) or with tumor-associated stromal cells (PS1) at different E:T ratios. After 72 hours, the T cells were removed, and an MTT assay was performed to assess tumor cell survival. However, the decrease in tumor survival was minimal when the target cells were co-cultured with UT T cells or NKG2D-expressing T cells, even at low E:T ratios. Figure 5 , 7 A and B), N1012 + T cells exhibited effective lysis against all target cell lines. Analysis of the co-culture supernatant by ELISA showed that N1012... +T cells secrete large amounts of interferon-γ (IFN-γ) and interleukin-2 (IL-2), but UT T cells or cells expressing the control NKG2D structure did not secrete interferon-γ (IFN-γ) and interleukin-2 (IL-2). Figure 6A And B). These data prove N1012 + T cells have the ability to recognize and lyse multiple tumor types, including tumor-associated stromal cells.

[0170] Example 4: An example of co-culturing N1012 T cells to destroy tumor cells and stromal cells.

[0171] To determine N1012 + To determine whether T cells retain their ability to lyse tumor cells when grown in the presence of stromal cells, they were co-cultured with a monolayer containing both tumor cells and stromal cells. To achieve this, 5 × 10⁶ cells were co-cultured with the monolayer. 4 Tumor cells and 5×10 4 PS1 stellate cells were mixed and seeded into three wells of a 24-well plate. After 24 hours, 1×10⁻⁶ cells were added. 5 T cells were collected and the plates were cultured for 72 hours. The T cells were then removed, and the survival rates of tumor cell and stromal cell monolayers were assessed using the MTT assay as described in Example 3. N1012 + T cells can effectively lyse tumor cells and stromal cells, but are less effective against NKG2D or UT T cells ( Figure 7 When co-cultured with CD, the survival rate of target cells decreased slightly. N1012 + T cells and equivalent lysed B×PC3_LT cells expressing A2028z CAR were grown in isolated cultures. a2028zCAR is a pCAR targeting αvβ6 integrin. The targeting moiety of this CAR consists of a VPI-derived A20FMDV2 20mer peptide (SEQ ID NO:80), which binds to αvβ6 integrin. It is located downstream of the CD124 signal peptide (aa1-25, Uniprot index P24394, SEQ ID NO:81). The targeting moiety is fused via an AAA linker to a portion of the extracellular, transmembrane, and intracellular domains of CD28 (aa114-220, Uniprot index P10747, SEQ ID NO:82), wherein the B7 binding residues of CD28 (aa117-122) are replaced by aa410-419 of human c-myc (Uniprot index P01106, SEQ ID NO:52). It is fused with CD247 aa52-164 (Uniprot index P20963, SEQ ID NO:83).

[0172] In comparison, although N1012 +T cells maintain effective lysis of monolayers containing B×PC3_LT tumor cells and PS1 stellate cells, but the efficacy of A2028z T cells ( Figure 7 EF) and cytokine secretion ( Figure 7 The GH content was significantly reduced.

[0173] Example 5: An example of N1012 T cells destroying tumor spheroids

[0174] To evaluate N1012 + The ability of T cells to mediate target cell lysis in a 3D system was demonstrated by co-culturing them with tumor spheroids. After spheroid formation, 6 × 10⁶ cells were added to each well. 3 CellTracker violet-labeled T cells. The survival of tumor cells and stellate cells was assessed using fluorescence microscopy at 72 and 192 hours, respectively, by measuring GFP and RFP. GFP and RFP signals were quantified using ImageJ software and expressed as a percentage of fluorescence readings of stellate cells grown in the absence of T cells. (N1012) + Effective dissolution of globules was observed in T cells, but not in NKG2D or UT T cells. Figure 7 (IJ). In addition, only N1012 + T cells showed IFN-γ secretion ( Figure 7 (K).

[0175] Spheroid viability and T cell proliferation were assessed alternately using flow cytometry. To do this, spheroids were removed from the culture dish 72 or 192 hours after T cell addition and placed into flow cytometry tubes, with up to five spheroids treated with the same CAR T cells added to the same tube. Spheroid depolymerization was achieved using Accutase solution and vigorous resuspending via pipette tip. The resulting single-cell suspension was washed in RPMI 1640 medium + 5% normal human AB serum and then resuspended in PBS containing counting beads. The same number of counting beads were collected from each tube, and the resulting number of tumor cells (assessed by GFP and RFP fluorescence) and T cells (assessed by CellTracker violet fluorescence) were determined. Data showed the percentage of total GFP and RFP cells present in spheroids grown in the absence of T cells.

[0176] Example 6: An example of N1012 T cell recognition of sequence targets

[0177] To evaluate N1012 + The ability of T cells to continuously lyse target cells (“restimulation”) was demonstrated by co-culturing them twice weekly with a fresh monolayer until no monolayer disruption was observed. UT or NKG2D +T cells mediated minimal target cell destruction, showing no evidence of proliferation, while N1012 + T cells respond to multiple rounds of restimulation ( Figure 8A -B) mediates effective lysis. Target cell destruction is also associated with N1012. + It is related to the massive proliferation and expansion of T cells. Figure 8C -D).

[0178] Compared with CYAD-01NKG2D CAR and control T cells, N1012 + T cells underwent significantly more rounds of restimulation on B×PC3_LT cells. Figure 14 (A). In addition, N1012 + T cell proliferation was also significantly higher than that of CYAD-01 T cells or the control group. Figure 14 (B).

[0179] Example 7: Efficacy of N1012 T cells in a living model of pancreatic cancer

[0180] To determine N1012 + The ability of T cells to target tumor cells in vivo resulted in T cells expressing N1012 and NKG2D, or two different pan-ErbB-targeting CAR iterations (T4 and TMY). At a 1:1 ratio ( Figure 9B After co-culturing for 72 hours, the expression of various constructs in primary human T cells in vitro was confirmed for three cell lines. Figure 9A The efficacy of firefly luciferase (ffLUC)-labeled B×PC3 tumors was assessed. To evaluate in vivo function, intraperitoneal firefly luciferase (ffLUC)-labeled B×PC3 tumors were established in NSG mice for 12 days. Tumor-bearing mice were intraperitoneally injected with PBS, 4×10 6 (N1012(lo), T4 or TMY) T cells or 1×10 7 T cells transduced with (N1012(hi) or NKG2D). Tumor growth was measured weekly by bioluminescence imaging, and mice were weighed three times weekly. Data showed the mean total flux (photons / second) for each treatment group. Figure 10A Or the total flux per mouse (photons / second) Figure 10B Accept NKG2D + T4 + Or TMY + The tumor burden in mice treated with T cells was the same as that in mice receiving PBS, indicating a lack of efficacy. In contrast, tumors were completely eradicated in 2 / 5 and 4 / 5 mice treated with N1012(lo) or N1012(hi), respectively. These mice remained tumor-free 76 days after T cell administration. No signs of toxicity were observed when assessed by measuring the percentage change in body weight. Figure 10C ).

[0181] To determine N1012 + Can T cells be transplanted into NSG mice and provide immune memory? Mice that completely rejected the tumor received a second intraperitoneal injection of 1×10⁻⁶ cells 88 days after the initial tumor inoculation (76 days after T cell infusion). 5 B×PC3 cells labeled with ffLUC. Although increased luminescence was observed in BLI 24 hours after tumor restimulation, 4 / 5 mice subsequently showed a significant reduction in tumor size, thus indicating that N1012 + T cells ( Figure 10D The cells were reactivated. The experiment ended after 145 days, and survival curves were generated, confirming that N1012 T cells have potent anti-tumor efficacy. Figure 10E ).

[0182] To further confirm N1012 + The efficacy of T-cell therapy in an in vivo pancreatic cancer model was replicated. 1×10⁶ cells were inoculated. 5 After 12 days, ffLUC-labeled B×PC3 cells were injected with 1×10⁻⁶ cells. 7 CAR + Or, control NSG mice without transformed T cells were intraperitoneally injected. Tumor growth was monitored weekly by bioluminescence imaging, and data showed the mean total flux (photons / second) for each treatment group. Figure 11A ) and total flux per mouse (photons / second) Figure 11B ). In N1012 + Significant and sustained tumor regression was again observed in mice treated with T-cell therapy. In fact, in mice treated with N1012... + In 5 / 6 mice treated with T cells, tumors were completely eradicated. In contrast, the tumor growth kinetics in mice treated with UT T cells were identical to those in mice treated with PBS. These data confirm that tumor eradication is N1012. + Specific.

[0183] To investigate the potential formation of memory T cells, tumor-free mice were injected with 1×10⁻⁶ cells on day 41 (29 days after T cell infusion). 5 Fresh B×PC3 cell clusters labeled with ffLUC were restimulated. Mice were imaged on day 42 to confirm tumorigenesis. Subsequent imaging showed that 5 / 5 of the restimulated mice reduced tumor burden to undetectable levels, with 3 / 5 of the mice showing long-term tumor control. Figure 11B These data suggest that N1012 CAR T cells are able to form memories and be reactivated when target cells reappear.

[0184] Example 8: Efficacy of N1012 T cells in an in vivo model of malignant mesothelioma

[0185] To confirm the efficacy of N1012 in another in vivo model, NSG mice were intraperitoneally injected with 1×10⁻⁶ N1012. 6 H226 malignant mesothelioma cells labeled with ffLUC. Eight days after tumor inoculation, mice were given PBS or 4×10⁻⁶ malignant mesothelioma cells. 6 N1012 + T-cell therapy. As a control, a group of mice were treated alone with 4 × 10⁻⁶ T cells expressing NKG2D. 6 T-cell therapy. Tumor growth was monitored weekly using bioluminescence imaging, and the data were presented as the average total flux (photons / second) per treatment. Figure 12A ) and total flux per mouse (photons / second) Figure 12B Tumor growth was observed in mice receiving PBS, while in mice receiving N1012... + 100% tumor clearance was observed in mice with T cells.

[0186] To confirm the persistence and maintenance of T cell function, all tumor-free mice were intraperitoneally injected with an additional 1×10⁻⁶ T cells 91 days after initial tumor inoculation. 6 H226 cells labeled with ffLUC. After 24 hours, bioluminescence imaging confirmed the presence of tumors in all mice. No tumors were developed in any of the restimulated mice, confirming the presence of N1012 tumors. + The persistence of T cells and their ability to mediate long-term tumor control.

[0187] Example 9: Comparison of N1012 T cells and CYAD-01 T cells

[0188] The restimulation and proliferative potential of N1012 T cells and CYAD-01 T cells were compared. As previously described, the CYAD-01 CAR consists of a fusion of NKG2D and CD3ζ (Zhang et al., 2005, Blood 106:1544-1551). Although nominally a first-generation CAR, it is associated with endogenous DAP10 in T cells, meaning that both signals 1 and 2 are provided. Celyad SA is currently conducting clinical development of this CAR under the name CYAD-01, therefore this CAR is provided in these examples for comparison only.

[0189] When evaluated by flow cytometry, the surface expression of CYAD-01 was confirmed in primary human T cells. Figure 13 ).

[0190] In short, co-culture N1012 or CYAD-01T cells with a fresh monolayer twice a week until no monolayer disruption is observed. To achieve this, use 1×102 5Tumor cells were placed in three wells of a 24-well plate and cultured at 37°C and 5% CO2 for 24 hours. After 24 hours, a final concentration of 1×10⁻⁶ cells was added to each well. 5 CAR+ / mL 1×10 5 CAR+T cells. After 72 hours, the T cells were gently removed, and the wells were washed with 1 mL of PBS. After removing the PBS, 1 mL of MTT (final concentration 500 μg / mL) was added to each well, and the plates were incubated at 37°C and 5% CO2 for approximately 1 hour. The plates were counted and the tumor cell viability was calculated according to the above instructions. If the tumor cell viability was less than 50%, the restimulation was considered successful.

[0191] To investigate the response of T cell proliferation to target cell recognition, T cells removed from the culture plate were rotated at 400×g for 5 minutes, and the supernatant was removed. The pellet was resuspended in 3.2 mL of R5 medium, and 1 mL was added to each well of a fresh tumor monolayer, in triplicate. The total T cell count was assessed by tryingan blue exclusion assay on a small fraction of the remaining 200 μL.

[0192] Compared with CYAD-01, NKG2D CAR and control T cells, N1012 + T cells in B×PC3_LT cells ( Figure 14 The N1012 T cells underwent significantly more rounds of restimulation than the CYAD-01 T cells or the control group. Furthermore, the proliferation of N1012 T cells was significantly higher than that of CYAD-01 T cells or the control group. Figure 14 (B).

[0193] N1012 was observed when co-cultured with tumor spheroids. + The survival rate of T cell spheroids was significantly reduced, but the survival rates of UT control T cells and cells expressing functional CYAD-01CAR were not significantly reduced. Figure 15A Compared to UT or CYAD-01T cells, N1012 T cells also showed significant proliferation. Figure 15B ).

[0194] SEQ ID NO:1 (Human DAP10 full sequence)

[0195] MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVASLLIVGAVFL CARPRRSPAQ EDGKVYINMP GRG

[0196] SEQ ID NO:2(DAP10 aa19-93 - Lack of guide sequence)

[0197] QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV FLCARPRRSPAQEDGKVYIN MPGRG

[0198] SEQ ID NO:3(DAP10 aa19-69-Extracellular / Transmembrane Domain)

[0199] QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV F

[0200] SEQ ID NO:4(DAP10 aa1-71)

[0201] MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVASLLIVGAVFL C

[0202] SEQ ID NO:5(DAP10 aa19-71)

[0203] QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV FLC

[0204] SEQ ID NO:6(DAP10 aa70-93-intracellular domain)

[0205] LCARPRRSPA QEDGKVYINM PGRG

[0206] SEQ ID NO:7(DAP10 aa49-93-Transmembrane and Intracellular Domains)

[0207] LLAGLVAADA VASLLIVGAV FLCARARPRRSP AQEDGKVYIN MPGRG

[0208] SEQ ID NO:8(DAP10 aa49-69-transmembrane domain)

[0209] LLAGLVAADA VASLLIVGAV F

[0210] SEQ ID NO:9 (Human DAP12 full sequence)

[0211] MGGLEPCSRL LLLPLLLAVS GLRPVQAQAQ SDCSCSTVSP GVLAGIVMGD LVLTVLIALAVYFLGRLVPR GRGAAEAATR KQRITETESP YQELQGQRSD VYSDLNTQRP YYK

[0212] SEQ ID NO:10(DAP12 aa22-113 - Lack of guide sequence)

[0213] LRPVQAQAQS DCSCSTVSPG VLAGIVMGDL VLTVLIALAV YFLGRLVPRG RGAAEAATRKQRITETESPY QELQGQRSDV YSDLNTQRPY YK

[0214] SEQ ID NO:11(DAP12 aa62-113-Cytoplasmic / Intracellular Domain)

[0215] YFLGRLVPRG RGAAEAATRK QRITETESPY QELQGQRSDV YSDLNTQRPY YK

[0216] SEQ ID NO:12(DAP12 aa41-61-transmembrane domain)

[0217] GVLAGIVMGD LVLTVLIALA V

[0218] SEQ ID NO:13(DAP12 aa22-61-Extracellular and transmembrane domains)

[0219] LRPVQAQAQS DCSCSTVSPG VLAGIVMGDL VLTVLIALAV

[0220] SEQ ID NO:14 (Human NKG2D full sequence)

[0221] MGWIRGRRSR HSWEMSEFHN YNLDLKKSDF STRWQKQRCP VVKSKCRENA SPFFFCCFIAVAMGIRFIIM VAIWSAVFLN SLFNQEVQIP LTESYCGPCP KNWICYKNNC YQFFDESKNW YESQASCMSQNASLLKVYSK EDQDLLKLVK SYHWMGLVHI PTNGSWQWED GSILSPNLLT IIEMQKGDCA LYASSFKGYIENCSTPNTYI CMQRTV

[0222] SEQ ID NO: 15 (human NKG2D aa 73-216 - extracellular domain)

[0223] IWSAVFLNSL FNQEVQIPLT ESYCGPCPKN WICYKNNCYQ FFDESKNWYE SQASCMSQNASLLKVYSKED QDLLKLVKSY HWMGLVHIPT NGSWQWEDGS ILSPNLLTII EMQKGDCALY ASSFKGYIENCSTPNTYICM QRTV

[0224] SEQ ID NO: 16 (human NKG2D aa 82-216 - extracellular domain)

[0225] LFNQEVQIPL TESYCGPCPK NWICYKNNCY QFFDESKNWY ESQASCMSQN ASLLKVYSKEDQDLLKLVKS YHWMGLVHIP TNGSWQWEDG SILSPNLLTI IEMQKGDCAL YASSFKGYIE NCSTPNTYICMQRTV

[0226] SEQ ID NO: 17 (human NKG2D aa 52-216 - transmembrane and extracellular domain)

[0227] PFFFCCFIAV AMGIRFIIMV AIWSAVFLNS LFNQEVQIPL TESYCGPCPK NWICYKNNCYQFFDESKNWY ESQASCMSQN ASLLKVYSKE DQDLLKLVKS YHWMGLVHIP TNGSWQWEDG SILSPNLLTIIEMQKGDCAL YASSFKGYIE NCSTPNTYIC MQRTV

[0228] SEQ ID NO:18 (connector)

[0229] GSG

[0230] SEQ ID NO:19 (connector)

[0231] GSGGG

[0232] SEQ ID NO:20 (connector)

[0233] GSGG

[0234] SEQ ID NO:21 (connector)

[0235] SGGG

[0236] SEQ ID NO:22 (connector)

[0237] GGGGS

[0238] SEQ ID NO:23 (connector)

[0239] GGGGSGGGGS GGGGSGGGGGS

[0240] SEQ ID NO:24 (connector)

[0241] GGGGSGGGGS GGGGS

[0242] SEQ ID NO:25 (connector)

[0243] GPPGS

[0244] SEQ ID NO:26 (connector)

[0245] GGGS

[0246] SEQ ID NO:27 (connector)

[0247] GGGGS

[0248] SEQ ID NO:28 (connector)

[0249] GYS

[0250] SEQ ID NO:29 (connector)

[0251] GS

[0252] SEQ ID NO:30 (connector)

[0253] SGGGG

[0254] SEQ ID NO:31 (connector)

[0255] SGGG

[0256] SEQ ID NO:32 (connector)

[0257] SGG

[0258] SEQ ID NO:33 (connector)

[0259] SGSG

[0260] SEQ ID NO:34 (connector)

[0261] SG

[0262] SEQ ID NO:35 (connector)

[0263] GGGGA

[0264] SEQ ID NO:36 (connector)

[0265] GGGA

[0266] SEQ ID NO:37 (connector)

[0267] EAAAK

[0268] SEQ ID NO:38 (furanase cleavage site)

[0269] RRKR

[0270] SEQ ID NO:39 (P2A ribosomal jumping peptide)

[0271] ATNFSLLKQA GDVEENPGP

[0272] SEQ ID NO:40 (T2A ribosomal jumping peptide)

[0273] EGRGSLLTCG DVEENPGP

[0274] SEQ ID NO:41(SGSG+P2A)

[0275] SGSGATNFSL LKQAGDVEEN PGP

[0276] SEQ ID NO:42(SGSG+T2A)

[0277] SGSGEGRGSL LTCGDVEENP GP

[0278] SEQ ID NO:43 (furin protease cleavage site + SGSG + P2A)

[0279] RRKRSGSGAT NFSLLKQAGD VEENPGP

[0280] SEQ ID NO:44 (furin protease cleavage site +SGSG+T2A)

[0281] RRKRSGSGEG RGSLLTCGDV EENPGP

[0282] SEQ ID NO:45 (F2A ribosomal jumping peptide)

[0283] VKQTLNFDLL KLAGDVESNP GP

[0284] SEQ ID NO:46 (E2A ribosomal jumping peptide)

[0285] QCTNYALLKL AGDVESNPGP

[0286] SEQ ID NO:47 (His tag)

[0287] HHHHHH

[0288] SEQ ID NO:48 (FLAG tag)

[0289] DYKDDDDK

[0290] SEQ ID NO:49 (Avi tag)

[0291] GLNDIFEAQK IEWHE

[0292] SEQ ID NO:50 (V5 tag)

[0293] GKPIPNPLLG LDST

[0294] SEQ ID NO:51 (V5 tag)

[0295] IPNPLLGLD

[0296] SEQ ID NO:52 (Myc tag)

[0297] EQKLISEEDL

[0298] SEQ ID NO:53 (AHF tag)

[0299] GLNDIFEAQK IEWHEGGHHH HHHDYKDDDD K

[0300] SEQ ID NO:54 (FHA tag)

[0301] DYKDDDDKHH HHHHGGGLND IFEAQKIEWH E

[0302] SEQ ID NO:55 (CD8α guide sequence)

[0303] MALPVTALLL PLALLLHAAR P

[0304] SEQ ID NO:56 (4-1BB intracellular domain)

[0305] KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL

[0306] SEQ ID NO:57 (CD27 intracellular domain)

[0307] QRRKYRSNKG ESPVEPAEPC HYSCPREEEG STIPIQEDYR KPEPACSP

[0308] SEQ ID NO:58 (Human IgG1 hinge)

[0309] EPKSCDKTHT CP

[0310] SEQ ID NO:59 (Cut-down CD8α hinge)

[0311] TTTPAPRPPT PAPTIASQPL SLRPEACRPA AGGAVHTRGL DFACD

[0312] SEQ ID NO:60

[0313]

[0314] SEQ ID NO:61

[0315]

[0316] SEQ ID NO:62

[0317]

[0318] SEQ ID NO:63

[0319]

[0320] SEQ ID NO:64 (Constructor 1 / N012)

[0321]

[0322] SEQ ID NO:65 (Constructor 3)

[0323]

[0324] SEQ ID NO:66 (Constructor 8)

[0325]

[0326]

[0327] SEQ ID NO:67 (Constructor 9)

[0328]

[0329] SEQ ID NO:68 (Constructor 10)

[0330]

[0331] SEQ ID NO:69 (Constructor 11)

[0332]

[0333]

[0334] SEQ ID NO:70 (encoding SEQ ID NO:60 polypeptide)

[0335]

[0336] SEQ ID NO:71 (encoding SEQ ID NO:61 polypeptide)

[0337]

[0338] SEQ ID NO:72 (encoding SEQ ID NO:62 polypeptide)

[0339]

[0340] SEQ ID NO:73 (encoding SEQ ID NO:63 polypeptide)

[0341]

[0342] SEQ ID NO:74 (encoding SEQ ID NO:64 peptide / constructor 1 / N1012)

[0343]

[0344]

[0345] SEQ ID NO:75 (encoding SEQ ID NO:65 peptide / constructor 3)

[0346]

[0347] SEQ ID NO:76 (encoding SEQ ID NO:66 peptide / constructor 8)

[0348]

[0349]

[0350] SEQ ID NO:77 (encoding SEQ ID NO:67 peptide / constructor 9)

[0351]

[0352] SEQ ID NO:78 (encoding SEQ ID NO:68 peptide / constructor 10)

[0353]

[0354]

[0355] SEQ ID NO:79 (encoding SEQ ID NO:69 peptide / constructor 11)

[0356]

[0357] SEQ ID NO:80 (A20FMDV2 polypeptide)

[0358] NAVPNLRGDL QVLAQKVART

[0359] SEQ ID NO:81 (CD124 monopeptide)

[0360] MGWLCSGLLF PVSCLVLLQV ASSGN

[0361] SEQ ID NO:82(CD28 aa114-220)

[0362] IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVAFIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRS

[0363] SEQ ID NO: 83 (CD247 aa52-164)

[0364] RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPR RKNPQEGLYNELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT YDALHMQALP PR

[0365] SEQ ID NO: 84 (SEQ ID NO: 1 of WO 2019 / 182425)

[0366] MGWSCIILFL VATATGVHSQ IQLVQSGPEL KKPGETVKIS CKTSGYTFTD YSMHWVNQAPGKGLKWMGWI NTETGEPTYT DDFKGRFAFS LETSASTAYL QINNLKNEDT ATYFCARTAV YWGQGTTLTVSSGSTSGSGK PGSGEGSDIQ MTQSPSSLSA SLGERVSLTC RASQEISGSL SWLQQKPDGT IKRLIYAASTLNSGVPKRFS GRRSGSDYSL TISSLESEDF VDYYCLQYSS YPWSFGGGTK LEIKEPKSPD KTHTCPPCPSHTQPLGVFLF PPKPKDQLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVVSVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFYPSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVLHEALH NHYTQKSLSLSLGKFWVLVV VGGVLACYSL LVTVAFIIFW VARPRRSPAQ EDGKVYINMP GRGGRLVPRG RGAAEAATRKQRITETESPY QELQGQRSDV YSDLNTQRPY YKRVKFSRSADAPAYQQGQN QLYNELNLGR REEYDVLDKRRGRDPEMGGK PQRRKNPQEG LYNELQKDKM AEAYSEIGMK

[0367] SEQ ID NO: 85 (SEQ ID NO: 9 of WO 2019 / 182425)

[0368] ARPRRSPAQE DGKVYINMPG RG

[0369] SEQ ID NO: 86 (SEQ ID NO: 11 of WO 2019 / 182425)

[0370] GRLVPRGRGA AEAATRKQRI TETESPYQEL QGQRSDVYSD LNTQRPYYK SEQUENCE LISTING <110> King's College London <120> DAP10 / DAP12 fusion peptide <130> P22111550WP <150> GB1913697.7 <151> 2019-09-23 <160> 86 <170> PatentIn version 3.5 <210> 1 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 1 Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala 1 5 10 15 Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30 Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 50 55 60 Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln 65 70 75 80 Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly 85 90 <210> 2 <211> 75 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 2 Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly 1 5 10 15 Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu 20 25 30 Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly 35 40 45 Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp 50 55 60 Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly 65 70 75 <210> 3 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 3 Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly 1 5 10 15 Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu 20 25 30 Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly 35 40 45 Ala Val Phe 50 <210> 4 <211> 71 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 4 Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala 1 5 10 15 Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30 Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 50 55 60 Val Gly Ala Val Phe Leu Cys 65 70 <210> 5 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 5 Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly 1 5 10 15 Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu 20 25 30 Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly 35 40 45 Ala Val Phe Leu Cys 50 <210> 6 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 6 Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val 1 5 10 15 Tyr Ile Asn Met Pro Gly Arg Gly 20 <210> 7 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 7 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 1 5 10 15 Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln 20 25 30 Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly 35 40 45 <210> 8 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 8 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 1 5 10 15 Val Gly Ala Val Phe 20 <210> 9 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 9 Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu Leu 1 5 10 15 Leu Ala Val Ser Gly Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp 20 25 30 Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met 35 40 45 Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu 50 55 60 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 65 70 75 80 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 85 90 95 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 100 105 110 Lys <210> 10 <211> 92 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 10 Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp Cys Ser Cys Ser Thr 1 5 10 15 Val Ser Pro Gly Val Leu Ala Gly Ile Val Met Gly Asp Leu Val Leu 20 25 30 Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu Gly Arg Leu Val Pro 35 40 45 Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr 50 55 60 Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val 65 70 75 80 Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys 85 90 <210> 11 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 11 Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala 1 5 10 15 Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu 20 25 30 Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg 35 40 45 Pro Tyr Tyr Lys 50 <210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 12 Gly Val Leu Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu 1 5 10 15 Ile Ala Leu Ala Val 20 <210> 13 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 13 Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp Cys Ser Cys Ser Thr 1 5 10 15 Val Ser Pro Gly Val Leu Ala Gly Ile Val Met Gly Asp Leu Val Leu 20 25 30 Thr Val Leu Ile Ala Leu Ala Val 35 40 <210> 14 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 14 Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser 1 5 10 15 Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr 20 25 30 Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu 35 40 45 Asn Ala Ser Pro Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly 50 55 60 Ile Arg Phe Ile Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn 65 70 75 80 Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys 85 90 95 Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln 100 105 110 Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met 115 120 125 Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp 130 135 140 Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile 145 150 155 160 Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro 165 170 175 Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr 180 185 190 Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr 195 200 205 Tyr Ile Cys Met Gln Arg Thr Val 210 215 <210> 15 <211> 144 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 15 Ile Trp Ser Ala Val Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln 1 5 10 15 Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile 20 25 30 Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp 35 40 45 Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys 50 55 60 Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr 65 70 75 80 His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp 85 90 95 Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met 100 105 110 Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile 115 120 125 Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val 130 135 140 <210> 16 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 16 Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly 1 5 10 15 Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe 20 25 30 Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser 35 40 45 Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu 50 55 60 Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro 65 70 75 80 Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn 85 90 95 Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala 100 105 110 Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr 115 120 125 Ile Cys Met Gln Arg Thr Val 130 135 <210> 17 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> isolated <400> 17 Pro Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly Ile Arg Phe 1 5 10 15 Ile Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn Ser Leu Phe 20 25 30 Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys 35 40 45 Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp 50 55 60 Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn 65 70 75 80 Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys 85 90 95 Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn 100 105 110 Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu 115 120 125 Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser 130 135 140 Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys 145 150 155 160 Met Gln Arg Thr Val 165 <210> 18 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 18 Gly Ser Gly 1 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 19 Gly Ser Gly Gly Gly 1 5 <210> 20 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 20 Gly Ser Gly Gly 1 <210> twenty one <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> twenty one Ser Gly Gly Gly 1 <210> twenty two <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 22 Gly Gly Gly Gly Ser 1 5 <210> 23 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 23 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 24 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 24 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 25 Gly Pro Pro Gly Ser 1 5 <210> 26 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 26 Gly Gly Gly Ser 1 <210> 27 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 27 Gly Gly Gly Gly Ser 1 5 <210> 28 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 28 Gly Tyr Ser 1 <210> 29 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 29 Gly Ser 1 <210> 30 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 30 Ser Gly Gly Gly Gly 1 5 <210> 31 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 31 Ser Gly Gly Gly 1 <210> 32 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 32 Ser Gly Gly 1 <210> 33 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 33 Ser Gly Ser Gly 1 <210> 34 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 34 Ser Gly 1 <210> 35 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 35 Gly Gly Gly Gly Ala 1 5 <210> 36 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 36 Gly Gly Gly Ala 1 <210> 37 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 37 Glu Ala Ala Ala Lys 1 5 <210> 38 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 38 Arg Arg Lys Arg 1 <210> 39 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 39 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 40 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 40 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 41 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 41 Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp 1 5 10 15 Val Glu Glu Asn Pro Gly Pro 20 <210> 42 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 42 Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> 43 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 43 Arg Arg Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 1 5 10 15 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25 <210> 44 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 44 Arg Arg Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr 1 5 10 15 Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25 <210> 45 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 45 Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 <210> 46 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 46 Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 47 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 47 His His His His His His 1 5 <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 48 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 49 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 49 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 1 5 10 15 <210> 50 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 50 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 51 Ile Pro Asn Pro Leu Leu Gly Leu Asp 1 5 <210> 52 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 52 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 53 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 53 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly 1 5 10 15 Gly His His His His His Asp Tyr Lys Asp Asp Asp Asp Lys 20 25 30 <210> 54 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Separate <400> 54 Asp Tyr Lys Asp Asp Asp Asp Lys His His His His His His Gly Gly 1 5 10 15 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 20 25 30 <210> 55 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 55 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 56 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 56 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 57 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 57 Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro 1 5 10 15 Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr 20 25 30 Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro 35 40 45 <210> 58 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 58 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 1 5 10 <210> 59 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 59 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 60 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 60 Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala 1 5 10 15 Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30 Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 50 55 60 Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln 65 70 75 80 Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Tyr Phe Leu 85 90 95 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 100 105 110 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 115 120 125 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 130 135 140 Lys 145 <210> 61 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 61 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Gln Thr Thr 20 25 30 Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly Thr Ser Gly 35 40 45 Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu Ala Gly Leu 50 55 60 Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly Ala Val Phe 65 70 75 80 Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala 85 90 95 Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu 100 105 110 Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg 115 120 125 Pro Tyr Tyr Lys 130 <210> 62 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 62 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Glu Pro Lys 20 25 30 Ser Cys Asp Lys Thr His Thr Cys Pro Leu Leu Ala Gly Leu Val Ala 35 40 45 Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly Ala Val Phe Leu Cys 50 55 60 Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val Tyr Ile 65 70 75 80 Asn Met Pro Gly Arg Gly Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly 85 90 95 Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr 100 105 110 Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser 115 120 125 Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys 130 135 <210> 63 <211> 171 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 63 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Thr Thr Thr 20 25 30 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 35 40 45 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 50 55 60 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Leu Leu Ala Gly Leu Val 65 70 75 80 Ala Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly Ala Val Phe Leu 85 90 95 Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val Tyr 100 105 110 Ile Asn Met Pro Gly Arg Gly Tyr Phe Leu Gly Arg Leu Val Pro Arg 115 120 125 Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu 130 135 140 Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr 145 150 155 160 Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys 165 170 <210> 64 <211> 388 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 64 Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala 1 5 10 15 Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30 Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 50 55 60 Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln 65 70 75 80 Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Tyr Phe Leu 85 90 95 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 100 105 110 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 115 120 125 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 130 135 140 Lys Arg Arg Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 145 150 155 160 Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Trp Ile 165 170 175 Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser Glu Phe His Asn 180 185 190 Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr Arg Trp Gln Lys 195 200 205 Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu Asn Ala Ser Pro 210 215 220 Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly Ile Arg Phe Ile 225 230 235 240 Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn Ser Leu Phe Asn 245 250 255 Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro 260 265 270 Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu 275 280 285 Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala 290 295 300 Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu 305 310 315 320 Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly 325 330 335 Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr 340 345 350 Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe 355 360 365 Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met 370 375 380 Gln Arg Thr Val 385 <210> 65 <211> 494 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 65 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Gln Thr Thr 20 25 30 Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly Thr Ser Gly 35 40 45 Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu Ala Gly Leu 50 55 60 Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly Ala Val Phe 65 70 75 80 Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala 85 90 95 Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu 100 105 110 Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg 115 120 125 Pro Tyr Tyr Lys Arg Arg Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly 130 135 140 Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ile 145 150 155 160 His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala Ala Ala 165 170 175 Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly 180 185 190 Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu 195 200 205 Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly 210 215 220 Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp 225 230 235 240 Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Arg Arg Lys Arg Ser 245 250 255 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 260 265 270 Glu Glu Asn Pro Gly Pro Met Gly Trp Ile Arg Gly Arg Arg Ser Arg 275 280 285 His Ser Trp Glu Met Ser Glu Phe His Asn Tyr Asn Leu Asp Leu Lys 290 295 300 Lys Ser Asp Phe Ser Thr Arg Trp Gln Lys Gln Arg Cys Pro Val Val 305 310 315 320 Lys Ser Lys Cys Arg Glu Asn Ala Ser Pro Phe Phe Phe Cys Cys Phe 325 330 335 Ile Ala Val Ala Met Gly Ile Arg Phe Ile Ile Met Val Ala Ile Trp 340 345 350 Ser Ala Val Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro 355 360 365 Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr 370 375 380 Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu 385 390 395 400 Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr 405 410 415 Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp 420 425 430 Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp 435 440 445 Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys 450 455 460 Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn 465 470 475 480 Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val 485 490 <210> 66 <211> 527 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 66 Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala 1 5 10 15 Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30 Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 50 55 60 Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln 65 70 75 80 Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Tyr Phe Leu 85 90 95 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 100 105 110 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 115 120 125 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 130 135 140 Lys Arg Arg Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 145 150 155 160 Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Trp Ile 165 170 175 Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser Glu Phe His Asn 180 185 190 Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr Arg Trp Gln Lys 195 200 205 Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu Asn Ala Ser Pro 210 215 220 Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly Ile Arg Phe Ile 225 230 235 240 Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn Ser Leu Phe Asn 245 250 255 Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro 260 265 270 Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu 275 280 285 Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala 290 295 300 Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu 305 310 315 320 Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly 325 330 335 Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr 340 345 350 Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe 355 360 365 Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met 370 375 380 Gln Arg Thr Val Arg Arg Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly 385 390 395 400 Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ile 405 410 415 His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala Ala Ala 420 425 430 Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly 435 440 445 Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu 450 455 460 Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly 465 470 475 480 Ala Val Phe Leu Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 485 490 495 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 500 505 510 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 515 520 525 <210> 67 <211> 538 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 67 Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala 1 5 10 15 Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30 Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 50 55 60 Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln 65 70 75 80 Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Tyr Phe Leu 85 90 95 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 100 105 110 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 115 120 125 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 130 135 140 Lys Arg Arg Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 145 150 155 160 Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Trp Ile 165 170 175 Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser Glu Phe His Asn 180 185 190 Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr Arg Trp Gln Lys 195 200 205 Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu Asn Ala Ser Pro 210 215 220 Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly Ile Arg Phe Ile 225 230 235 240 Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn Ser Leu Phe Asn 245 250 255 Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro 260 265 270 Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu 275 280 285 Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala 290 295 300 Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu 305 310 315 320 Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly 325 330 335 Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr 340 345 350 Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe 355 360 365 Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met 370 375 380 Gln Arg Thr Val Arg Arg Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly 385 390 395 400 Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala 405 410 415 Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala 420 425 430 Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Gln Thr Thr Pro Gly 435 440 445 Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly Thr Ser Gly Ser Cys 450 455 460 Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu Ala Gly Leu Val Ala 465 470 475 480 Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly Ala Val Phe Leu Cys 485 490 495 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 500 505 510 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 515 520 525 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 530 535 <210> 68 <211> 533 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 68 Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala 1 5 10 15 Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30 Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 50 55 60 Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln 65 70 75 80 Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Tyr Phe Leu 85 90 95 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 100 105 110 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 115 120 125 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 130 135 140 Lys Arg Arg Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 145 150 155 160 Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Trp Ile 165 170 175 Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser Glu Phe His Asn 180 185 190 Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr Arg Trp Gln Lys 195 200 205 Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu Asn Ala Ser Pro 210 215 220 Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly Ile Arg Phe Ile 225 230 235 240 Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn Ser Leu Phe Asn 245 250 255 Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro 260 265 270 Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu 275 280 285 Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala 290 295 300 Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu 305 310 315 320 Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly 325 330 335 Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr 340 345 350 Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe 355 360 365 Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met 370 375 380 Gln Arg Thr Val Arg Arg Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly 385 390 395 400 Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ile 405 410 415 His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala Ala Ala 420 425 430 Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly 435 440 445 Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu 450 455 460 Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly 465 470 475 480 Ala Val Phe Leu Cys Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu 485 490 495 Ser Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu 500 505 510 Glu Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu 515 520 525 Pro Ala Cys Ser Pro 530 <210> 69 <211> 544 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 69 Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala 1 5 10 15 Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30 Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45 Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 50 55 60 Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln 65 70 75 80 Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly Tyr Phe Leu 85 90 95 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 100 105 110 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 115 120 125 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 130 135 140 Lys Arg Arg Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 145 150 155 160 Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Trp Ile 165 170 175 Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser Glu Phe His Asn 180 185 190 Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr Arg Trp Gln Lys 195 200 205 Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu Asn Ala Ser Pro 210 215 220 Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly Ile Arg Phe Ile 225 230 235 240 Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn Ser Leu Phe Asn 245 250 255 Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro 260 265 270 Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu 275 280 285 Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala 290 295 300 Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu 305 310 315 320 Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly 325 330 335 Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr 340 345 350 Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe 355 360 365 Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met 370 375 380 Gln Arg Thr Val Arg Arg Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly 385 390 395 400 Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala 405 410 415 Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala 420 425 430 Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Gln Thr Thr Pro Gly 435 440 445 Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly Thr Ser Gly Ser Cys 450 455 460 Ser Gly Cys Gly Ser Leu Ser Leu Pro Leu Leu Ala Gly Leu Val Ala 465 470 475 480 Ala Asp Ala Val Ala Ser Leu Leu Ile Val Gly Ala Val Phe Leu Cys 485 490 495 Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro 500 505 510 Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr 515 520 525 Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro 530 535 540 <210> 70 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> isolated <400> 70 atgatccacc tgggccacat cctgttcctg ctgctgctgc ccgtggccgc tgcccagacc 60 acccctggcg agcggagcag cctgcctgcc ttctaccctg gcaccagcgg cagctgcagc 120 ggctgcggca gcctgagcct gcccctgctg gccggcctgg tggccgccga cgccgtggcc 180 agcctgctga tcgtgggcgc cgtgttcctg tgcgccaggc ccaggcggag ccctgcccag 240 gaggacggca aggtgtacat caacatgccc ggccggggct acttcctggg caggctggtg 300 cccaggggca ggggcgctgc cgaggctgcc acccggaagc agcggatcac cgagaccgag 360 agcccctacc aggagctgca gggccagcgg agcgacgtgt acagcgacct gaacacccag 420 aggccctact acaag 435 <210> 71 <211> 396 <212> DNA <213> Artificial Sequence <220> <223> Isolated <400> 71 atggctctgc ctgtgacagc tctgctgctg cctctggctc tgctgctgca cgccgctaga 60 cccgattata aggacgacga cgacaagcag accacccctg gcgagcggag cagcctgcct 120 gccttctacc ctggcaccag cggcagctgc agcggctgcg gcagcctgag cctgcccctg 180 ctggctggcc tggtggccgc cgacgccgtg gccagcctgc tgatcgtggg cgccgtgttc 240 tacttcctgg gcaggctggt gcccaggggc aggggcgctg ccgaggctgc cacccggaag 300 cagcggatca ccgagaccga gagcccctac caggagctgc agggccagcg gagcgacgtg 360 tacagcgacc tgaacaccca gaggccctac tacaag 396 <210> 72 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> Isolated <400> 72 atggctctgc ctgtgacagc tctgctgctg cctctggctc tgctgctgca cgccgctaga 60 cccgattata aggacgacga cgacaaggag cccaagagct gcgacaagac acacacatgc 120 cctcttctgg ccggcctggt ggccgccgac gccgtggcca gcctgctgat cgtgggcgcc 180 gtgttcctgt gcgccaggcc caggcggagc cctgcccagg aggacggcaa ggtgtacatc 240 aacatgcccg gccggggcta cttcctgggc aggctggtgc ccaggggcag gggcgctgcc 300 gaggctgcca cccggaagca gcggatcacc gagaccgaga gcccctacca ggagctgcag 360 ggccagcgga gcgacgtgta cagcgacctg aacacccaga ggccctacta caag 414 <210> 73 <211> 513 <212> DNA <213> Artificial Sequence <220> <223> Isolated <400> 73 atggctctgc ctgtgacagc tctgctgctg cctctggctc tgctgctgca cgccgctaga 60 cccgattata aggacgacga cgacaagacc acaacacctg ctcctagacc tcccacccct 120 gctcccacca tcgccagcca gcccctgagc ctgagacccg aggcctgcag acccgctgct 180 ggcggcgctg tgcataccag aggcctggat ttcgcctgcg accttctggc cggcctggtg 240 gccgccgacg ccgtggccag cctgctgatc gtgggcgccg tgttcctgtg cgccaggccc 300 aggcggagcc ctgcccagga ggacggcaag gtgtacatca acatgcccgg ccggggctac 360 ttcctgggca ggctggtgcc caggggcagg ggcgctgccg aggctgccac ccggaagcag 420 cggatcaccg agaccgagag cccctaccag gagctgcagg gccagcggag cgacgtgtac 480 agcgacctga acacccagag gccctactac aag 513 <210> 74 <211> 1164 <212> DNA <213> Artificial Sequence <220> <223> Isolated <400> 74 atgatccacc tgggccacat cctgttcctg ctgctgctgc ccgtggccgc tgcccagacc 60 acccctggcg agcggagcag cctgcctgcc ttctaccctg gcaccagcgg cagctgcagc 120 ggctgcggca gcctgagcct gcccctgctg gccggcctgg tggccgccga cgccgtggcc 180 agcctgctga tcgtgggcgc cgtgttcctg tgcgccaggc ccaggcggag ccctgcccag 240 gaggacggca aggtgtacat caacatgccc ggccggggct acttcctggg caggctggtg 300 cccaggggca ggggcgctgc cgaggctgcc acccggaagc agcggatcac cgagaccgag 360 agcccctacc aggagctgca gggccagcgg agcgacgtgt acagcgacct gaacacccag 420 aggccctact acaagaggcg gaaaaggtct gggagtgggg ctaccaattt ctctctcctc 480 aagcaagccg gagacgttga ggaaaaccct ggacccatgg gctggatccg gggacggagg 540 agccggcaca gctgggagat gagcgagttc cacaactaca acctggacct gaagaagagc 600 gacttcagca cccggtggca gaagcagcgg tgccccgtgg tgaagagcaa gtgccgggag 660 aacgccagcc ccttcttctt ctgctgcttc atcgccgtgg ctatgggcat ccggttcatc 720 atcatggtgg ccatctggag cgccgtgttc ctgaacagcc tgttcaacca ggaggtgcag 780 atccccctga ccgagagcta ctgcggcccc tgccccaaga actggatctg ctacaagaac 840 aactgctacc agttcttcga cgagagcaag aactggtacg agagccaggc cagctgcatg 900 agccagaacg ccagcctgct gaaggtgtac agcaaggagg accaggacct gctgaagctg 960 gtgaagagct accactggat gggcctggtg cacatcccca ccaacggcag ctggcagtgg 1020 gaggacggca gcatcctgag ccccaacctg ctgaccatca tcgagatgca gaagggcgac 1080 tgcgccctgt acgccagcag cttcaagggc tacatcgaga actgcagcac ccccaacacc 1140 tacatctgca tgcagcggac cgtg 1164 <210> 75 <211> 1482 <212> DNA <213> Artificial Sequence <220> <223> Isolated <400> 75 atggctctgc ctgtgacagc tctgctgctg cctctggctc tgctgctgca cgccgctaga 60 cccgattata aggacgacga cgacaagcag accacccctg gcgagcggag cagcctgcct 120 gccttctacc ctggcaccag cggcagctgc agcggctgcg gcagcctgag cctgcccctg 180 ctggctggcc tggtggccgc cgacgccgtg gccagcctgc tgatcgtggg cgccgtgttc 240 tacttcctgg gcaggctggt gcccaggggc aggggcgctg ccgaggctgc cacccggaag 300 cagcggatca ccgagaccga gagcccctac caggagctgc agggccagcg gagcgacgtg 360 tacagcgacc tgaacaccca gaggccctac tacaagcgga gaaagcgctc cggctccggc 420 gagggccgcg gcagcctgct gacctgcggc gacgtggaag agaaccccgg acccatgatc 480 cacctgggcc acatcctgtt cctgctgctg ctgcccgtgg ccgctgccca aacaacaccc 540 ggcgagagat cctccttgcc cgctttctat cccggaacat ccggaagctg ttccggatgt 600 ggatcccttt ctttgccttt gcttgctgga ttggtcgcag ctgacgctgt cgcttccctc 660 cttattgtcg gagctgtctt cctgtgcgcc aggcccaggc ggagccctgc ccaggaggac 720 ggcaaggtgt acatcaacat gcccggccgg ggcaggcgga agcgctccgg gagtggggct 780 accaatttct ctctcctcaa gcaagccgga gacgttgagg aaaaccctgg acccatgggc 840 tggatccggg gacggaggag ccggcacagc tgggagatga gcgagttcca caactacaac 900 ctggacctga agaagagcga cttcagcacc cggtggcaga agcagcggtg ccccgtggtg 960 aagagcaagt gccgggagaa cgccagcccc ttcttcttct gctgcttcat cgccgtggct 1020 atgggcatcc ggttcatcat catggtggcc atctggagcg ccgtgttcct gaacagcctg 1080 ttcaaccagg aggtgcagat ccccctgacc gagagctact gcggcccctg ccccaagaac 1140 tggatctgct acaagaacaa ctgctaccag ttcttcgacg agagcaagaa ctggtacgag 1200 agccaggcca gctgcatgag ccagaacgcc agcctgctga aggtgtacag caaggaggac 1260 caggacctgc tgaagctggt gaagagctac cactggatgg gcctggtgca catccccacc 1320 aacggcagct ggcagtggga ggacggcagc atcctgagcc ccaacctgct gaccatcatc 1380 gagatgcaga agggcgactg cgccctgtac gccagcagct tcaagggcta catcgagaac 1440 tgcagcaccc ccaacaccta catctgcatg cagcggaccg tg 1482 <210> 76 <211> 1581 <212> DNA <213> Artificial Sequence <220> <223> Isolated <400> 76 atgatccacc tgggccacat cctgttcctg ctgctgctgc ccgtggccgc tgcccagacc 60 acccctggcg agcggagcag cctgcctgcc ttctaccctg gcaccagcgg cagctgcagc 120 ggctgcggca gcctgagcct gcccctgctg gccggcctgg tggccgccga cgccgtggcc 180 agcctgctga tcgtgggcgc cgtgttcctg tgcgccaggc ccaggcggag ccctgcccag 240 gaggacggca aggtgtacat caacatgccc ggccggggct acttcctggg caggctggtg 300 cccaggggca ggggcgctgc cgaggctgcc acccggaagc agcggatcac cgagaccgag 360 agcccctacc aggagctgca gggccagcgg agcgacgtgt acagcgacct gaacacccag 420 aggccctact acaagaggcg gaaaaggtct gggagtgggg ctaccaattt ctctctcctc 480 aagcaagccg gagacgttga ggaaaaccct ggacccatgg gctggatccg gggacggagg 540 agccggcaca gctgggagat gagcgagttc cacaactaca acctggacct gaagaagagc 600 gacttcagca cccggtggca gaagcagcgg tgccccgtgg tgaagagcaa gtgccgggag 660 aacgccagcc ccttcttctt ctgctgcttc atcgccgtgg ctatgggcat ccggttcatc 720 atcatggtgg ccatctggag cgccgtgttc ctgaacagcc tgttcaacca ggaggtgcag 780 atccccctga ccgagagcta ctgcggcccc tgccccaaga actggatctg ctacaagaac 840 aactgctacc agttcttga cgagagcaag aactggtacg agagccaggc cagctgcatg 900 agccagaacg ccagcctgct gaaggtgtac agcaaggagg accaggacct gctgaagctg 960 gtgaagagct accactggat gggcctggtg cacatcccca ccaacggcag ctggcagtgg 1020 gaggagca gcatcctgag ccccaacctg ctgaccatca tcgagatgca gaagggcgac 1080 tgcgccctgt acgccagcag cttcaagggc tacatcgaga actgcagcac ccccaacacc 1140 tacatctgca tgcagcggac cgtgagaaga aagagaagcg gcagcggcga gggcagaggc 1200 agcctgctga cctgcggcga cgtgggagg aaccccggac ccatgattca tctcggagacat 1260 attctctttc tcttgctctt gcctgtcgct gccgctcaaa caactcccgg agaaagatct 1320 tctctccccg ctttttatcc cggaacatct ggatcttgtt ctggatgtgg atctttgtct 1380 ctccctctcc tcgctggact cgtcgcagct gatgctgtcg cttctctctt gattgtcgga 1440 gctgtctttt tgtgtaagag aggcagaaag aagctgctgt acatcttcaa gcagcccttc 1500 atgagacccg tgcagaccac ccaggaggag gacggctgca gctgcagatt ccccgaggag 1560 gaggagggcg gctgcgagct g 1581 <210> 77 <211> 1614 <212> DNA <213> Artificial Sequence <220> <223> isolated <400> 77 atgatccacc tgggccacat cctgttcctg ctgctgctgc ccgtggccgc tgcccagacc 60 acccctggcg agcggagcag cctgcctgcc ttctaccctg gcaccagcgg cagctgcagc 120 ggctgcggca gcctgagcct gcccctgctg gccggcctgg tggccgccga cgccgtggcc 180 agcctgctga tcgtgggcgc cgtgttcctg tgcgccaggc ccaggcggag ccctgcccag 240 gaggacggca aggtgtacat caacatgccc ggccggggct acttcctggg caggctggtg 300 cccaggggca ggggcgctgc cgaggctgcc acccggaagc agcggatcac cgagaccgag 360 agcccctacc aggagctgca gggccagcgg agcgacgtgt acagcgacct gaacacccag 420 aggccctact aaagaggcg gaaaaggtct gggagtgggg ctaccaattt ctctctcctc 480 aagcaagccg gagacgttga ggaaaaccct ggaaccatgg gctggatccg gggacggagg 540 agccggcaca gctgggagat gagcgagttc cacaactaca acctggacct gaagaagc 600 gacttcagca cccggtggca gaagcagcgg tgccccgtgg tgaagagcaa gtgccgggag 660 aacgccagcc ccttcttctt ctgctgcttc atcgccgtgg ctatgggcat ccggttcatc 720 atcatggtgg ccatctggag cgccgtgttc ctgaacagcc tgttcaacca ggaggtgcag 780 atccccctga ccgagagcta ctgcggcccc tgccccaaga actggatctg ctacaagaac 840 aactgctacc agttcttga cgagagcaag aactggtacg agagccaggc cagctgcatg 900 agccagaacg ccagcctgct gaaggtgtac agcaaggagg accaggacct gctgaagctg 960 gtgaagagct accactggat gggcctggtg cacatcccca ccaacggcag ctggcagtgg 1020 gaggacggca gcatcctgag ccccaacctg ctgaccatca tcgagatgca gaagggcgac 1080 tgcgccctgt acgccagcag cttcaagggc tacatcgaga actgcagcac ccccaacacc 1140 tacatctgca tgcagcggac cgtgagaaga aagagaagcg gcagcggcga gggcagaggc 1200 agcctgctga cctgcggcga cgtggaggag aaccccggac ctatggctct gcctgtgaca 1260 gctctgctgc tgcctctggc tctgctgctg cacgccgcta gacccgatta taaggacgac 1320 gacgacaagc aaacaactcc cggagaaaga tcttctctcc ccgcttttta tcccggaaca 1380 tctggatctt gttctggatg tggatctttg tctctccctc tcctcgctgg actcgtcgca 1440 gctgatgctg tcgcttctct cttgattgtc ggagctgtct ttttgtgtaa gagaggcaga 1500 aagaagctgc tgtacatctt caagcagccc ttcatgagac ccgtgcagac cacccaggag 1560 gaggacggct gcagctgcag attccccgag gaggaggagg gcggctgcga gctg 1614 <210> 78 <211> 1599 <212> DNA <213> Artificial Sequence <220> <223> Isolated <400> 78 atgatccacc tgggccacat cctgttcctg ctgctgctgc ccgtggccgc tgcccagacc 60 acccctggcg agcggagcag cctgcctgcc ttctaccctg gcaccagcgg cagctgcagc 120 ggctgcggca gcctgagcct gcccctgctg gccggcctgg tggccgccga cgccgtggcc 180 agcctgctga tcgtgggcgc cgtgttcctg tgcgccaggc ccaggcggag ccctgcccag 240 gaggacggca aggtgtacat caacatgccc ggccggggct acttcctggg caggctggtg 300 cccaggggca ggggcgctgc cgaggctgcc acccggaagc agcggatcac cgagaccgag 360 agcccctacc aggagctgca gggccagcgg agcgacgtgt acagcgacct gaacacccag 420 aggccctact aaagaggcg gaaaaggtct gggagtgggg ctaccaattt ctctctcctc 480 aagcaagccg gagacgttga ggaaaaccct ggaaccatgg gctggatccg gggacggagg 540 agccggcaca gctgggagat gagcgagttc cacaactaca acctggacct gaagaagc 600 gacttcagca cccggtggca gaagcagcgg tgccccgtgg tgaagagcaa gtgccgggag 660 aacgccagcc ccttcttctt ctgctgcttc atcgccgtgg ctatgggcat ccggttcatc 720 atcatggtgg ccatctggag cgccgtgttc ctgaacagcc tgttcaacca ggaggtgcag 780 atccccctga ccgagagcta ctgcggcccc tgccccaaga actggatctg ctacaagaac 840 aactgctacc agttcttcga cgagagcaag aactggtacg agagccaggc cagctgcatg 900 agccagaacg ccagcctgct gaaggtgtac agcaaggagg accaggacct gctgaagctg 960 gtgaagagct accactggat gggcctggtg cacatcccca ccaacggcag ctggcagtgg 1020 gaggacggca gcatcctgag ccccaacctg ctgaccatca tcgagatgca gaagggcgac 1080 tgcgccctgt acgccagcag cttcaagggc tacatcgaga actgcagcac ccccaacacc 1140 tacatctgca tgcagcggac cgtgagaaga aagagaagcg gcagcggcga gggcagaggc 1200 agcctgctga cctgcggcga cgtggaggag aaccccggac ccatgattca tctcggacat 1260 attctctttc tcttgctctt gcctgtcgct gccgctcaaa caactcccgg agaaagatct 1320 tctctccccg ctttttatcc cggaacatct ggatcttgtt ctggatgtgg atctttgtct 1380 ctccctctcc tcgctggact cgtcgcagct gatgctgtcg cttctctctt gattgtcgga 1440 gctgtctttt tgtgtcagag gcggaagtac cggagcaaca agggcgagag ccccgtggag 1500 cctgccgagc cctgccacta cagctgtccc cgggaggagg agggcagcac catccccatc 1560 caggaggact accggaagcc cgagcctgcc tgcagcccc 1599 <210> 79 <211> 1632 <212> DNA <213> Artificial Sequence <220> <223> isolated <400> 79 atgatccacc tgggccacat cctgttcctg ctgctgctgc ccgtggccgc tgcccagacc 60 acccctggcg agcggagcag cctgcctgcc ttctaccctg gcaccagcgg cagctgcagc 120 ggctgcggca gcctgagcct gcccctgctg gccggcctgg tggccgccga cgccgtggcc 180 agcctgctga tcgtgggcgc cgtgttcctg tgcgccaggc ccaggcggag ccctgcccag 240 gaggacggca aggtgtacat caacatgccc ggccggggct acttcctggg caggctggtg 300 cccaggggca ggggcgctgc cgaggctgcc acccggaagc agcggatcac cgagaccgag 360 agcccctacc aggagctgca gggccagcgg agcgacgtgt acagcgacct gaacacccag 420 aggccctact aaagaggcg gaaaaggtct gggagtgggg ctaccaattt ctctctcctc 480 aagcaagccg gagacgttga ggaaaaccct ggaaccatgg gctggatccg gggacggagg 540 agccggcaca gctgggagat gagcgagttc cacaactaca acctggacct gaagaagc 600 gacttcagca cccggtggca gaagcagcgg tgccccgtgg tgaagagcaa gtgccgggag 660 aacgccagcc ccttcttctt ctgctgcttc atcgccgtgg ctatgggcat ccggttcatc 720 atcatggtgg ccatctggag cgccgtgttc ctgaacagcc tgttcaacca ggaggtgcag 780 atccccctga ccgagagcta ctgcggcccc tgccccaaga actggatctg ctacaagaac 840 aactgctacc agttcttga cgagagcaag aactggtacg agagccaggc cagctgcatg 900 agccagaacg ccagcctgct gaaggtgtac agcaaggagg accaggacct gctgaagctg 960 gtgaagagct accactggat gggcctggtg cacatcccca ccaacggcag ctggcagtgg 1020 gaggacggca gcatcctgag ccccaacctg ctgaccatca tcgagatgca gaagggcgac 1080 tgcgccctgt acgccagcag cttcaagggc tacatcgaga actgcagcac ccccaacacc 1140 tacatctgca tgcagcggac cgtgagaaga aagagaagcg gcagcggcga gggcagaggc 1200 agcctgctga cctgcggcga cgtggaggag aaccccggac ctatggctct gcctgtgaca 1260 gctctgctgc tgcctctggc tctgctgctg cacgccgcta gacccgatta taaggacgac 1320 gacgacaagc aaacaactcc cggagaaaga tcttctctcc ccgcttttta tcccggaaca 1380 tctggatctt gttctggatg tggatctttg tctctccctc tcctcgctgg actcgtcgca 1440 gctgatgctg tcgcttctct cttgattgtc ggagctgtct ttttgtgtca gaggcggaag 1500 taccggagca acaagggcga gagccccgtg gagcctgccg agccctgcca ctacagctgt 1560 ccccgggagg aggagggcag caccatcccc atccaggagg actaccggaa gcccgagcct 1620 gcctgcagcc cc 1632 <210> 80 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 80 Asn Ala Val Pro Asn Leu Arg Gly Asp Leu Gln Val Leu Ala Gln Lys 1 5 10 15 Val Ala Arg Thr 20 <210> 81 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 81 Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val 1 5 10 15 Leu Leu Gln Val Ala Ser Ser Gly Asn 20 25 <210> 82 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 82 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 35 40 45 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 50 55 60 Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn 65 70 75 80 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 85 90 95 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 100 105 <210> 83 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 83 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 84 <211> 695 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 84 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys 20 25 30 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ser Met His Trp Val Asn Gln Ala Pro Gly Lys Gly Leu 50 55 60 Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Thr 65 70 75 80 Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 85 90 95 Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Thr Ala Val Tyr Trp Gly Gln Gly Thr Thr Leu 115 120 125 Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly 130 135 140 Glu Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 145 150 155 160 Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile 165 170 175 Ser Gly Ser Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys 180 185 190 Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asn Ser Gly Val Pro Lys Arg 195 200 205 Phe Ser Gly Arg Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser 210 215 220 Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ser Ser 225 230 235 240 Tyr Pro Trp Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Glu Pro 245 250 255 Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ser His Thr 260 265 270 Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu 275 280 285 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455 460 Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 465 470 475 480 Ser Leu Gly Lys Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 485 490 495 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Ala 500 505 510 Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val Tyr Ile Asn 515 520 525 Met Pro Gly Arg Gly Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala 530 535 540 Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr 545 550 555 560 Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr 565 570 575 Gln Arg Pro Tyr Tyr Lys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 580 585 590 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 595 600 605 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 610 615 620 Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly 625 630 635 640 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 645 650 655 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 660 665 670 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 675 680 685 Met Gln Ala Leu Pro Pro Arg 690 695 <210> 85 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Isolated <400> 85 Ala Arg Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val Tyr Ile 1 5 10 15 Asn Met Pro Gly Arg Gly 20 <210> 86 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> separation <400> 86 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 1 5 10 15 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 20 25 30 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 35 40 45 Lys

Claims

1. A complex comprising a fusion polypeptide and an NKG2D polypeptide, said fusion polypeptide comprising (i) a DNAX-activated protein 10 (DAP10) polypeptide and (ii) a DNAX-activated protein 12 (DAP12) polypeptide; The amino acid sequence of the fusion polypeptide is SEQ ID NO: 60, and the amino acid sequence of the NKG2D polypeptide is SEQ ID NO:

14.

2. The complex according to claim 1, characterized in that, The fusion polypeptide: (a) Does not include sequences as shown in SEQ ID NO: 84; and / or (b) Excluding anti-EpCAM peptide.

3. The complex according to claim 1 or 2, characterized in that, The fusion peptide is electrostatically linked to the NKG2D peptide.

4. The complex according to claim 1, characterized in that, The complex comprises or consists of a sequence as shown in SEQ ID NO:

64.

5. An isolated nucleic acid sequence encoding a complex as described in any one of claims 1-4.

6. The isolated nucleic acid sequence of claim 5, wherein the nucleic acid sequence encodes a linker, the linker comprising a cleavage site and / or a ribosomal jumping peptide, the linker being located between the fusion polypeptide and the NKG2D polypeptide.

7. The isolated nucleic acid sequence of claim 6, wherein the nucleic acid sequence encodes a linker having an amino acid sequence as shown in any of SEQ ID NO: 33 and 38-44, the linker being located between the fusion polypeptide and the NKG2D polypeptide.

8. The isolated nucleic acid sequence of claim 5, comprising a nucleotide sequence encoding SEQ ID NO: 60 or 64.

9. The isolated nucleic acid sequence of claim 5, comprising or consisting of a nucleotide sequence as shown in SEQ ID NO: 70 or 74.

10. A vector comprising the nucleic acid sequence as described in any one of claims 5-9.

11. The vector as described in claim 10, wherein it is a lentiviral vector or a retroviral vector.

12. A host cell comprising the complex as described in any one of claims 1-4.

13. The host cell as described in claim 12, characterized in that, The host cell further comprises nucleic acid molecules encoding the fusion protein and the NKG2D polypeptide.

14. A host cell comprising the nucleic acid sequence as described in any one of claims 5-9 or the vector as described in claim 10 or 11.

15. The host cell according to any one of claims 12-14, wherein it is a T cell or an NK cell.

16. A method for preparing a complex according to any one of claims 1-4, comprising maintaining a host cell as described in claim 14 under conditions suitable for expressing the nucleic acid sequence, thereby expressing the nucleic acid sequence and generating the complex.

17. A method for preparing immune-response cells, comprising the following steps: (i) Transform the nucleic acid sequence of any one of claims 5-9 or the vector of claim 10 or 11 into the immune response cells, and (ii) culture the immune response cells to express the fusion polypeptide and bind it to the NKG2D polypeptide to form a CAR.

18. A pharmaceutical composition comprising a complex as described in any one of claims 1-4, a nucleic acid sequence as described in any one of claims 5-9, a vector as described in claim 10 or 11, or a host cell as described in any one of claims 12-15.

19. The pharmaceutical composition of claim 18, further comprising a pharmaceutically or physiologically acceptable diluent and / or carrier.

20. The use of the complex of any one of claims 1-4, the nucleic acid sequence of any one of claims 5-9, the vector of any one of claims 10 or 11, the host cell of any one of claims 12-15, or the pharmaceutical composition of any one of claims 18 or 19 in the preparation of a medicament for treating cancer, wherein the cancer is selected from: malignant mesothelioma, breast cancer, pancreatic cancer, ovarian cancer, or any combination of the above cancers.

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