RU2839702C2 - Methods and compositions for enhancing multiplication and cytotoxicity of natural killer cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, particularly to methods for enhancing multiplication of natural killer (NK) cells (embodiments). In one embodiment, said method involves (a) co-cultivation in a culture medium of a population of NK cells with a population of feeder cells containing cells engineered to express 4-1BBL and membrane-bound interleukin-15 (mbIL15); (b) enriching the culture medium with interleukin-2 (IL2) and (c) enriching the culture medium with at least one soluble stimulant, which is a combination (i) soluble interleukin-12 and soluble interleukin-18 (IL18).

EFFECT: present invention provides intensified reproduction of natural killers and their cytotoxicity against target cells.

24 cl, 37 dwg, 1 tbl, 4 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/881,311, filed July 31, 2019, and U.S. Provisional Patent Application No. 62/932,342, filed November 7, 2019, the entire contents of each of which are incorporated herein by reference.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] Some embodiments of the methods and compositions disclosed herein relate to enhancing the proliferation and/or enhancing the cytotoxicity of engineered immune cells, such as natural killer (NK) cells and/or T cells.

PREREQUISITES FOR THE CREATION OF THE INVENTION

[0003] The use of engineered cells for cellular immunotherapy provides the opportunity to treat types of cancer or other diseases by recruiting various components of the immune system to target and destroy diseased or damaged cells. Such therapies require engineered cells in quantities sufficient to provide therapeutically relevant doses.

INCLUSION OF MATERIAL BY LINK IN THE FORM OF AN ASCII TEXT FILE

[0004] The sequence listing contained in the following ASCII text file, which is filed concurrently with this application, is incorporated by reference into this application. File name: NKT034WO_ST25.txt; created July 20, 2020, size 123 KB.

ESSENCE OF THE INVENTION

[0005] Several embodiments provide various methods for enhancing the expansion of immune cells for use in cellular immunotherapy. For example, several embodiments provide a method in which immune cells are co-cultured with a feeder cell line in a medium enriched with one or more soluble cytokines, wherein the cytokines are added to the medium at least once during the co-culture. In several embodiments, the immune cells are NK cells. In several embodiments, the expanded NK cells are unexpectedly amenable to cell engineering, such as engineering the cells to express a chimeric receptor (e.g., for use in cancer immunotherapy). In several embodiments, NK cells (or other immune cells) co-cultured with a medium enriched with a soluble interleukin express such chimeric receptors more robustly than NK cells not co-cultured in a medium enriched with a soluble interleukin. In addition, in several embodiments, the engineered NK cells exhibit unexpectedly enhanced cytotoxicity.

[0006] In several embodiments, a method for enhancing the expansion of natural killer cells for use in immunotherapy is provided, comprising co-culturing in a culture medium a population of natural killer (NK) cells with a population of feeder cells, enriching the culture medium with interleukin-2 (IL2), and enriching the culture medium with at least one soluble stimulatory agent selected from interleukin-12 (IL12), interleukin-18 (IL18), interleukin-21 (IL21), or a combination thereof. In several embodiments, the population of feeder cells comprises cells engineered to express 4-1BBL and membrane-bound interleukin-15 (mbIL15);

[0007] In several embodiments, methods are provided for enhancing the cytotoxicity of natural killer (NK) cells, comprising contacting NK cells with a nucleic acid encoding a chimeric antigen receptor (CAR) to cause the NK cells to express the CAR, co-cultivating in a culture medium a population of NK cells with a population of feeder cells, enriching the culture medium with interleukin-2, enriching the culture medium with at least one soluble stimulatory agent, wherein the soluble stimulatory agent is selected from interleukin-12, interleukin-18, interleukin-21, and combinations thereof, wherein enriching the medium with at least one soluble stimulatory agent results in enhanced cytotoxicity in the CAR-expressing NK cells compared to NK cells co-cultured with the feeder cells in the absence of the at least one soluble stimulatory agent.

[0008] In some embodiments, enriching the medium with at least one soluble stimulant results in increased NK cell expansion compared to co-culturing the NK cells with feeder cells in the absence of the at least one soluble stimulant.

[0009] In some embodiments, enriching the medium with at least one soluble stimulatory agent results in enhanced NK cell expansion compared to co-culturing the NK cells with feeder cells in the absence of the at least one soluble stimulatory agent. In some embodiments, one or more additional characteristics of the NK cells are enhanced, such as, for example, activity (e.g., cytotoxicity against a target cell or cells), lifespan (either in culture or in vivo), potency (e.g., activity or duration of activity is enhanced), etc. For example, in some embodiments, the culturing methods result in enhanced one or more of persistence and/or cytotoxicity of the NK cells compared to the persistence and/or cytotoxicity of NK cells co-cultivated with feeder cells in the absence of the at least one soluble stimulatory agent. In some embodiments, the obtained NK cells exhibit a memory cell-like phenotype characterized by (i) increased expression of NKG2C in NK cells and/or (ii) decreased or equivalent expression of CD62 ligand in NK cells, wherein the expression in both (i) and (ii) is compared to NK cells cultured under the same conditions but without one or more soluble stimulatory molecules. Preferably, in some embodiments, upon administration to a subject, the obtained NK cells exhibit reduced signs of cytokine withdrawal compared to NK cells cultured in a medium containing at least one soluble stimulatory agent but lacking feeder cells. This is in contrast to other methods of NK cell expansion that result in NK cells that exhibit a dependence on high concentrations of the cytokines applied. In such methods, NK cells exhibit reduced viability when removed from culture conditions, such as when administered to a patient, which may limit the utility and/or effectiveness of such cells in killing tumor cells.

[0010] In some embodiments, the soluble stimulant used to enrich the medium is a combination of IL12 and IL18. In some embodiments, when IL12 and IL18 are used in combination, IL21 is not used. In some embodiments, IL21 is not used. In some embodiments, the concentration of at least one soluble stimulant is from 0.01 ng/mL to 50 ng/mL at a time within 1, 2, 4, 6, 8, 10, 12, 16, 18, 20, or 24 hours of the start of co-cultivation. In some embodiments, the concentration of at least one soluble stimulatory agent is from 0.01 ng/mL to 30 ng/mL at a time within 1, 2, 4, 6, 8, 10, 12, 16, 18, 20, or 24 hours of the start of co-cultivation. In some embodiments, the concentration of at least one soluble stimulatory agent is from 0.01 ng/mL to 50 ng/mL at a time within 120 hours of the start of co-cultivation. In some embodiments, the at least one stimulatory agent comprises soluble IL12 at a concentration of less than 10 ng/mL at a time within 1, 2, 4, 6, 8, 10, 12, 16, 18, 20, or 24 hours of the start of co-cultivation. In some embodiments, the at least one stimulatory agent comprises soluble IL18 at a concentration of less than 50 ng/mL at a time within 24 hours of the start of co-cultivation. In some embodiments, the concentration of the at least one soluble stimulatory agent is from 0.01 ng/mL to 30 ng/mL at a time within 120 hours of the start of co-cultivation. In some embodiments, the at least one stimulatory agent comprises soluble IL12 at a concentration of less than 10 ng/mL at a time within 120 hours of the start of co-cultivation. In some embodiments, the at least one stimulatory agent comprises soluble IL18 at a concentration of less than 50 ng/mL at a time within 120 hours of the start of co-cultivation. In several embodiments, the at least one stimulatory agent comprises (i) soluble IL12 at a concentration of 0.01 ng/ml to 8 ng/ml and (ii) soluble IL18 at a concentration of 0.01 ng/ml to 30 ng/ml, and wherein the culture medium is enriched a second time with interleukin-2 at a concentration that exceeds that during the first enrichment of the culture medium with IL2, wherein said concentrations are present at a time point within 1, 2, 4, 6, 8, 10, 12, 16, 18, 20, or 24 hours from the start of the co-cultivation. In several embodiments, the at least one stimulatory agent comprises (i) soluble IL12 at a concentration of 0.01 ng/mL to 8 ng/mL and (ii) soluble IL18 at a concentration of 0.01 ng/mL to 30 ng/mL, and wherein the culture medium is enriched a second time with IL2 at a concentration that exceeds the concentration of IL2 in the culture medium enriched a first time, wherein said concentrations are present at a time point within 120 hours of the start of co-cultivation.

[0011] In some embodiments, the feeder cell population comprises K562 cells. In some embodiments, the feeder cell population is not the 721.221 cell line. In some embodiments, the feeder cells (e.g., the K562 cells) are irradiated prior to co-culture. In some embodiments, the feeder cells (e.g., the K562 cells) express both 4-1BBL and mbIL15. In some embodiments, the feeder cells (e.g., the K562 cells) express both 4-1BBL and mbIL15 and are irradiated prior to co-culture.

[0012] According to several embodiments, the at least one stimulatory agent comprises (i) soluble IL12 at a concentration of 0.01 ng/mL to 8 ng/mL and (ii) soluble IL18 at a concentration of 0.01 ng/mL to 30 ng/mL. In several embodiments where IL12 is used, IL12 is added to the cell culture medium at a concentration of less than 7 ng/mL. In several embodiments where IL18 is used, IL18 is added to the cell culture medium at a concentration of less than 40 ng/mL. In several embodiments, when multiple stimulatory cytokines are used, the concentration of IL12 is less than 7 ng/mL, the concentration of IL18 is less than 40 ng/mL. In some such embodiments, IL2 is present at an initial concentration and additional IL2 is added later. In some such embodiments, the initial concentration of IL2 is from 50 IU/mL to 500 IU/mL. In some embodiments, the medium is first enriched with IL2 to a concentration of less than 500 IU/mL. In further embodiments, the medium is first enriched with IL2 to a concentration of less than 50 IU/mL. In some embodiments, the initial concentration of IL2 is less than 50 IU/mL. In some embodiments, the medium is later enriched with additional IL2 to a concentration of less than 500 IU/mL.

[0013] In some embodiments, the concentration of the at least one soluble stimulatory agent is between 0.01 ng/mL and 50 ng/mL at a time point within 120 hours of said co-culture. In some embodiments, the feeder cell population comprises cells engineered to express 4-1BBL and membrane-bound IL-15 (mbIL15). In some embodiments, the at least one soluble stimulatory agent comprises a combination of said interleukin-12 and said interleukin-18. In some embodiments, the concentration of the at least one soluble stimulatory agent is between 0.01 ng/mL and 30 ng/mL at a time point within 120 hours of co-culture. In some embodiments, the at least one stimulatory agent comprises soluble IL12 at a concentration of less than 10 ng/mL at a time point within 120 hours of co-culture. In some embodiments, the at least one stimulatory agent comprises soluble IL18 at a concentration of less than 50 ng/mL at a time point within 120 hours of co-culture. In some embodiments, the at least one stimulatory agent comprises (i) soluble IL12 at a concentration of 0.01 ng/mL to 8 ng/mL and (ii) soluble IL18 at a concentration of 0.01 ng/mL to 30 ng/mL, and wherein the culture medium is enriched a second time with interleukin-2 at a concentration that exceeds the concentration of IL2 in the culture medium enriched a first time, wherein each concentration is present at a time point within 120 hours of co-culture. In some embodiments, the methods described herein further comprise enriching the medium with an additional amount of the at least one of the soluble stimulatory agents. In some embodiments, enriching the medium a second time is performed between 12 hours and 120 hours from enriching the first time. In further embodiments, further enrichment of the medium is performed at later time points. In some embodiments, the concentrations of the soluble agents, such as IL12 and/or IL18, are the same at a first time point as at a corresponding second time point. In some embodiments, their subsequent concentrations are different (e.g., greater).

[0014] In several embodiments, a population of engineered natural killer cells is provided that comprises an engineered chimeric receptor configured to bind a marker on a target cancer cell and, upon binding, induce an NK cell to develop a cytotoxic effect against the target cancer cell, wherein the NK cell has been expanded in culture in the presence of at least one soluble stimulatory agent, wherein the soluble stimulatory agent is selected from interleukin-12, interleukin-18, interleukin-21, and combinations thereof, and wherein the population of engineered NK cells at least in part exhibits a memory cell-like phenotype characterized by (i) increased expression of NKG2C in the NK cells and/or (ii) decreased or equivalent expression of CD62 ligand in the NK cells, wherein the expression in both (i) and (ii) compared to NK cells cultured under the same conditions but without the soluble stimulating agent.

[0015] In several embodiments, the engineered chimeric receptor is encoded by a sequence that is at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. In several embodiments, the engineered chimeric receptor has an amino acid sequence that is at least 85%, 90%, 95%, 86%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28.

[0016] In some embodiments, the methods further comprise contacting the NK cells with a vector encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR is configured to target one or more of CD19, CD 123, CD70, BCMA, or natural killer cell receptor group D (NKG2D) ligand. In some embodiments, the CAR does not include a DAP 10 or DAP12 subdomain.

[0017] In several embodiments, NK cells obtained by the methods disclosed herein are used in the production of a medicament for treating cancer. In several embodiments, NK cells obtained by the methods disclosed herein are for treating cancer. Also provided are methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of engineered NK cells expanded using any of the methods disclosed herein.

[0018] In several embodiments, a cell propagation culture medium is also provided, wherein the culture medium comprises IL2 provided at a concentration of less than 500 IU/mL; IL12 provided at a concentration of less than 10 ng/mL; and IL18 provided at a concentration of 30 ng/mL.

[0019] In several embodiments, a combination culture medium for cell expansion is also provided, wherein the combination comprises IL2 provided at a concentration of less than 500 IU/mL, IL12 provided at a concentration of less than 10 ng/mL, IL18 provided at a concentration of 30 ng/mL, and IL15 that is associated with the surface of a cell membrane (mbIL15). In several embodiments, mbIL15 is associated with the surface of a cell membrane of a feeder cell. In several embodiments, the culture medium and/or the combination culture medium further comprise at least one amino acid, at least one inorganic salt, and at least one vitamin.

[0020] In several embodiments, a method for enhancing the expansion of natural killer cells for use in immunotherapy is provided, comprising co-culturing in a culture medium a population of natural killer (NK) cells with a population of feeder cells, enriching the culture medium at a first time with at least one soluble stimulatory agent, wherein the soluble stimulatory agent is selected from interleukin-12, interleukin-18, interleukin-21, and combinations thereof, and enriching the culture medium at a second time with an additional amount of at least one of the soluble stimulatory agents. In several embodiments, the NK are co-cultured with the feeder cells for a second period of time. In several embodiments, enriching the medium with at least one soluble stimulatory agent results in enhanced expansion of the NK cells compared to co-culturing the NK cells with the feeder cells in the absence of the at least one soluble stimulatory agent.

[0021] In some embodiments, the concentration of the at least one soluble stimulating agent is between 0.01 ng/mL and 100 ng/mL. In some embodiments, the feeder cell population comprises cells engineered to express one or more of 4-1BBL and membrane-bound IL-15. In some embodiments, the method also involves enriching the culture medium with interleukin-2. In some embodiments, the first and second time points are separated by more than 12 hours and less than 120 hours. In some embodiments, the concentrations provided herein are the final concentrations of the subject molecule or agent in the culture medium. In some embodiments, the concentrations provided herein are the concentrations of the molecule or agent at dilution (if applicable) prior to addition to a given volume of medium. In some embodiments, the concentration is present at a time point within 12, 24, 72, or 120 hours. In some embodiments, when more than one agent is used, the concentration of each agent is from 0.01 ng/mL to 100 ng/mL or from 1 IU/mL to 1000 IU/mL (and, for example, is present at a time within 12, 24, 72, or 120 hours). In other embodiments, when more than one agent is used, the concentration of all agents is from 0.01 ng/mL to 100 ng/mL or from 1 IU/mL to 1000 IU/mL (and, for example, is present at a time within 12, 24, 72, or 120 hours).

[0022] In several embodiments, the at least one soluble stimulatory agent comprises a combination of IL12 and IL18. In several embodiments, the first time point is at the beginning of the co-culture of the NK cells with the feeder cell, or where the second time point is at the beginning of the second period of time. In several embodiments, the first time point and the second time point are separated by 24 to 120 hours, and the concentration of the stimulatory agent is from 0.01 ng/mL to 30 ng/mL

[0023] In some embodiments, the at least one stimulatory agent comprises (i) soluble IL12 at a concentration of 10 ng/mL to 30 ng/mL and (ii) soluble IL18 at a concentration of 0.01 ng/mL to 30 ng/mL. In some embodiments, the at least one stimulatory agent comprises (i) soluble IL12 at a concentration of 0.01 ng/mL to 10 ng/mL and (ii) soluble IL18 at a concentration of 0.01 ng/mL to 30 ng/mL. In some embodiments, the concentration of each of soluble IL12 and soluble IL18 is the same at a first time point as at a respective second time point. In some embodiments, the concentration of each of soluble IL12 and soluble IL18 is different at a first time point as at a respective second time point. In some embodiments, the concentrations of soluble IL12 and soluble IL18 are equivalent to each other.

[0024] In some embodiments, the method also includes transducing the expanded NK cells with a nucleic acid construct encoding a chimeric receptor, wherein expression of the chimeric receptor is enhanced relative to expression of the chimeric receptor on NK cells co-cultured with feeder cells in the absence of at least one soluble stimulant. In some embodiments, the cytotoxic activity of the chimeric receptor is unexpectedly enhanced relative to the cytotoxic activity of the chimeric receptor on NK cells co-cultured with feeder cells in the absence of at least one soluble stimulant.

[0025] This document also provides the use of NK cells expanded using the method disclosed herein for the treatment of cancer and/or for the production of a medicament for the treatment of cancer.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0026] The descriptions of the figures below refer to experiments and results that represent non-limiting embodiments of the present invention disclosed herein.

[0027] Figs. 1A and 1B depict a non-limiting example of an expansion protocol used to enhance the expansion of NK cells according to the embodiments disclosed herein.

[0028] Fig. 2 depicts data comparing the NK cell expansion using various expansion methods, including non-limiting embodiments of the methods disclosed herein.

[0029] Figures 3A-3B depict data relating to the expansion of NK cells obtained from four different donors under various conditions. Figure 3A shows flow cytometric data measuring NKG2D expression on the surface of NK cells when expanded with feeder cells alone (top row) or using cytokine enrichment (bottom row). Figure 3B shows the measurement of mean fluorescence intensity (representative of transduction by a chimeric receptor construct (NKX101) carrying NKG2D) under various conditions.

[0030] Figure 4 shows data relating to NK cell cytotoxicity at various time points after the onset of expansion under conditions using feeder cells alone or with cytokine enrichment.

[0031] Figs. 5A-5B depict data relating to the expression of certain markers characteristic of the memory cell phenotype in NK cells.

[0032] Fig. 6 shows in vivo data relating to the antitumor activity of NK cells expanded with or without the indicated cytokine stimulation during expansion.

[0033] Figs. 7A-7B relate to NK cell expansion under various conditions. Fig. 7A shows various concentrations determined to be supersaturating, saturating, or subsaturating for IL12/18. Fig. 7B shows NK cell proliferation data under various culture conditions.

[0034] Fig. 8 shows data relating to the release of interferon gamma from NK cells cultured with varying concentrations of IL12 and/or IL18 in the culture medium.

[0035] Figs. 9A-9H relate to the assessment of NK cell expansion after seven days of culture under the indicated conditions. Fig. 9A shows the summary data for each of the culture groups. Fig. 9B shows the statistical comparisons between the groups. Fig. 9C shows the fold expansion data (at day 7) for a specific titration data set including different concentrations of IL12 with IL18 at 4 ng/mL. Fig. 9D shows similar data with IL18 at 20 ng/mL. Fig. 9E shows the viability of engineered NK cells at day 7 of culture with 20 ng/mL IL18, 40 IU/mL IL-2, and the indicated concentrations of IL12. Fig. 9F shows the viability of engineered NK cells on day 8 in culture with 20 ng/ml IL18, 400 IU/ml IL-2, and the indicated concentrations of IL12. Fig. 9G shows the viability of engineered NK cells on day 7 in culture with 4 ng/ml IL18, 40 IU/ml IL-2, and the indicated concentrations of IL12. Fig. 9H shows the viability of engineered NK cells on day 8 in culture with 4 ng/ml IL18, 400 IU/ml IL-2, and the indicated concentrations of IL12.

[0036] Figs. 10A-10B relate to the assessment of NK cell cytotoxicity. Fig. 10A shows the summary data on NK cell cytotoxicity in each of the culture groups after 8 days of culture. Fig. 10B shows the statistical comparisons of cytotoxicity.

[0037] Figs. 11A-11B relate to the evaluation of NK cell cytotoxicity. Fig. 11A shows the summary data on NK cell cytotoxicity in each of the culture groups after 15 days of culture. Fig. 11B shows the statistical comparisons of cytotoxicity.

[0038] Fig. 12 shows expression data for NK cells obtained from two donors that were transduced with the chimeric receptor construct and cultured under different conditions.

[0039] Fig. 13 shows expression data for NK cells obtained from two additional donors that were transduced with the chimeric receptor construct and cultured under different conditions.

[0040] Figs. 14A-14B show cytotoxicity data. Fig. 14A shows summary data relating to the cytotoxicity of NK cells transduced with a chimeric receptor targeting NKG2D ligands and cultured under the indicated conditions. Fig. 14B shows statistical comparisons between groups.

[0041] Figs. 15A-15D relate to the cytotoxic effects of NK cells transduced with a chimeric receptor targeting NKG2D after culture under the indicated conditions. Figs. 15A and 15B show cytotoxicity data for NK cells obtained from two different donors 13 days after transduction with either a vector encoding GFP or a vector encoding a chimeric receptor targeting NKG2D ligands. Figs. 15C and 15D show the corresponding cytotoxicity data for cells from the same two donors at day 21 after transduction.

[0042] Figs. 16A-16B show data relating to the phenotype of NK cells. Fig. 16A shows data relating to the expression of markers associated with a memory cell-like phenotype in NK cells over time under the indicated culture conditions. Fig. 16B shows flow cytometric data showing the progression of marker expression over time in culture.

[0043] Figures 17A-17D show summary expression data related to selected markers in NK cells under various culture conditions. Figure 17A shows expression data related to CD62 ligand, Figure 17B shows NKG2C expression, Figure 17C shows CD57 expression, and Figure 17D shows both CD62L and NKG2C expression.

[0044] Fig. 18 shows cytotoxicity data for NK cells expressing either GFP and/or the chimeric receptor targeting NKG2D ligand at day 21 post-transduction.

[0045] Fig. 19 shows cell viability and proliferation data for NK cells grown under varying culture conditions.

[0046] Fig. 20 shows expression data (based on the Flag tag) for NK cells transduced with a CD19-binding CAR and cultured using the indicated conditions. These data were collected on day 15 of expansion.

[0047] Fig. 21 shows expression data (based on the Flag tag) for NK cells transduced with the CB19-binding CAR and cultured using the indicated conditions. These data were collected on day 22 of expansion.

[0048] Figures 22A-22C show data relating to the cytotoxicity of NK cells expressing the CB19-binding CAR. NK cells were expanded using the indicated conditions and tested with Nalm6 cells using the E:T ratios indicated in Figure 22A (average of 3 donors). Figure 22B shows the pooled cytotoxicity data. Figure 22C shows the cytotoxicity data as a function of the effector to target ratio.

[0049] Fig. 23 shows a schematic of the experimental conditions for assessing the cytotoxicity of NK cells expressing a chimeric receptor targeting NKG2D ligands in a hepatocellular carcinoma xenograft model.

[0050] Figure 24 shows a summary of tumor burden in mice for the indicated treatments over time.

[0051] Fig. 25 shows a schematic of the experimental conditions for assessing the effect of culture conditions during expansion on NK cell cytotoxicity in vivo.

[0052] Figures 26A-26F show cytotoxicity, survival data, NK cell persistence data, and CAR expression data in fresh or cryopreserved NK cells. Figure 26A shows cytotoxicity data of NK cells expanded under the indicated conditions against Nalm6 cells in a xenograft model. Figure 26B shows the survival curve for mice receiving the indicated treatments. Figure 26C shows human NK cell detection data in mouse blood 18 days post injection, stratified based on expansion culture conditions. Figure 26D shows CAR-positive NK cell detection data in mouse blood 18 days post injection, stratified based on expansion culture conditions. Figure 26E shows expression data relating to the percentage of NK cells (either fresh or cryopreserved) expressing a non-limiting embodiment of a CD19-binding CAR at day 15 of expansion and in the presence or absence of additional stimulatory molecules. Fig. 26F shows expression data relating to the percentage of NK cells (either fresh or cryopreserved) expressing a non-limiting embodiment of a CD19-binding CAR at day 22 of expansion and in the presence or absence of additional stimulatory molecules.

[0053] Figs. 27A-27C relate to the in vivo efficacy of various CD19-targeted CARs according to embodiments disclosed herein. Fig. 27A shows a schematic diagram of an experimental protocol for evaluating the in vivo efficacy of humanized NK cells expressing various CD19-targeted CAR constructs. The various experimental groups tested are indicated. For cells designated "IL12/IL18," the cells were expanded in the presence of soluble IL12 and/or IL18 according to embodiments disclosed herein. Figs. 27B and 27C show bioluminescence data from animals dosed with Nalm6 tumor cells and treated with the construct.

[0054] FIGS. 28A-28J are graphical representations of the bioluminescence data shown in FIGS. 27B-27C. FIG. 28A shows the bioluminescence (as photon flux per second) from animals receiving untransduced NK cells. FIG. 28B shows the flux measured in animals receiving PBS as the vehicle. FIG. 28C shows the flux measured in animals receiving pre-frozen NK cells expressing the NK19 NF2 CAR (as a non-limiting example of a CAR). FIG. 28D shows the flux measured in animals receiving pre-frozen NK cells expressing the NK19 NF2 CAR (as a non-limiting example of a CAR) expanded with IL12 and/or IL18. FIGS. 28E and FIG. 28F shows the flux measured in animals receiving fresh NK cells expressing the NK19 NF2 CAR (as a non-limiting example of a CAR). Fig. 28G and Fig. 28H show the flux measured in animals receiving fresh NK cells expressing the NK19 NF2 CAR (as a non-limiting example of a CAR) expanded with IL12 and/or IL18. Fig. 28I shows a line graph depicting the bioluminescence measured in different groups over the first 30 days after tumor inoculation. Fig. 28J shows a line graph depicting the bioluminescence measured in different groups over the first 56 days after tumor inoculation.

[0055] Fig. 29 shows data relating to body weight of mice over time while receiving the indicated therapy.

[0056] Figs. 30A-30C show data relating to data characterizing NK cells engineered to express CARs (disclosed herein) and expanded in the presence or absence of one or more stimulatory cytokines. Fig. 30A shows data relating to the percentage of NK cells expressing CARs in the blood of animals over time. Fig. 30B shows data relating to the percentage of NK cells expressing CARs in the blood of animals over a period of 50 days. Fig. 30C shows data relating to the percentage of NK cells expressing CARs over time and based on the number of live cells tested.

[0057] Figs. 31A-31C show data obtained from three different mice (31A, 31B, and 31C, respectively) relating to the expression of CD19-binding CAR and determination of which cells express the CAR.

[0058] Figs. 32A-32C show data obtained from three different mice (32A, 32B, and 32C, respectively) relating to the expression of CD19-binding CAR and determination of which cells express the CAR.

[0059] Figures 33A-33C show summary expression data obtained for blood samples collected 4 days after in vivo administration (protocol in Figure 27A). Figure 33A shows the percentage of CD3-CD56 ⁺ NK cells obtained from whole blood samples for the indicated experimental groups. Figure 33B shows the percentage of NK cells expressing a specific CD19-binding CAR for each experimental group. Figure 33C shows data relating to the number of GFP-positive cancer cells detected for each experimental group.

[0060] Figures 34A-34C show summary expression data obtained for blood samples collected 12 days after in vivo administration (protocol in Figure 27A). Figure 34A shows the percentage of CD3-CD56 ⁺ NK cells obtained from whole blood samples for the indicated experimental groups. Figure 34B shows the percentage of NK cells expressing a specific CD19-binding CAR for each experimental group. Figure 34C shows data relating to the number of GFP-positive cancer cells detected for each experimental group.

[0061] Figures 35A-35E show summary expression data obtained for blood samples collected 18 days after in vivo administration (protocol in Figure 27A). Figure 35A shows the percentage of CD3-CD56 ⁺ NK cells obtained from whole blood samples for the indicated experimental groups. Figure 35B shows the percentage of CD19-positive tumor cells for each experimental group as measured using a phycoerythrin (PE)-conjugated antibody. Figure 35C shows data relating to the number of GFP-positive cancer cells detected for each experimental group. Figure 35D shows the percentage of NK cells expressing a specific CD19-binding CAR for each experimental group as measured using an anti-CD19-FC antibody. Figure 35E shows the percentage of NK cells expressing CD19-binding CAR in each treatment group.

[0062] Fig. 36 shows data collected over 4 weeks regarding the half-life of NK cells expressing the CD19-binding CAR for each of two doses of NK cells, as measured by the number of NK cells per 10,000 white blood cells. The two doses represented (i) 2 million NK cells expressing the CD19-binding CAR and (ii) 5 million NK cells expressing the CD19-binding CAR. These data were collected after administration of the third dose of NK cells.

[0063] Fig. 37 shows data collected to determine the half-life of cryopreserved NK cells engineered to express a CAR targeting NKG2D ligands and expanded without the use of an additional stimulatory cytokine.

DETAILED DESCRIPTION

[0064] While cancer immunotherapy or cell therapy for other diseases has advanced greatly in terms of the ability to engineer cells to express structures of interest, there is still a need for a clinically relevant amount of such cells to be administered to a patient. This is especially important when the intended native immune cell to be engineered and subsequently administered is less abundant than other types of immune cells. This requires either starting with a larger amount of starting material, which may be impractical, or developing more efficient methods and compositions for expanding (in some cases, preferentially) the immune cell of interest, such as an NK cell. Accordingly, in several embodiments, provided herein are methods and compositions that advantageously provide the ability to enhance the expansion of NK cells (or other immune cells), but also provide the ability to enhance the cytotoxicity of such cells.

[0065] Several embodiments provide populations of expanded and activated NK cells obtained by co-cultivating a modified feeder cell disclosed herein with a parent population of immune cells and enriching the co-culture with various cytokines at specific time points during expansion.

Cells for use in immune cell expansion

[0066] In some embodiments, cell lines are used in co-culture with the immune cell population to be expanded. Such cell lines are referred to herein as "stimulator cells," which may also be referred to as "feeder cells." In some embodiments, the entire immune cell population is expanded, while in some embodiments, a selected subpopulation of immune cells is expanded. For example, in some embodiments, NK cells are expanded relative to other immune cell subpopulations (such as T cells). In other embodiments, both NK cells and T cells are expanded. In some embodiments, the feeder cells themselves are genetically modified. In some embodiments, the feeder cells do not express MHC class I molecules that have an inhibitory effect on NK cells. In some embodiments, the feeder cells do not necessarily completely lack MHC class I molecule expression, but may express MHC class I molecules at a lower level than a wild-type cell. For example, in some embodiments, if a wild-type cell expresses an MHC molecule at a level of X, the cell lines used may express the MHC molecule at a level of less than 95% of X, less than 90% of X, less than 85% of X, less than 80% of X, less than 70% of X, less than 50% of X, less than 25% of X, and at any expression level between (and including) the above. In some embodiments, the stimulating cells are immortalized, such as a cancer cell line. However, in some embodiments, the stimulating cells are primary cells.

[0067] Depending on the embodiment, various types of cells can be used as feeder cells. These include, but are not limited to, K562 cells, certain Wilms tumor cell lines (e.g., the HFWT Wilms tumor cell line), endometrial tumor cells (e.g., HHUA), melanoma cells (e.g., HMV-II), hepatoblastoma cells (e.g., HuH-6), small cell lung carcinoma cells (e.g., Lu-130 and Lu-134-A), neuroblastoma cells (e.g., NB19 and NB69), testicular embryonal carcinoma cells (e.g., NEC14), cervical carcinoma cells (TCO-2), neuroblastoma cells (e.g., TNB1), EBV-transformed B cell line, 721.221, among others.

[0068] In additional embodiments, the feeder cells also have reduced (or absent) expression of MHC class II molecules, as well as reduced (or absent) expression of MHC class I molecules. In some embodiments, other cell lines that can natively express MHC class I molecules can be used in combination with genetic modification of such cells to reduce or knock out the expression of MHC class I molecules. Genetic modification can be accomplished through the use of gene editing techniques (e.g., the crispr/cas system; RNA editing using adenosine deaminases acting on RNA (ADARs), zinc finger domains, TALENs, etc.), inhibitory RNA (e.g., siRNA), or other molecular methods to disrupt and/or reduce the expression of MHC class I molecules on the surface of cells.

[0069] As discussed in more detail below, in several embodiments, feeder cells are engineered to express certain stimulatory molecules (e.g., interleukins, CD3, 4-1BBL, etc.) to promote the expansion and activation of immune cells. Engineered feeder cells are disclosed, for example, in International Patent Application PCT/SG2018/050138, which is incorporated herein by reference in its entirety. In several embodiments, stimulatory molecules, such as interleukin-12, 18, and/or 21, are added to the co-culture medium individually, such as at specific times and in specific amounts, to effect an enhancement of the expansion of the desired immune cell subpopulation(s).

Stimulating molecules

[0070] As discussed briefly above, certain molecules promote the expansion of immune cells, such as NK cells or T cells, including engineered NK or T cells. Depending on the embodiment, the stimulatory molecule or molecules can be expressed on the surface of feeder cells used to expand the immune cell population. For example, in several embodiments, the K562 feeder cell population is engineered to express 4-1BBL and/or membrane-bound interleukin-15 (mbIL15). Additional embodiments relate to additional membrane-bound interleukins or stimulatory agents. Examples of such additional membrane-bound stimulatory molecules can be found in International Patent Application PCT/SG2018/050138, which is incorporated herein by reference in its entirety.

[0071] In some embodiments, the methods disclosed herein relate to adding one or more stimulatory molecules to a culture medium in which engineered feeder cells and engineered NK cells are co-cultured. In some embodiments, one or more interleukins are added. For example, in some embodiments, IL2 is added to the medium. In some embodiments, IL12 is added to the medium. In some embodiments, IL18 is added to the medium. In some embodiments, IL21 is added to the medium. In some embodiments, combinations of two or more of IL2, IL12, IL18, and/or IL21 are added to the medium. In some embodiments, instead of using a feeder cell with mbIL15, soluble IL15 (alone or in combination with any of IL2, IL12, IL18, and IL21) is added to the medium.

[0072] In some embodiments, the medium comprises one or more vitamins, inorganic salts, and/or amino acids. In some embodiments, the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of glycine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine (e.g., L-cysteine 2HCl), L-glutamic acid, L-glutamine, L-histidine, L-hydroxyproline, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine (e.g., disodium L-tyrosine dihydrate), and L-valine. In some embodiments, the medium comprises 1, 2, 3, 4 or more of biotin, choline chloride, calcium D-patotenate, folic acid, i-inositol, niacinamide, para-aminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and vitamin B12. In some embodiments, the medium comprises 1, 2, 3, 4 or more _of calcium nitrate (Ca( _NO3 ) _24H2O ), magnesium sulfate ( _MgSO4 ) (e.g., magnesium sulfate ( _MgSO4 ) (anhydrous)), potassium chloride (KCl), sodium bicarbonate ( _NaHCO3 ), sodium chloride (NaCl), and dibasic sodium phosphate _{( Na2HPO4} ) (e.g., dibasic sodium phosphate ( _Na2HPO4 ) _anhydrous ).

[0073] In some embodiments, the medium further comprises D-glucose and/or glutathione (optionally reduced glutathione). In some embodiments, the medium further comprises blood serum (e.g., fetal bovine serum) in an amount ranging from about 1% to about 20%. In some embodiments, the serum is heat-inactivated. In some embodiments, the medium does not comprise blood serum. In some embodiments, the medium does not comprise foreign elements.

[0074] Depending on the embodiment, IL2 is used to enrich the culture medium and enhance the proliferation or other characteristics of NK cells. In some embodiments, the concentration of IL2 used is in the range of about 1 IU/mL to about 1000 IU/mL, including, for example, about 1 IU/mL to about 5 IU/mL (e.g., 1, 2, 3, 4, and 5), about 5 IU/mL to about 10 IU/mL (e.g., 5, 6, 7, 8, 9, and 10), about 10 IU/mL to about 20 IU/mL (e.g., about 10, 12, 14, 16, 18, and 20), about 20 IU/mL to about 30 IU/mL (e.g., about 20, 22, 24, 26, 28, and 30), about 30 IU/mL to about 40 IU/mL (e.g., 30, 32, 34, 36, 38 and 40), from about 40 to about 50 IU/mL (e.g., 40, 42, 44, 46, 48, 50), from about 50 IU/mL to about 75 IU/mL (e.g., 50, 55, 60, 65, 70 and 75), from about 75 IU/mL to about 100 IU/mL (e.g., 75, 80, 85, 90, 95 and 100), from about 100 IU/mL to about 200 IU/mL (e.g., 100, 125, 150, 175 and 200), from about 200 IU/mL to about 300 IU/mL (e.g., 200, 225, 250, 275 and 300), from about 300 IU/mL to about 400 IU/mL (e.g., 300, 325, 350, 375 and 400), from about 400 IU/mL to about 500 IU/mL (e.g., 400, 425, 450, 475 and 500), from about 500 IU/mL to about 750 IU/mL (e.g., 500, 550, 600, 650, 700 and 750) or from about 750 IU/mL to about 1000 IU/mL (e.g., 750, 800, 850, 900, 950 and 1000) and any concentrations therebetween, including cutoff points. In some embodiments, IL2 may be added at multiple time points during the culturing. In some such embodiments, the concentration of IL2 applied at the selected time points is different.

[0075] Depending on the embodiment, IL12A and/or IL12B are used to enrich the culture medium and enhance the expansion or other characteristics of NK cells. In some embodiments, the concentration of IL12 (either IL12A or IL12B) used is in the range of from about 0.01 ng/mL to about 100 ng/mL, including, for example, from about 0.01 ng/mL to about 0.05 ng/mL (e.g., 0.01, 0.02, 0.03, 0.04, and 0.05), from about 0.05 ng/mL to about 0.1 ng/mL (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1), from about 0.1 ng/mL to about 0.5 ng/mL (e.g., 0.1, 0.2, 0.3, 0.4, and 0.5), from about 0.5 ng/mL to about 1.0 ng/mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0), from about 1.0 ng/mL to about 2.0 ng/mL (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0), from about 2.0 ng/mL to about 5.0 ng/mL (e.g., 2.0, 3.0, 4.0, and 5.0), from about 5.0 ng/mL to about 10.0 ng/mL (e.g., 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0), from about 10.0 ng/mL to about 15.0 ng/mL (e.g., 10.0, 11.0, 12.0, 13.0, 14.0 and 15.0), from about 15.0 ng/mL to about 20.0 ng/mL (e.g., 15.0, 16.0, 17.0, 18.0, 19.0 and 20.0), from about 20.0 ng/mL to about 25.0 ng/mL (e.g., 20.0, 21.0, 22.0, 23.0, 24.0 and 25.0), from about 25.0 ng/mL to about 30.0 ng/mL (e.g., 25.0, 26.0, 27.0, 28.0, 29.0 and 30.0), from about 30.0 ng/mL to about 50.0 ng/mL (e.g., 30.0, 35.0, 40.0, 45.0, and 50.0), from about 50.0 ng/mL to about 75.0 ng/mL (e.g., 50.0, 55.0, 60.0, 65.0, 70.0, and 75.0), from about 75.0 ng/mL to about 100.0 ng/mL (e.g., 75.0, 80.0, 85.0, 90.0, 95.0, and 100.0), and any concentrations in between, including cutoff points. In some embodiments, the IL12 concentration is from about 0.01 ng/mL to about 8 ng/mL, including any concentrations in between, including cutoff points.

[0076] In some embodiments, a mixture of IL12A and IL12B is used. In some embodiments, a specific ratio of IL12A:IL12B is used, such as 1:10, 1:50, 1:100, 1:150, 1:200, 1:250:, 1:500, 1:1000, 1:10000, 10000:1, 1000:1, 500:1, 250:1, 150:1, 100:1, 10:1, and any ratio in between, including cutoff points.

[0077] In some embodiments, interleukin-18 (IL18) is used to enhance the proliferation or other characteristics of NK cells. In some embodiments, the concentration of IL18 used is in the range of about 0.01 ng/mL to about 100 ng/mL, including, for example, about 0.01 ng/mL to about 0.05 ng/mL (e.g., 0.01, 0.02, 0.03, 0.04, and 0.05), about 0.05 ng/mL to about 0.1 ng/mL (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1), about 0.1 ng/mL to about 0.5 ng/mL (e.g., 0.1, 0.2, 0.3, 0.4, and 0.5), about 0.5 ng/mL to about 1.0 ng/mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0), from about 1.0 ng/mL to about 2.0 ng/mL (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0), from about 2.0 ng/mL to about 5.0 ng/mL (e.g., 2.0, 3.0, 4.0 and 5.0), from about 5.0 ng/mL to about 10.0 ng/mL (e.g., 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0), from about 10.0 ng/mL to about 15.0 ng/mL (e.g., 10.0, 11.0, 12.0, 13.0, 14.0 and 15.0), from about 15.0 ng/mL to about 20.0 ng/mL (e.g., 15.0, 16.0, 17.0, 18.0, 19.0 and 20.0), from about 20.0 ng/mL to about 25.0 ng/mL (e.g., 20.0, 21.0, 22.0, 23.0, 24.0 and 25.0), from about 25.0 ng/mL to about 30.0 ng/mL (e.g., 25.0, 26.0, 27.0, 28.0, 29.0 and 30.0), from about 30.0 ng/mL to about 50.0 ng/mL (e.g., 30.0, 35.0, 40.0, 45.0, and 50.0), from about 50.0 ng/mL to about 75.0 ng/mL (e.g., 50.0, 55.0, 60.0, 65.0, 70.0, and 75.0), from about 75.0 ng/mL to about 100.0 ng/mL (e.g., 75.0, 80.0, 85.0, 90.0, 95.0, and 100.0), and any concentrations therebetween, including cutoff points.

[0078] In some embodiments, interleukin-21 (IL21) is used to enhance the proliferation or other characteristics of NK cells. In some embodiments, the concentration of IL21 used is in the range of about 0.01 ng/mL to about 100 ng/mL, including, for example, about 0.01 ng/mL to about 0.05 ng/mL (e.g., 0.01, 0.02, 0.03, 0.04, and 0.05), about 0.05 ng/mL to about 0.1 ng/mL (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1), about 0.1 ng/mL to about 0.5 ng/mL (e.g., 0.1, 0.2, 0.3, 0.4, and 0.5), about 0.5 ng/mL to about 1.0 ng/mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0), from about 1.0 ng/mL to about 2.0 ng/mL (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0), from about 2.0 ng/mL to about 5.0 ng/mL (e.g., 2.0, 3.0, 4.0 and 5.0), from about 5.0 ng/mL to about 10.0 ng/mL (e.g., 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0), from about 10.0 ng/mL to about 15.0 ng/mL (e.g., 10.0, 11.0, 12.0, 13.0, 14.0 and 15.0), from about 15.0 ng/mL to about 20.0 ng/mL (e.g., 15.0, 16.0, 17.0, 18.0, 19.0 and 20.0), from about 20.0 ng/mL to about 25.0 ng/mL (e.g., 20.0, 21.0, 22.0, 23.0, 24.0 and 25.0), from about 25.0 ng/mL to about 30.0 ng/mL (e.g., 25.0, 26.0, 27.0, 28.0, 29.0 and 30.0), from about 30.0 ng/mL to about 50.0 ng/mL (e.g., 30.0, 35.0, 40.0, 45.0, and 50.0), from about 50.0 ng/mL to about 75.0 ng/mL (e.g., 50.0, 55.0, 60.0, 65.0, 70.0, and 75.0), from about 75.0 ng/mL to about 100.0 ng/mL (e.g., 75.0, 80.0, 85.0, 90.0, 95.0, and 100.0), and any concentrations therebetween, including cutoff points.

[0079] In some embodiments, interleukin-15 (IL15) is used in a soluble format (either instead of or in addition to mbIL15 on feeder cells) to enhance proliferation or other characteristics of NK cells. In some embodiments, the concentration of IL15 used is in the range of about 0.01 ng/mL to about 100 ng/mL, including, for example, about 0.01 ng/mL to about 0.05 ng/mL (e.g., 0.01, 0.02, 0.03, 0.04, and 0.05), about 0.05 ng/mL to about 0.1 ng/mL (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1), about 0.1 ng/mL to about 0.5 ng/mL (e.g., 0.1, 0.2, 0.3, 0.4, and 0.5), about 0.5 ng/mL to about 1.0 ng/mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0), from about 1.0 ng/mL to about 2.0 ng/mL (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0), from about 2.0 ng/mL to about 5.0 ng/mL (e.g., 2.0, 3.0, 4.0 and 5.0), from about 5.0 ng/mL to about 10.0 ng/mL (e.g., 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0), from about 10.0 ng/mL to about 15.0 ng/mL (e.g., 10.0, 11.0, 12.0, 13.0, 14.0 and 15.0), from about 15.0 ng/mL to about 20.0 ng/mL (e.g., 15.0, 16.0, 17.0, 18.0, 19.0 and 20.0), from about 20.0 ng/mL to about 25.0 ng/mL (e.g., 20.0, 21.0, 22.0, 23.0, 24.0 and 25.0), from about 25.0 ng/mL to about 30.0 ng/mL (e.g., 25.0, 26.0, 27.0, 28.0, 29.0 and 30.0), from about 30.0 ng/mL to about 50.0 ng/mL (e.g., 30.0, 35.0, 40.0, 45.0, and 50.0), from about 50.0 ng/mL to about 75.0 ng/mL (e.g., 50.0, 55.0, 60.0, 65.0, 70.0, and 75.0), from about 75.0 ng/mL to about 100.0 ng/mL (e.g., 75.0, 80.0, 85.0, 90.0, 95.0, and 100.0), and any concentrations therebetween, including cutoff points.

[0080] In some embodiments, interleukin-22 (IL22) is used to facilitate the proliferation of NK cells. In some embodiments, the concentration of IL22 used is in the range of about 0.01 ng/mL to about 100 ng/mL, including, for example, about 0.01 ng/mL to about 0.05 ng/mL (e.g., 0.01, 0.02, 0.03, 0.04, and 0.05), about 0.05 ng/mL to about 0.1 ng/mL (e.g., 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1), about 0.1 ng/mL to about 0.5 ng/mL (e.g., 0.1, 0.2, 0.3, 0.4, and 0.5), about 0.5 ng/mL to about 1.0 ng/mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0), from about 1.0 ng/mL to about 2.0 ng/mL (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0), from about 2.0 ng/mL to about 5.0 ng/mL (e.g., 2.0, 3.0, 4.0 and 5.0), from about 5.0 ng/mL to about 10.0 ng/mL (e.g., 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0), from about 10.0 ng/mL to about 15.0 ng/mL (e.g., 10.0, 11.0, 12.0, 13.0, 14.0 and 15.0), from about 15.0 ng/mL to about 20.0 ng/mL (e.g., 15.0, 16.0, 17.0, 18.0, 19.0 and 20.0), from about 20.0 ng/mL to about 25.0 ng/mL (e.g., 20.0, 21.0, 22.0, 23.0, 24.0 and 25.0), from about 25.0 ng/mL to about 30.0 ng/mL (e.g., 25.0, 26.0, 27.0, 28.0, 29.0 and 30.0), from about 30.0 ng/mL to about 50.0 ng/mL (e.g., 30.0, 35.0, 40.0, 45.0, and 50.0), from about 50.0 ng/mL to about 75.0 ng/mL (e.g., 50.0, 55.0, 60.0, 65.0, 70.0, and 75.0), from about 75.0 ng/mL to about 100.0 ng/mL (e.g., 75.0, 80.0, 85.0, 90.0, 95.0, and 100.0), and any concentrations therebetween, including cutoff points.

[0081] When two stimulants are used, the relative ratio between the two agents may be in a range including ratios of 1:10, 1:20, 1:50, 1:100, 1:150, 1:200, 1:250, 1:500, 1:750, 1:1000, 1:10000, 1:50000, 1:100000, 100000:1, 50000:1, 10000:1, 1000:1, 750:1, 500:1, 250:1, 200:1, 150:1, 100:1, 50:1, 20:1, 10:1, any ratio in between, including cutoff points. Similarly, if three or more agents are used, any of the above ratios may be used as the ratio between such additional agents and the other agents.

[0082] As discussed in more detail below, depending on the embodiment, the stimulatory molecules may be added at a certain point (or points) during the propagation process or may be added such that they are present as a component of the culture medium throughout the co-cultivation process.

Methods for co-cultivation and expansion of immune cells

[0083] In some embodiments, NK cells isolated from a donor's peripheral blood sample are co-cultured with K562 cells modified to express 4-1BBL and mbIL15. While other approaches involve expressing other membrane-bound cytokines, generating a feeder cell with multiple stimulatory molecules can be difficult to accomplish (e.g., ensuring the desired expression levels of the various stimulatory molecules, expression at the right time during expansion, etc.). As such, several embodiments disclosed herein relate to enriching the culture medium with specific concentrations of the various stimulatory agents at specific time points. In several embodiments, feeder cells are seeded into culture vessels and allowed to grow until substantially confluent. Immune cells can then be added to the culture at a desired concentration, which, in some embodiments, is in the range of from about 0.5×10 ⁶ cells/cm ² to about 5×10 ⁶ cells/cm ² , including any density in between, including cutoff points.

[0084] In some embodiments, immune cells are isolated from a peripheral blood sample. In some embodiments, the immune cells may then be expanded collectively or an isolated subpopulation of cells, such as NK cells, may be used.

[0085] The NK cells are then seeded with feeder cells, optionally one or more cytokines (either present in the culture medium or supplied as an external supplement), and cultured for a first period of time, such as about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or any period of time in between, including cutoff points.

[0086] In some embodiments, after the first period of expansion, the expanded cells (e.g., NK cells) are transduced with an engineered construct, such as a chimeric antigen receptor. Any of a variety of chimeric antigen receptors may be expressed in engineered cells, such as NK cells, including those described in International Application filed under the PCT, PCT/US2018/024650, PCT/IB2019/000141, PCT/IB2019/000181 and/or PCT/US2020/020824, PCT/US2020, 035752, U.S. Provisional Patent Application Nos. 62/924967, 62/960285 and/or 623/038645, each of which is incorporated herein by reference in its entirety.

[0087] Following viral transduction, the engineered cells are cultured for a second period of time, such as about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or any period of time in between, including cutoff points. It should be noted that certain data presented herein relate to viral expression of a chimeric receptor complex expressing the ligand binding domain of NKG2D (e.g., NKX101) or CD19 (e.g., NK19-1 or NKX101). However, any suitable chimeric receptor or chimeric antigen receptor can be used.

[0088] Enrichment of the medium with one or more stimulatory agents, such as IL12 and/or IL18, can occur at any time during the culturing process. For example, one or more stimulatory agents can be added at the beginning of the culturing, such as at time zero (e.g., at the beginning of the culturing). The agent or agents can be added a second, third, fourth, fifth, or more times. Subsequent additions may or may not be at the same concentration as the previous addition. The interval between multiple additions can vary, such as a time interval of about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours or more, and any time period in between, including cutoff points.

[0089] If multiple additions of the stimulant are used, the concentration in the first enrichment addition may be the same as the second (and/or any enrichment addition), or a different concentration. For example, in some embodiments, when the stimulant is added over multiple time points, its concentration may increase, decrease, remain constant, or vary between multiple nonequivalent concentrations.

[0090] In some embodiments, certain ratios of feeder cells to cells to be expanded are used. For example, in some embodiments, a feeder cell:target cell ratio of about 5:1 is used. In some embodiments, 1:1 ratios are used, while in further embodiments, it can be in a range including about: 1:10, 1:20, 1:50, 1:100, 1:1000, 1:10000, 1:50000, 1:100000, 100000:1, 50000:1, 10000:1, 1000:1, 100:1, 50:1, 20:1, 10:1, and any other ratio between the above, including cutoff points.

EXAMPLES

[0091] The materials and methods disclosed in the examples are non-limiting examples of materials and methods (including reagents and conditions) applicable to the various embodiments presented herein.

Example 1. - Initial assessment of breeding conditions

[0092] Fig. 1A shows a non-limiting example of a replication process. In this example, stimulatory cytokines are added on day 0 and the same dose is added again on day 4, as was used in certain embodiments discussed herein. Fig. 1B shows a non-limiting embodiment of a process using a single dose, as was used in certain embodiments discussed herein.

[0093] Figure 2 shows data relating to fold expansion of NK cells using different methods. The leftmost data set shows NK cell expansion using K562 feeder cells (expressing mbIL15 and 4-1BBL) alone, while each of the three data sets to the right shows an increase in fold expansion when the medium is enriched with IL12 and IL18 at different concentrations. The presence of any amount of enrichment IL12 and IL18 resulted in a significant increase in NK cell expansion, thus demonstrating that additional stimulants can enhance NK cell expansion.

[0094] Figure 3A shows flow cytometry data relating to NKG2D expression in NK cells obtained from four different donors that were expanded with either K562 cells alone (top row) or with IL12/18 enrichment. As can be seen based on the increased height of the right-shifted curve (which relates to cells transduced with NKX101), NKG2D expression was increased. The designation NKX101 refers to an engineered NK cell that expresses a truncated extracellular domain of NKG2D capable of binding NKG2D receptor ligands. In several embodiments, the truncated NKG2D domain is linked to the CD8alpha hinge and the TM domain of CD8alpha. In some embodiments, the truncated NKG2D domain is linked to an OX40 costimulatory domain and a CD3zeta signaling domain. In some embodiments, the construct further comprises membrane-bound IL15. In some embodiments, NKX101 has the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence set forth in SEQ ID NO: 2. Further supporting the enhancement of NKG2D expression is Fig. 3A, which demonstrates a greater presence of NKG2D on the cell by means of a higher mean fluorescence intensity (MFI) when using enriched soluble IL12/18. Thus, enrichment of the feeder cell with soluble IL12/18 not only enhances NK cell expansion, but it also enhances the expression of the chimeric receptors on such NK cells. This is an unexpected benefit because more NK cells now express more of the receptor that will target an unwanted cell, such as a tumor cell.

[0095] Other receptors can be used to target NK cells to tumors. For example, in some embodiments, the receptor is a chimeric antigen receptor that targets CD19 on tumor cells. In some embodiments, a CD19-binding CAR comprises an scFv that binds CD19 (e.g., an FMC63 scFv or variant thereof) fused to an OX40 costimulatory domain and a CD3-zeta signaling domain. In some embodiments, the nucleic acid sequence encoding the CAR further encodes IL15. In some embodiments, IL15 is configured to be expressed in a host cell (e.g., an NK cell or a T cell) in a membrane-bound form. In some embodiments, the CAR is encoded by a nucleotide sequence having at least 95%, 97%, 98%, 99%, or more sequence identity to the sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. In some embodiments, the CAR has an amino acid sequence having at least 95%, 97%, 98%, 99%, or more sequence identity to the sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28. In some embodiments, the CAR uses a humanized CD19 binding molecule.

[0096] Figure 4 depicts data showing that the use of enriched soluble IL12/18 during NK cell expansion does indeed result in enhanced cytotoxicity in these expanded NK cells. Figure 4 shows data for two different donors at two time points, 14 days and 21 days after viral transduction. The culture conditions used for NK cell expansion were either soluble IL12/18 (dashed lines) or K562 (expressing 4-1BBL and mbIL15) alone (solid lines). Cells transduced with GFP were used as a control - the curves for NKX101 are indicated by arrows in Figure 4. As indicated by the data for expansion in K562 cells alone, IL12/18 enhances NK cell cytotoxicity at 21 days after transduction (lower panels). Although in this particular experiment the effect at 14 days was limited, in several embodiments, possibly depending on the donor and/or specific IL concentrations, in several embodiments the enhancement of cytotoxicity was achieved at earlier time points such as 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 days after viral transduction. Regardless of the time, it was surprising that the use of soluble interleukins during the expansion process can significantly enhance the cytotoxicity of the expanded cells.

[0097] In some embodiments, the enhanced cytotoxicity of the engineered NK cells is at least in part due to the cells evolving toward a specific phenotype. Figures 5A and 5B depict data regarding the development of certain markers associated with memory NK cells over time. Figure 5A shows the expression of CD57, NKG2C, and CD62L in NK cells expanded on feeder cells alone, while Figure 5B shows the use of feeder cells in conjunction with soluble IL12/18. NKG2C expression was increased at day 21 in NK cells expanded with IL12/18. NKG2C is a marker of cytokine-induced memory NK cells. Increased CD67L levels were also observed at later time points in NK cells expanded with soluble IL12/18. CD67L is associated with increased lymphocyte extravascular egress (indicative of increased cellular activity). Taken together, these data suggest that administration of soluble interleukins during NK cell expansion may trigger distinct signaling pathways that are associated with antigen-specific NK cell memory and enhanced cytotoxicity against cells bearing such antigens.

[0098] Figure 6 depicts data relating to the anti-tumor effect of NK cells expressing NKX101 in vivo, when parental NK cells were expanded using K562 cells alone versus expansion medium supplementation with soluble IL12/18. The animal model involves administering to mice a dose of 4 x 10 ⁶ SNU499 hepatocellular carcinoma cells (i.p.) on day 0, followed by 3 x 10 ⁶ NK cells expressing NKX101 that had been expanded with or without IL12/18 expansion medium supplementation (or control). As shown in the left panels, control mice have significant tumor burden already at day 7, with tumor signal present and slightly elevated in some mice at days 14 and 21. In vivo bioluminescence imaging (BLI) is shown below the photographs. The right panel shows an experiment performed with NK cells expressing NKX101. As shown in the photographs, tumor burden was present at day 7, but was largely undetectable at day 14 and persisted at day 21. The center panel shows photographs for an experiment with NK cells expressing NKX101, where NK cells were expanded with soluble IL12/18. The effect on tumor burden was at least as effective as with NKX101 cells ("standard" expansion), although the significant degree of potency of NKX101 may make it difficult to detect an improvement in effect with IL12/18. Nevertheless, in several embodiments disclosed herein, the use of soluble IL12/18 to enrich the NK cell expansion medium results in not only enhanced expansion, but also enhanced expression of the chimeric receptor and enhanced cytotoxicity.

Example 2. - Additional evaluations of reproduction and efficiency

[0099] As discussed above, in several embodiments disclosed herein, one or more soluble stimulatory factors are used to enhance the expansion and/or cytotoxicity of engineered immune cells, such as NK cells, T cells, or combinations thereof. Experiments conducted within the framework of the present example were performed to evaluate the effectiveness of various concentrations of selected stimulatory molecules compared to a conventional expansion system. Although other stimulatory agents may be used depending on the embodiment, soluble interleukin-12 and soluble interleukin-18 were used in this example. These cytokines were added (at various concentrations described below) and the resulting expanded cells were compared to cells expanded using K562 cells modified to express membrane-bound interleukin-15 and 4-1BBL (described in more detail in U.S. Patent Nos. 7,435,596 and 8,026,097, the entire contents of each of which are incorporated herein by reference in their entireties). The expanded cells were assessed for proliferation, cytokine secretion, cytotoxicity, and phenotype.

[00100] Experiments were set up using NK cells obtained from multiple donors and expanded under different conditions. One set of NK cells was expanded on mbIL15-expressing feeder cells (K562/4-1BBL/mbIL15). Another set of NK cells was expanded on mbIL15-expressing cells that were further modified to express IL12 and IL18 on the cell surface. For all other sets, different culture conditions were used and proliferation assays were performed to determine the effects of different concentrations of stimulatory cytokines. For example, one set of cells was exposed to a fixed concentration of IL12 (5 ng/mL) and varying concentrations of IL18. An additional set was exposed to another fixed concentration of IL12 (2.5 ng/mL) and varying concentrations of IL18. It should be noted that those cultures exposed to IL12 and IL18 in soluble form were exposed to IL12/18 on day 0 of culture (and again on day 4). As discussed above, addition of soluble cytokines on day 0 and day 4 was used in the experiments providing the data shown in Figures 2-18 and Figures 23-24. Other experiments used exposure to soluble cytokines on day 0 only.

[00101] Fig. 7A is a schematic table of the various culture conditions used to expand NK cells. Fig. 7B shows the data regarding the cell number after 72 hours of exposure to the various conditions. As can be seen from the lower curve, the addition of IL18 alone at any concentration had a limited effect on NK cell proliferation. In contrast, the addition of IL12 alone increased NK cell proliferation in a dose-dependent manner. The combination of IL12 (at either 2.5 ng/mL or 5 ng/mL) at varying concentrations further enhanced NK cell proliferation, indicating a synergistic interaction between the two interleukins. Data for IL12 at both 2.5 ng/mL and 5 ng/mL demonstrate robust NK cell expansion, with near maximal levels achieved when IL18 was present at concentrations between approximately 0.1 and approximately 1 ng/mL. Addition of IL18 at higher concentrations was still able to positively enhance NK cell expansion, with the highest concentration of IL18 at 50 ng/mL in combination with IL12 at 5 ng/mL resulting in a small increase in expansion compared to IL12 at 2.5 ng/mL. Data for expansion with supersaturating concentrations of IL12 or IL18 were out of scale and are not shown.

[00102] Figure 8 shows data relating to IFNg concentrations after 72 hours of culture with varying concentrations of either IL12 or IL18. The graph plots IFNg concentration (measured by absorbance in an ELISA assay) against increasing concentrations of the selected interleukin. Similar to the proliferation data, addition of IL12 resulted in greater IFNg production than addition of IL18. However, addition of increasing concentrations of IL18 still resulted in increased IFNg production. On the other hand, IL12 resulted in greater IFNg production in NK cells at nearly every concentration tested. Similar to proliferation, combination of either concentration of IL12 with IL18 concentrations of approximately 1 ng/mL (or greater) resulted in increased IFNg production. The combination of IL12 (at one concentration) with IL18 at concentrations below approximately 0.5 ng/ml resulted in IFNg production similar to that achieved with IL12 alone. On the other hand, inclusion of IL18 at approximately 1 ng/ml or greater resulted in a significant increase in IFNg production, again indicating synergistic stimulation of NK cells.

[00103] Figures 9A-9B show data relating to the expansion of NK cells (non-transduced) after 7 days of expansion under the indicated culture conditions. The first group was expanded using saturating concentrations of both IL12 (20 ng/mL) and IL18 (25 ng/mL). The second group was expanded using saturating concentrations of IL12 (20 ng/mL) and subsaturating concentrations of IL18 (0.05 ng/mL). The third group was expanded using feeder cells engineered to express membrane-bound forms of each of IL15, IL12, and IL18 (further details regarding this feeder cell line can be found in International Patent Application PCT/SG2018/0501387, which is incorporated herein by reference in its entirety). A fourth group, serving as a control, was expanded on a stable feeder cell line (K562 cells expressing mbIL15 and 4-1BBL). Figure 9A shows the calculated expansion data, and Figure 9B shows the statistical analysis. Figures 9C and 9D show the determined titration curve data and NK cell expansion. Figure 9C shows the data for varying IL12 concentrations, holding IL18 constant at 4 ng/mL. Figure 9D shows similar data with varying IL12 and holding IL18 constant at 20 ng/mL. Taken together, these data indicate that the addition of IL12 and IL18, whether in a soluble format or membrane-bound on feeder cells (such as mbIL15-expressing K562 cells), provides a significant enhancement of NK cell expansion. Interestingly, IL12 appears to be the primary stimulus for expansion, with its activity being enhanced by the inclusion of IL18 even at low concentrations (see, for example, similar numbers of cells expanded at saturating and subsaturating concentrations of IL18). These data indicate that combinations of IL12 and IL18 reliably enhance NK cell expansion.

[00104] Figures 10A-10B show cytotoxicity data for untransduced NK cells after 8 days of expansion under the indicated conditions (and supplementation of the medium with IL-2 at 40 IU/mL). The target cells were Reh acute lymphocytic leukemia cells (non-T cells; non-B cells) at an effector:target ratio of 1:1. Regardless of culture conditions, all cells exhibited cytotoxicity ranging from approximately 40% to approximately 65%. Cells expanded on mbIL15-expressing feeder cells without either IL12 or IL18 exhibited the highest degree of cytotoxicity, significantly greater than either group cultured with soluble IL12/IL18. When feeder cells with membrane-bound IL12 and IL18 were used, higher degrees of cytotoxicity were observed than when soluble cytokines were used.

[00105] Figures 11A-11B show cytotoxicity data for untransduced NK cells against Reh cells at a 1:1 effector:target ratio at day 15 of culture (IL2 concentration was 400 IU/mL). These data demonstrate not only higher levels of cytotoxicity for all groups tested, but also limited differences between groups. In other words, all groups showed increased levels of cytotoxicity, meaning there was no significant difference between culture conditions. In some embodiments, administration of IL12 and IL18 induces a signaling pathway or cascade that influences expansion early in culture. In several embodiments, such pathway or cascade (or pathways/cascades) had a delayed effect on enhancing cytotoxicity. In some embodiments, the use of certain stimulatory factors induced a phenotypic change in NK cells, such as a memory cell-like phenotype, which prepares the NK cells to induce cytotoxic effects against a target cell. In some embodiments, the induction of such a phenotypic change may take 1-2, 3-4, 5-6, 7-8, or more days before it is recognized, depending on the NK cell characteristic being assessed.

[00106] Although the above experiments were performed with non-transduced NK cells, they demonstrate that inclusion of IL12 and IL18 at varying concentrations can enhance NK cell expansion and cytotoxicity. Additional experiments were undertaken with NK cells transduced with the chimeric receptor (compared to GFP-transduced cells or non-transduced (NT) NK cells). As a non-limiting example, the chimeric receptor used comprises a truncated NKG2D domain linked to the CDSalpha hinge and the TM domain of CDSalpha, an OX40 costimulatory domain, a CD3zeta signaling domain, and membrane-bound IL15. In Fig. 12 shows flow cytometric data assessing the expression of the chimeric receptor (labeled as 45_4) on NK cells obtained from different donors that were cultured under different conditions. The left column of Fig. 12 shows data for NK cells cultured on mbIL15-expressing feeder cells for two donors (227 at the top, 732 at the bottom). The curve labeled “45_4” shows higher NKG2D expression (as expected for cells that were transduced with the NKG2D-containing chimeric receptor). The right column shows the expression results for NK cells cultured on mBIL15-expressing feeder cells with soluble IL12 and soluble IL18 added to the medium on day 0 at 20 ng/ml and 25 ng/ml, respectively. Fig. 13 shows the corresponding data for two additional donors. As can be seen from the MFI data in both Fig. 12 and Fig. 13, the application of IL12 and IL18 resulted in increased NKG2D expression, further supporting previous findings that certain stimulatory factors can reliably drive NK cell expansion. These data also support that the application of stimulatory molecules such as IL12 and IL18 is appropriate for transduced NK cells.

[00107] After confirming that stimulatory cytokines enhance the expansion of transduced NK cells, cytotoxicity was assessed. Figures 14A and 14B show data relating to the cytotoxicity of NK cells transduced with the indicated constructs and expanded using the indicated culture conditions. The groups were as follows: GFP-transduced NK cells grown on mbIL-15-expressing feeder cells; GFP-transduced NK cells expanded on mbIL-15-expressing feeder cells and exposed to IL12 and IL18, NKX101-transduced NK cells expanded on mbIL-15-expressing feeder cells, and NKX101-transduced NK cells expanded on mbIL-15-expressing feeder cells and exposed to IL12 and IL18. Target cells were Reh cells at an E:T ratio of 1:1. Cytotoxicity was assessed at day 13 after the start of expansion using cells obtained from four different donors. As shown, both GFP-transduced and NKX101-transduced NK cells exhibited cytotoxicity, with NKX101-expressing cells showing the highest effects against target cells. No significant differences were found in terms of the culture conditions used during propagation (see Fig. 14B).

[00108] Figures 15A-15B show additional cytotoxicity data for cells obtained from two donors where different E:T ratios were tested. These data show a pattern consistent with that shown in Figure 14. Figure 15A shows data for the four culture conditions for the first donor, and Figure 15B shows the corresponding data for the second donor. It should be noted that donor 543 (Figure 15A) was cytomegalovirus negative and donor 224 (Figure 15B) was CMV positive. CMV positive individuals have a subpopulation of NK cells that have a memory cell-like phenotype, meaning that they have a more rapid response to target cells. The data in Figures 15A-15B were collected at day 13 after the start of expansion. These curves are similar to the data described above, and at this relatively early time point the presence or absence of IL12/IL18 has a limited effect on NK cell-induced cytotoxicity. Figures 15C and 15D show data with the same donors/conditions but at 21 days post-expansion. Notably, IL12/IL18 treatment results in enhanced cytotoxicity against target cells at most E:T ratios tested. These data are consistent with those described above for untransduced NK cells in that there is a delay in the induction of enhanced cytotoxicity but it can be detected at later time points. As discussed above, this effect may be due to the time required to induce a phenotypic change in NK cells.

[00109] Figs. 16A-16B relate to the assessment of the phenotype of NK cells cultured under different conditions over time. Fig. 16A shows the expression levels of NKG2C and CD62L (L-selectin) over 5 weeks of culture under the indicated conditions. The expression levels of neither CD62L nor NKG2C varied significantly over 5 weeks of culture when mbIL 15-expressing feeder cells were used. In contrast, however, the use of such feeder cells and enrichment of the medium with IL12 and IL18 on day 0 had a significant effect on the expression of both NKG2C and CD62L. CD62L was initially present on approximately 50% of NK cells after 1 week of culture. Although its presence increased after one week, there was a significant decline in CD62L expression thereafter, with limited detection possible after 4 weeks of culture. In contrast, NKG2C expression was slightly increased after one week of culture, with NKG2C expression being increased on NK cells, with more than 40% of cells expressing NKG2C after 5 weeks. Thus, after 5 weeks, the culture could be characterized as having increased NKG2C levels compared to NK cells grown without the stimulatory cytokine and having reduced or equivalent CD62L expression compared to NK cells grown without the stimulatory cytokine. Figure 16B shows additional data supporting the formation of an altered, memory-like phenotype in NK cells. Figure 16B shows FACS expression data from donor NK cells at day 14 (top row) and day 21 (bottom row) cultured with mbIL15-expressing cells (left column) or mbIL15-expressing cells plus IL12 and IL18 on day 0 (right column). CD57 expression is also shown, with the relatively low percentage of cells positive for expression confirming the tendency for this marker to be lost during NK cell culture (fresh NK cells would have higher levels of CD57 expression). As can be seen in the mbIL15 column, NKG2C expression (x-axis) is not significantly altered. In contrast, the percentage of cells expressing NKG2C after an additional week of culture increased by 40% (as indicated by the arrow) following initial exposure to soluble IL12 and soluble IL18.

[00110] Figures 17A-17D show summary data regarding the expression of markers on NK cells after 14 days of culture under the indicated conditions. As shown in Figure 17A, at this time point, CD62L expression was enhanced by the use of IL12 and IL18, regardless of whether they were in soluble or membrane-bound formats. As discussed above, this expression was reduced during additional culture time. Figure 17B shows the enhancement of NKG2D expression by the addition of IL12 and IL18 to the medium on day 1. As with the other data, it should be noted that the effects on NK cell phenotype (such as expansion and cytotoxicity) were approximately equivalent across varying concentrations of IL18 (e.g., effects were seen at saturating or subsaturating concentrations of IL18). CD57 expression levels were relatively low under all conditions, which is typical for cultured (rather than freshly isolated) cells, as shown in Fig. 17D. Fig. 17D shows double positive expression of the CD62L and NKG2C markers, with expression levels again increased in the presence of IL12 and IL18 in culture. These data reflect a shift in the phenotype of NK cells cultured with IL12 and IL18 (whether soluble or membrane-bound) toward a more memory-like phenotype. In some embodiments, this phenotype provides NK cells, particularly those engineered to express a chimeric receptor, with enhanced proliferative capacity and/or enhanced cytotoxicity, making them a more potent cancer immunotherapy product.

[00111] Figure 18 shows that the application of IL12 and IL18 enhances the cytotoxicity of the engineered NK cells even at later time points (cytotoxicity at 21 days post-expansion is shown). Notably, the two central dots in the figure represent NKX101-transduced NK cells, which exhibit the greatest cytotoxic effect compared to any other group. Importantly, NKX101-transduced NK cells cultured with soluble IL12 and 18 on mbIL15-expressing feeder cells exhibit the greatest degree of cytotoxicity toward the target cells (as a non-limiting example, the target in this case was Reh leukemia cells). Thus, in several embodiments, the use of soluble stimulatory factors such as IL12, IL18, IL21, etc., in culturing NK cells provides an unexpected enhancement of cell proliferation (which is very important for obtaining clinically significant amounts of cells), as well as an unexpected enhancement of cytotoxicity against target cells.

Example 3 - Evaluation of Proliferation, Cryopreservation and Cytotoxicity

[00112] As disclosed herein, in several embodiments, the engineered NK cells that were expanded are intended for use in an autologous situation. In several embodiments, an allogeneic approach was used. In several embodiments, the NK cells are designed as "stock," meaning there is a pre-existing population of NK cells that have been expanded and engineered and then stored for dosing to a patient at a later time. In several embodiments, the storage occurs by cryopreservation. As with any freeze-thaw cycle, cell viability and activity can be an issue. Figure 19 shows data relating to the characteristics of NK cells obtained from three different donors that were cultured with mbIL15-expressing feeder cells or mbIL15-expressing feeder cells enriched for soluble IL12/18 at the start of the culture. The bottom three rows of the table provide evidence of the positive effect of soluble IL12 and 18 on NK cells in culture. Six days after the start of expansion, the viability of NK cells in the IL12/18-enriched medium was slightly higher, while the total cell number and, as a result, the multiplication were significantly higher with IL12/18.

[00113] Based on these data, cells were transduced with CD19-binding chimeric antigen receptor and cultured in the presence or absence of soluble IL12 and 18 (using mbIL15-expressing feeder cells). A portion of the cells were cryopreserved and then compared to the corresponding fresh cells. NK cells were assessed for FLAG expression (a tag within the NK19-1 construct, although it should be understood that the corresponding constructs without the tag are presented herein) by FACS. As shown in Fig. 20, both fresh and cryopreserved NK cells from 3 donors retained expression of the CD19-binding CAR. The presence of IL12/18 appeared to have a limited effect on CAR expression. Fig. 21 shows cells from the same donors at day 22 of expansion. Interestingly, the percentage of cells expressing the CD19-binding CAR was reduced at day 14 compared to day 21. Expression of the constructs at day 21 was approximately the same as that of fresh NK cells (e.g., unfrozen) (compare lanes 3-4 with lanes 7-8). These data indicate that NK cells cultured according to the methods disclosed herein are a robust cell population and can survive cryopreservation and still remain viable and maintain significant levels of expression of cytotoxicity-inducing constructs.

[00114] Further analysis of the effects of cryopreservation on NK cells was undertaken. The nuclear red Nalm6 cell line was used as a target cell and was targeted with an NK cell line expressing a CB19-binding CAR. As a non-limiting example, the CAR encoded by SEQ ID NO: 1 was used in this experiment. The results of the analysis are shown in Figs. 22A-22B. Fig. 22A shows the cell number curves (average of three donors) over the time course of the assay. As shown, untransduced NK cells and Nalm6 cells alone showed similar degrees of increase in Nalm6 target cells. Untransduced NK cells grown with soluble IL12/18 showed little cytotoxic effect (downward shift in the cell number per well curve). Notably, cryopreserved cells at day 14 of culture showed a significant cytotoxic effect on Nalm6 cells, limiting their growth until the last hours of the experiment. Importantly, at day 14, cryopreserved cells grown in culture with soluble IL12/18 completely limited the growth of Nalm6 cells. In several embodiments, cells expanded for longer periods of time (either fresh or cryopreserved) were also able to significantly reduce tumor growth. The summary data for 14 days are shown in Fig. 22B. Regarding untransduced NK cells, expansion of NK cells with soluble IL12/18 added at day 0 of culture significantly increased the cytotoxicity of NK cells against target tumor cells. Similar data are shown for the CAR-expressing NK cell. Even in the presence of a CAR, which resulted in nearly 80% cytotoxicity against target cells, culturing CAR-expressing NK cells with soluble IL12/18 significantly enhanced the cytotoxicity. Figure 22C shows additional cytotoxicity data for NK cells cultured in the presence or absence of IL12/18 in the culture medium during expansion, at various E:T ratios. As shown, cells engineered to express a non-limiting embodiment of a CD19-binding CAR exhibit enhanced cytotoxicity at nearly all E:T ratios. As the number of target cells increases, the cytotoxicity of NK cells expanded with the IL12 and IL18 disclosed herein exhibits increasing cytotoxic effects compared to cells expanded on feeder cells alone. Taken together, these data provide evidence that the use of IL12/18 in the culture medium results in enhanced NK cell proliferation as well as enhanced cytotoxicity. In addition, these data provide important additional evidence that cell activity is maintained even after cryopreservation of the cells. These data indicate that some embodiments have produced a thawed engineered NK cell product with robust antitumor effects.

[00115] Figure 23 shows a schematic of an in vivo experiment where hepatocellular carcinoma cells were injected into donor mice and NK cells grown under different culture conditions were introduced. Tumor burden was then monitored using bioluminescence. The cells introduced were either untransduced NK cells grown in soluble IL12/IL18-enriched medium on day 1, NKX101-expressing NK cells grown with IL2, or NKX101-expressing NK cells grown in soluble IL12/IL18-enriched medium on day 1. All cells were grown on mbIL15-expressing feeder cells. Figure 24 shows the results of the tumor burden assay over time. Control animals as well as animals receiving untransduced NK cells showed moderate tumor growth over time. In contrast, animals receiving NK cells expressing NKX101 and expanded with IL12/18 or IL2 showed significantly greater antitumor effects. Tumor burden was reduced in both groups, with only a slight increase at days 14-21 in the IL2 group. These data further support the use of stimulatory cytokines such as IL12, IL18 or IL21 in the culture medium during expansion to enhance the cytotoxicity of cultured NK cells.

[00116] Figure 25 shows similar experimental conditions, this time with a Nalm6 cell xenograft and treatment with NK cells expressing a CD19-binding CAR. Figure 26A shows the resulting bioluminescence data. As with the previous experiment, control animals and animals receiving untransduced NK cells showed a rapid increase in tumor burden, although it decreased at later time points. Animals receiving NK cells expressing NK19-1 (a CD19-binding CAR) showed effective tumor growth inhibition, limiting significant proliferation at later time points. Cells expressing NK19-1 and cultured with IL12/18 showed marked tumor growth control, limiting tumor expansion until late in the experiment and beyond, with a markedly lower overall tumor burden than the other groups. Figure 26B shows additional data related to survival. Mice receiving RV (control) or NT NK cells showed a rapid drop in survival after approximately 30 days. Animals receiving NK19-1 survived longer than these groups, and animals in the NK19-1 IL12/18 group still had 80% viability when there were no survivors in all other groups. Figures 26C and 26D show data related to the persistence of NK cells in vivo when they were cultured in IL12/18-enriched medium. Figure 26C shows the measurement of the percentage of human CD56+ cells (a marker of NK cells) from total mouse peripheral blood. As shown, expansion of NK cells using soluble IL12/18 results in a significantly higher percentage of human NK cells in mouse blood even 18 days after administration. This demonstrates the enhanced persistence conferred on NK cells by the application of stimulatory cytokines during expansion. Similarly, not only NK cells in general, which are persistent in vivo, but also CAR-expressing cells benefit from enhanced persistence by the application of soluble IL12/18 (or other stimulatory molecules). Fig. 26D shows the percentage of NK cells positive for CD19-binding CAR (from total mouse peripheral blood cells) 18 days after injection into recipient xenograft mice. As with the previous figure, these data demonstrate that engineered immune cells, such as NK cells expressing a chimeric antigen receptor, exhibit enhanced persistence in vivo when expanded using at least one stimulatory cytokine. An additional experiment was conducted to evaluate the effects of cytokines applied in expansion culture and cryopreservation (or the absence thereof) on CAR expression in NK cells. Fig. 26E shows that at day 15 of culture, the expression of a non-limiting embodiment of a CD19-binding CAR was not altered when cytokines were applied in expansion culture. That is, the enhanced effects demonstrated herein based on expansion culture using one or more additional stimulatory molecules are not counterbalanced by reduced CAR expression. Moreover, cryopreservation of NK cells did not negatively affect CAR expression in the engineered NK cells. Fig. 26F confirms that CAR expression was not impaired after additional culture time. These data again confirm the enhanced cytotoxicity, persistence and stable CAR expression in NK cells cultured under the influence of stimulatory cytokines such as IL12 and IL18, among others. Likewise, cryopreservation of the engineered NK cells did not significantly negatively impact these beneficial characteristics.

Example 4. - Additional experiments to evaluate the effects of cryopreservation and expansion on cytotoxicity, NK cell characteristics and NK cell survival

[00117] Further experiments were conducted to determine whether the cryopreservation and subsequent thawing process would have a negative impact on the engineered NK cells, such as by reducing their viability, persistence, or cytotoxicity. Figure 27A shows a schematic of the experimental protocol used, as well as the experimental groups and other conditions used. As described above, for the treatment groups designated "IL12/IL18", the cells were expanded in the presence of soluble IL12 and/or IL18 in accordance with the embodiments described herein. Treatment groups included fresh untransduced NK cells (G1) and PBS (G2) as controls. Experimental groups included cryopreserved and thawed NK cells engineered to express a non-limiting embodiment of a CD19-binding CAR and expanded without (G3) and in the presence of additional stimulatory cytokines (G4), and fresh NK cells engineered to express a non-limiting embodiment of a CD19-binding CAR and expanded without (G5, G6) and in the presence of additional stimulatory cytokines (G7, G8). Blood collection and imaging were performed at the time points indicated in Fig. 27A.

[00118] Figures 27B and 27C show in vivo bioluminescence imaging for the indicated experimental groups. Figures 28A-28H show line graphs representing bioluminescence intensity over time. These data are summarized in Figure 281, which shows data for the first 30 days after treatment, and Figure 28J, which shows data for 56 days. Although Figure 281 shows a clear difference between NK cells expressing CD19-binding CARs and the two control groups, each experimental group showed a limited to undetectable increase in BLI measured during the first 30 days of the experiment (an increase in BLI indicates increased tumor growth), indicating tumor growth control. Figure Figure 28J shows data over 56 days and shows greater separation of the experimental groups expressing the different CAR constructs and treated under the indicated conditions in tumor growth inhibition. The control groups (G1 and G2) showed significantly increased tumor growth, leading to termination of the experiment for these groups after 30 days. The group receiving fresh NK cells expressing the CD19-binding CAR and expanded without soluble interleukins (G5) showed a sharp increase in BLI between days 30 and 56. Another experimental replicate of this group (G6) showed a more pronounced tumor growth inhibition capacity. The group receiving frozen NK cells expressing the CD19-binding CAR and expanded without soluble interleukins (G3) also showed an increase in BLI between days 30 and 56, but not to the same extent as seen with fresh cells. Experimental groups receiving NK cells expressing a CD19-binding CAR, whether fresh or frozen, that were expanded using additional stimulatory factors during expansion (in accordance with embodiments disclosed herein), showed the most robust prevention of tumor growth. Notably, groups 4 and 8, both of which were cryopreserved NK cells, showed the greatest inhibition of tumor growth. When combined with the data collected when fresh engineered NK cells were administered, these data indicate that, in several embodiments, engineered NK cells expressing a CD19-binding CAR are effective not only when prepared and administered in a fresh state, but also when prepared, frozen, then thawed, and administered (e.g., as in certain allogeneic administration embodiments).

[00119] Figure 29 shows a line graph of the body weight of mice treated with the constructs over the 56 days of the experiment. The decrease in body weight correlates with increased tumor growth, such that tumor progression results in poor health of the mice and corresponding weight loss (e.g., wasting). As shown, the control groups show significant weight loss over the 30 days, while all but one of the experimental groups increased body weight throughout most of the experiment. As with the bioluminescence data discussed above, a notable trend is that many of the fresh and frozen preparations compared exhibit substantially similar effects on body weight. In several embodiments, engineered NK cells expressing a CD19-binding CAR are effective not only when prepared and administered fresh. Additionally, in some embodiments, engineered NK cells expressing CD19-binding CARs are effective not only when prepared, frozen, then thawed and administered (e.g., as in the case of allogeneic administration).

[00120] Additional data were collected to characterize the properties of NK cells expanded with or without one or more additional stimulatory factors. Figure 30A shows data regarding the longevity (e.g., persistence) of NK cells in culture. These data show the percentage of NK cells (based on CD56 positivity) that were engineered (based on activating chimeric receptor (ACR) positivity). These data show that NK cells expanded with or without additional stimulatory factors such as IL12 and/or IL18 during expansion exhibit similar persistence profiles in vivo, with such engineered NK cells being present at a relatively constant level in the blood (approximately 5-10%) for approximately 7 days. Again, the measurement is based on detection of engineered CAR expression and CD56 positivity, and the percentage of NK cells present in the blood of the animals was measured over ~50 days and is shown in Fig. 30B. In contrast to similar profiles over the 7-day period, the number of NK cells expanded without one or more additional stimulatory factors began to decline after approximately 25-30 days. These cells continued to decline slowly until approximately 48 days, when the cell count approached zero. At the same time point of approximately 25-30 days, engineered NK cells expanded with additional stimulatory factors (e.g., IL12 and/or IL18 in several embodiments) continued to be present in the blood at a level of approximately 10% for 45 days. Only in the last three days was a slight decline observed (to approximately 5-7%). These data clearly indicate that the use of one or more additional stimulatory molecules, such as IL12, IL18, and/or IL21, confers enhanced persistence to the engineered NK cells in vivo compared to NK cells expanded in culture without the use of such stimulatory molecules. Figure 30C shows persistence data based on the number of engineered CAR-expressing NK cells per 10,000 live cells counted. These data reflect the general trend shown in Figure 30B, i.e., cells expanded with one or more stimulatory molecules (e.g., soluble IL12 and/or soluble IL18) persist in the blood in higher numbers for a longer period compared to engineered NK cells expanded without such stimulatory molecules. In some embodiments, the methods disclosed herein are particularly advantageous because they avoid the cytokine dependence that is common among certain cytokine-based expansion methods. In some methods, the use of high concentrations of soluble cytokines promotes cell growth, but the cells grow accustomed to such concentrations and exhibit withdrawal symptoms (e.g., apoptosis, decreased viability, or decreased other functions) when exposed to an environment without such artificial conditions, such as after administration to a patient. The lack of a need for continued high concentrations of cytokines exhibited by engineered NK cells expanded according to the methods disclosed herein contributes at least in part to the longer lifespan (and active lifespan) of the cells in vivo.

[00121] FIGS. 31A-31C show additional data characterizing engineered NK cells obtained according to embodiments disclosed herein. These data are collected from blood tests of three mice (day 51 post-administration) that were injected with fresh (not cryopreserved) engineered NK cells expressing a CD19-binding CAR and expanded using soluble IL12 and soluble IL18 according to several embodiments disclosed herein. The data depicts the proportion of cells in the whole blood sample that are CD56 positive (indicating NK cells) and CD19-Fc positive (indicating cells expressing the engineered CD19-binding CAR). As shown in each of FIGS. 31A, 31B, and 31C, the proportion of double-positive cells (boxed area at upper right) ranged from approximately 4.75% to approximately 6.7%. Figs. 32A-32C show whole blood analysis for the same mice as in Fig. 31, but identify cells that are CD19-Fc positive (indicating cells expressing the engineered CD19-binding CAR) and CD3 positive (indicating T cells). These data demonstrate that the vast majority of cells expressing the CD19-binding CAR are CD3 negative, meaning that they are not T cells. In several embodiments, certain methods of producing NK cells do include the steps of removing T cells from the original donor whole blood sample, but a trace amount of T cells may remain. However, in several embodiments, consistent with the data shown in Fig. 31A-32C, the majority of engineered cells expressing CD19-binding CAR exhibit NK cell features (CD56-positive) and no T cell features (CD3-negative).

[00122] Figs. 33, 34, and 35 relate to data further characterizing cells from whole blood of animals at various time points after tumor inoculation. Fig. 33 relates to data at day 4 after administration, Fig. 34 relates to data at day 12 after tumor inoculation, and Fig. 35 relates to data at day 18 after tumor inoculation. These data relate to cells from whole blood of animals treated as control animals and receiving either untransduced NK cells (NT NK cells), PBS, or one of the other groups that received engineered NK cells expanded with IL2 in culture or IL12/18 in culture, with fresh and frozen cell treatment groups for each condition. In Fig. 33A shows the percentage of NK cells (CD56-pol./CD3-neg.) from the whole blood of the animals at day 4. Each of the treatment groups was relatively similar in this regard, with approximately 3-5% of the cells in the whole blood being engineered NK cells. FIG. 33B shows data relating to the percentage of cells that specifically express a non-limiting embodiment of the CD19-binding CAR. Just as in FIG. 33A, the percentage of cells expressing the CD19-binding CAR in each of the treatment groups was in the range of approximately 3-5%. FIG. 33C shows data relating to the percentage of GFP-positive tumor cells present in the blood at day 4 post-administration. Consistent with the BLI imaging shown in the previous figures, there was little detectable presence of tumor cells in any of the treatment groups. It may be that the low level signal detected reflects migration of GFP+ tumor cells from the bloodstream to various tissues (making them potentially detectable by BLI imaging but not in the blood sample per se). Figures 34A-34C show the corresponding data 12 days after tumor inoculation. As was the case at the earlier time point, for each treatment group, approximately 3%-5% of the blood cells in the sample were NK cells (Figure 34A). Figure 34B shows the percentage of cells positive for the CD19-binding CAR construct. Although expression levels were similar for all treatment groups at this time point, each experimental group was present at levels markedly higher than that of the control groups. Also, at 12 days, the percentage of cells expressing the CD19-binding CAR (e.g., NK cells) was slightly higher (approximately 7-9% of blood cells), suggesting increased persistence of the engineered cells in the circulation. Figure 34C shows the number of tumor cells in whole blood. Interestingly, all groups showed little GFP expression, although BLI imaging showed increased luminescence, particularly in the control groups. Again, these data may reflect the physiological “resident” behavior that certain tumor cells exhibit in suspension.

[00123] Figure 35A shows the percentage of NK cells (based on CD56 positivity) 18 days after tumor inoculation. All experimental groups showed significantly higher percentages compared to the control groups, with values for the groups ranging from approximately 15% to approximately 25% of the cells in whole blood. This increase in percentage corresponded to the time interval of the increase in NK cells, as shown in Figures 30B and 30C. Although these data were not statistically different in this particular experiment, they did show that NK cells expanded in IL12/IL18 containing medium and cryopreserved had the greatest proliferation in the experimental groups. In several embodiments, the feeder cell, together with cytokine-based expansion, in combination with cryopreservation provides the most robust NK cell that can survive under more normal conditions of cytokine provision (e.g., without added cytokines) and can persist for longer periods of time in a healthy state. Figures 35B and 35C show two measurements of tumor burden at day 18. Figure 35B shows the percentage of cells in the blood that are positive for CD19 (the target of the engineered CAR in this non-limiting embodiment) as measured using an anti-CD19 antibody coupled to PE. These data show a trend toward increased tumor burden in the control groups, and in contrast, the ability of the engineered NK cells from the treatment groups to limit tumor growth. Figure 35C shows similar data but obtained through detection of a GFP signal (e.g., by BLI). These data, although different from the data in Figure 35B, due to the different sensitivity of detection by PE compared to GFP, show a similar trend. The experimental NK cells demonstrate an enhanced ability to prevent tumor cell proliferation compared to controls. Fig. 35D refers to data regarding the number of NK cells that express the engineered CD19-binding molecule (e.g., positive for both CD56 and CD19-Fc). Similar to the data presented in Fig. 35A, these data demonstrate that an increased percentage of NK cells in the blood sample are NK cells expressing the engineered CD19-binding CAR, reflecting their enhanced persistence. Fig. 35E shows data confirming that nearly the entire population of NK cells from each experimental group that are CAR positive are NK cells that were engineered to express the CD19-binding CAR disclosed herein.

[00124] To further examine the persistence of engineered NK cells expanded according to the embodiments disclosed herein, two doses of engineered NK cells expanded using soluble cytokines as disclosed herein were administered to mice and cell numbers were monitored for four additional weeks (administration protocol based on Fig. 27A). Fig. 36 shows a box plot for these data. Briefly, the x-axis of the box plot represents time in two formats: either i) time since third administration or ii) total time since tumor inoculation (shown in parentheses). The y-axis represents the number of NK cells expressing the CD19-binding CAR (per 10,000 leukocytes). The box plots for the 2 million NK cell dose are the bottom row of boxes (shown as hatched), while the 5 million cell dose is the top row (shown as solid). These data indicate that the half-life of engineered NK cells expanded under conditions of one or more stimulatory molecules (such as IL12 and/or IL18) (in combination with feeder cells, as described in several embodiments herein) is increased compared to engineered NK cells expanded under conditions of feeder cells alone. The half-life for the 2 million engineered NK cell dose is -15 days. Based on variance in one or more of clearance and/or volume of distribution, the half-life for the 5 million engineered NK cell dose is -18 days. This is in contrast to the data for a dose of other engineered NK cells expanded without the use of one or more additional stimulatory molecules, which are shown in Fig. 37 and indicate a half-life of -5 days for a dose of 5 million cells. Thus, according to several embodiments disclosed herein, expansion of engineered NK cells using one or more additional cytokines in combination with a feeder cell system provides for increased expansion of NK cells and imparts enhanced persistence and/or cytotoxicity to such cells.

[00125] It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made and still remain within the scope of one or more embodiments of the present invention. Furthermore, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, etc. in connection with an embodiment may be applied to all other embodiments set forth herein. Accordingly, it should be understood that the various features and aspects of the disclosed embodiments may be combined or substituted for one another to obtain various forms of embodiments of the disclosed invention. Thus, it is intended that the scope of the present invention disclosed herein should not be limited to the specific disclosed embodiments described above. Moreover, although the present invention is susceptible to various modifications and alternative forms, specific examples thereof have been shown by means of drawings and described in detail herein. However, it is to be understood that the present invention is not to be limited to the specific forms or methods disclosed, but on the contrary, the present invention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any methods disclosed herein are not required to be performed in the order listed. The methods disclosed herein include specific actions taken by a practitioner; however, they can also include any third-party instructions for these actions, either directly or indirectly mentioned. For example, actions such as "administering a population of expanded NK cells" include "instructions for administering a population of expanded NK cells." In addition, when features or aspects of the present invention are described in terms of Markush groups, those skilled in the art will understand that the present invention thereby describes any individual member or subgroup of members of the Markush group.

[00126] The ranges disclosed herein also encompass any possible overlaps, subranges, and combinations thereof. Expressions such as "up to," "at least," "more than," "less than," "between," and the like include the stated number. Numbers preceded by a term such as "approximately" or "about" include the stated numbers. For example, "90%" includes "90%." In some embodiments, a sequence having at least 95% sequence identity to a reference sequence means sequences that are 96%, 97%, 98%, 99%, or 100% identical to the reference sequence. Furthermore, when a sequence is described as "comprising" a nucleotide or amino acid sequence, such reference shall also include, unless otherwise indicated, that the sequence "comprises," "consists of," or "consists essentially of" the said sequence.

[00127] The singular terms may mean "one or more than one" unless the contrary is stated or otherwise obvious from the context. The phrase "and/or" as used herein in the description and claims shall be understood to mean "one of or both" of the elements so combined. Multiple elements listed with "and/or" shall be construed in the same way, i.e. "one or more" of the elements so combined. Other elements other than the elements specifically identified by the "and/or" clause may optionally be present. As used herein in the description and claims, "or" shall be understood to have the same meaning as "and/or" as defined above. For example, when used in a list of elements, "or" or "and/or" shall be interpreted as inclusive, i.e. including at least one, but not necessarily more than one, of the listed elements and optionally additional, unlisted elements. Only terms for which the contrary is clearly indicated, such as "only one of" or "exactly one of", will refer to the inclusion of exactly one element from a series or list of elements. Thus, claims that include an "or" between one or more members of a group are considered satisfied if one, more than one, or all of the members of the group are present, used, or otherwise related to the product or process, unless otherwise indicated. Embodiments are provided in which exactly one member of the group is present, used, or otherwise related to the product or process. Embodiments are provided in which more than one or all of the members of the group are present, used, or otherwise related to the product or process. Any one or more claims may be amended to expressly exclude any embodiment, aspect, feature, element, or characteristic, or any combination thereof. Any one or more claims may be amended to expressly exclude any agent, composition, amount, dose, route of administration, cell type, target, cellular marker, antigen, targeting moiety, or combinations thereof.

[00128] Several embodiments provide amino acid sequences that correspond to any of the nucleic acids disclosed herein, taking into account the degeneracy of the genetic code. Moreover, it is intended that those sequences (whether a nucleic acid sequence or an amino acid sequence) that differ from those explicitly disclosed herein, but have functional similarity or equivalence, are also within the scope of the present invention. The foregoing includes mutant forms, truncations, substitutions, or other types of modifications.

[00129] Any headings or subheadings used in this document are for organizational purposes and are not intended to limit the scope of the embodiments disclosed in this document.

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SEQUENCE LIST

<110> Nkarta, Inc.

<120> METHODS AND COMPOSITIONS FOR ENHANCED REPRODUCTION AND CYTOTOXICITY

NATURAL KILLER CELLS

<130>NKT.034WO

<150> 62/881311

<151> 2019-07-31

<150> 62/932342

<151> 2019-11-07

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 1797

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

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<400> 1

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ccgttattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 120

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 180

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 240

gaggaccagg atttacttaa actggtgaag tcatatcatt ggatgggact agtacacatt 300

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 360

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 420

gaaaactgtt caactccaaa tacgtacatc tgcatgcaaa ggactgtgac cacgacgcca 480

gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 540

gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt 600

gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 660

atcacccttt actgccggag ggaccagagg ctgccccccg atgcccacaa gccccctggg 720

ggaggcagtt tccggacccc catccaagag gagcaggccg acgcccactc caccctggcc 780

aagatcagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 840

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 900

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 960

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1020

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1080

gacacctacg acgcccttca catgcaggcc ctgccccctc gcggctctgg cgagggaagg 1140

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Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

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

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

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Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

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cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

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gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

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cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

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His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

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

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

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

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

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

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

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

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

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

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

195 200 205

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

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 5

<211> 2150

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223> DNA NK19H-no Flag-2

<400> 5

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga tattcagatg acccagagcc cgagcagcct gagcgcgagc 120

gtgggcgatc gcgtgaccat tacctgccgc gcgagccagg atattagcaa atatctgaac 180

tggtatcagc agaaaccggg cggcaccgtg aaactgctga tttatcatac cagccgcctg 240

catagcggcg tgccgagccg ctttagcggc agcggcagcg gcaccgattt taccctgacc 300

attagcagcc tgcagccgga agatattgcg acctattttt gccagcaggg caacaccctg 360

ccgtatacct ttggcggcgg caccaaactg gaaattaccg gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg aaagcggccc gggcctggtg 480

aaaccgagcc agaccctgag cctgacctgc accgtgagcg gcgtgagcct gccggattat 540

ggcgtgagct ggattcgcca gccgccgggc aaaggcctgg aatggattgg cgtgatttgg 600

ggcagcgaaa ccacctatta taacagcgcg ctgaaaagcc gcctgaccat tagcaaagat 660

aacagcaaaa accaggtgag cctgaaactg agcagcgtga ccgcggcgga taccgcggtg 720

tattattgcg cgaaacatta ttattatggc ggcagctatg cgatggatta ttggggccag 780

ggcaccagcg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1500

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1560

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1620

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1740

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1800

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1860

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1920

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 1980

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

<210> 6

<211> 706

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-2

<400> 6

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 7

<211> 2150

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223> DNA NK19H-no Flag-3

<400> 7

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga tattcagatg acccagagcc cgagcagcct gagcgcgagc 120

gtgggcgatc gcgtgaccat tacctgccgc gcgagccagg atattagcaa atatctgaac 180

tggtatcagc agaaaccgga tggcaccgtg aaactgctga tttatcatac cagccgcctg 240

catagcggcg tgccgagccg ctttagcggc agcggcagcg gcaccgatta taccctgacc 300

attagcagcc tgcagccgga agatattgcg acctattttt gccagcaggg caacaccctg 360

ccgtatacct ttggcggcgg caccaaactg gaaattaccg gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg aaagcggccc gggcctggtg 480

aaaccgagcc agaccctgag cctgacctgc accgtgagcg gcgtgagcct gccggattat 540

ggcgtgagct ggattcgcca gccgccgggc aaaggcctgg aatggattgg cgtgatttgg 600

ggcagcgaaa ccacctatta taacagcgcg ctgaaaagcc gcctgaccat tagcaaagat 660

aacagcaaaa accaggtgag cctgaaactg agcagcgtga ccgcggcgga taccgcggtg 720

tattattgcg cgaaacatta ttattatggc ggcagctatg cgatggatta ttggggccag 780

ggcaccagcg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1500

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1560

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1620

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1740

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1800

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1860

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1920

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 1980

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

<210> 8

<211> 706

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-3

<400> 8

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 9

<211> 2150

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223> DNA NK19H-no Flag-4

<400> 9

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga tattcagatg acccagagcc cgagcagcct gagcgcgagc 120

gtgggcgatc gcgtgaccat tacctgccgc gcgagccagg atattagcaa atatctgaac 180

tggtatcagc agaaaccggg cggcaccgtg aaactgctga tttatcatac cagccgcctg 240

catagcggcg tgccgagccg ctttagcggc agcggcagcg gcaccgattt taccctgacc 300

attagcagcc tgcagccgga agatattgcg acctattatt gccagcaggg caacaccctg 360

ccgtatacct ttggcggcgg caccaaactg gaaattaccg gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg aaagcggccc gggcctggtg 480

aaaccgagcc agaccctgag cctgacctgc accgtgagcg gcgtgagcct gccggattat 540

ggcgtgagct ggattcgcca gccgccgggc aaaggcctgg aatggctggg cgtgatttgg 600

ggcagcgaaa ccacctatta taacagcgcg ctgaaaagcc gcctgaccat tagcaaagat 660

aacagcaaaa gccaggtgag cctgaaactg agcagcgtga ccgcggcgga taccgcggtg 720

tattattgcg cgaaacatta ttattatggc ggcagctatg cgatggatta ttggggccag 780

ggcaccagcg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1500

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1560

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1620

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1740

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1800

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1860

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1920

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 1980

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

<210> 10

<211> 706

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-4

<400> 10

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 11

<211> 2153

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223> DNA NK19H-no Flag-5

<400> 11

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga tattcagatg acccagagcc cgagcagcct gagcgcgagc 120

gtgggcgatc gcgtgaccat tacctgccgc gcgagccagg atattagcaa atatctgaac 180

tggtatcagc agaaaccggg cggcaccgtg aaactgctga tttatcatac cagccgcctg 240

catagcggcg tgccgagccg ctttagcggc agcggcagcg gcaccgattt taccctgacc 300

attagcagcc tgcagccgga agatattgcg acctattttt gccagcaggg caacaccctg 360

ccgtatacct ttggcggcgg caccaaactg gaaattaccg gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg agtcaggacc tggcctggtg 480

aaaccctcac agactctgtc cctgacatgc actgtctcag gggtctcatt acccgactat 540

ggtgtaagct ggattcgcca gcctccaggt aagggtctgg agtggctggg agtaatatgg 600

ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat ctccaaggac 660

aactccaaga gccaagtttc cttaaaatta agtagtgtta ctgctgctga cacagccgtc 720

tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggccaa 780

ggaacctcag tcaccgtctc ctcaaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcaga 1140

tctagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1200

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1260

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1320

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440

atgcaggccc tgccccctcg cggctctggc gagggaaggg gttccctgct tacttgcggc 1500

gacgtcgaag agaatcccgg tccgatggcc ctcccagtaa ctgccctcct tttgcccctc 1560

gcactccttc ttcatgccgc tcgccccaac tgggtcaacg tgattagcga tttgaagaaa 1620

atcgaggacc ttatacagtc tatgcatatt gacgctacac tgtatactga gagtgatgta 1680

cacccgtcct gtaaggtaac ggccatgaaa tgctttcttc tggagctcca ggtcatcagc 1740

ttggagtctg gggacgcaag catccacgat acggttgaaa acctcatcat ccttgcgaac 1800

aactctctct catctaatgg aaacgttaca gagagtgggt gtaaggagtg cgaagagttg 1860

gaagaaaaaa acatcaaaga atttcttcaa tccttcgttc acatagtgca aatgttcatt 1920

aacacgtcca ctaccacacc cgccccgagg ccacctacgc cggcaccgac tatcgccagt 1980

caacccctct ctctgcgccc cgaggcttgc cggcctgcgg ctggtggggc ggtccacacc 2040

cggggcctgg attttgcgtg cgatatatac atctgggcac ctcttgccgg cacctgcgga 2100

gtgctgcttc tctcactcgt tattacgctg tactgctaag cggccgcgtc gac 2153

<210> 12

<211> 707

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-5

<400> 12

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Val Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu

500 505 510

Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Leu Tyr Cys

705

<210> 13

<211> 2150

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223> DNA NK19H-no Flag-6

<400> 13

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga catccagatg acacagagcc cgtcctccct gtctgcctct 120

gtgggagaca gagtcaccat cacctgcagg gcaagtcagg acattagtaa atatttaaat 180

tggtatcagc agaaaccaga cggaactgtt aaactcctga tctaccatac atcaagatta 240

cactcaggag tcccatcaag gttcagtggc agtgggtctg gaacagatta caccctcacc 300

attagcagcc tgcaaccgga agatattgcc acttacttct gccaacaggg taatacgctt 360

ccgtacacgt tcggaggggg gaccaagctg gagatcacag gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg agtcaggacc tggcctggtg 480

aaaccctcac agactctgtc cctgacatgc actgtctcag gggtctcatt acccgactat 540

ggtgtaagct ggattcgcca gcctccaggt aagggtctgg agtggctggg agtaatatgg 600

ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat ctccaaggac 660

aactccaaga gccaagtttc cttaaaatta agtagtgtta ctgctgctga cacagccgtc 720

tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggccaa 780

ggaacctcag tcaccgtctc ctcaaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1500

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1560

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1620

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1740

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1800

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1860

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1920

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 1980

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

<210> 14

<211> 706

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-6

<400> 14

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 15

<211> 2150

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223> DNA NK19H-no Flag-7

<400> 15

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga catccagatg acacagagcc cgtcctccct gtctgcctct 120

gtgggagaca gagtcaccat cacctgcagg gcaagtcagg acattagtaa atatttaaat 180

tggtatcagc agaaaccagg tggaactgtt aaactcctga tctaccatac atcaagatta 240

cactcaggag tcccatcaag gttcagtggc agtgggtctg gaacagatt caccctcacc 300

attagcagcc tgcaaccgga agatattgcc acttactact gccaacaggg taatacgctt 360

ccgtacacgt tcggaggggg gaccaagctg gagatcacag gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg agtcaggacc tggcctggtg 480

aaaccctcac agactctgtc cgtgacatgc actngtctcag gggtctcatt acccgactat 540

ggtgtaagct ggattcgcca gcctccaggt aagggtctgg agtggctggg agtaatatgg 600

ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat ctccaaggac 660

aactccaaga gccaagtttc cttaaaatta agtagtgtta ctgctgctga cacagccgtc 720

tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggccaa 780

ggaacctcag tcaccgtctc ctcaaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1500

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1560

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1620

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1740

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1800

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1860

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1920

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 1980

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

<210> 16

<211> 706

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-7

<400> 16

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 17

<211> 2150

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223> DNA NK19H-no Flag-8

<400> 17

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga catccagatg acacagagcc cgtcctccct gtctgcctct 120

gtgggagaca gagtcaccat cacctgcagg gcaagtcagg acattagtaa atatttaaat 180

tggtatcagc agaaaccagg tggaactgtt aaactcctga tctaccatac atcaagatta 240

cactcaggag tcccatcaag gttcagtggc agtgggtctg gaacagattt caccctcacc 300

attagcagcc tgcaaccgga agatattgcc acttacttct gccaacaggg taatacgctt 360

ccgtacacgt tcggaggggg gaccaagctg gagatcacag gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg agtcaggacc tggcctggtg 480

aaaccctcac agactctgtc cgtgacatgc actgtctcag gggtctcatt acccgactat 540

ggtgtaagct ggattcgcca gcctccaggt aagggtctgg agtggctggg agtaatatgg 600

ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat ctccaaggac 660

aactccaaga gccaagtttc cttaaaatta agtagtgtta ctgctgctga cacagccgtc 720

tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggccaa 780

ggaacctcag tcaccgtctc ctcaaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1500

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1560

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1620

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1740

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1800

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1860

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1920

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 1980

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

<210> 18

<211> 706

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-8

<400> 18

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 19

<211> 2150

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223>DNA NK19H-no Flag-9

<400> 19

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga catccagatg acacagagcc cgtcctccct gtctgcctct 120

gtgggagaca gagtcaccat cacctgcagg gcaagtcagg acattagtaa atatttaaat 180

tggtatcagc agaaaccaga cggaactgtt aaactcctga tctaccatac atcaagatta 240

cactcaggag tcccatcaag gttcagtggc agtgggtctg gaacagatta caccctcacc 300

attagcagcc tgcaaccgga agatattgcc acttacttct gccaacaggg taatacgctt 360

ccgtacacgt tcggaggggg gaccaagctg gagatcacag gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg agtcaggacc tggcctggtg 480

aaaccctcac agactctgtc cgtgacatgc actgtctcag gggtctcatt acccgactat 540

ggtgtaagct ggattcgcca gcctccaggt aagggtctgg agtggctggg agtaatatgg 600

ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat ctccaaggac 660

aactccaaga gccaagtttc cttaaaatta agtagtgtta ctgctgctga cacagccgtc 720

tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggccaa 780

ggaacctcag tcaccgtctc ctcaaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1500

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1560

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1620

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1740

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1800

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1860

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1920

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 1980

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

<210> 20

<211> 706

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-9

<400> 20

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 21

<211> 2150

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223>DNA NK19H-no Flag-10

<400> 21

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga tattcagatg acccagagcc cgagcagcct gagcgcgagc 120

gtgggcgatc gcgtgaccat tacctgccgc gcgagccagg atattagcaa atatctgaac 180

tggtatcagc agaaaccggg cggcaccgtg aaactgctga tttatcatac cagccgcctg 240

catagcggcg tgccgagccg ctttagcggc agcggcagcg gcaccgattt taccctgacc 300

attagcagcc tgcagccgga agatattgcg acctattatt gccagcaggg caacaccctg 360

ccgtatacct ttggcggcgg caccaaactg gaaattaccg gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg aaagcggccc gggcctggtg 480

aaaccgagcc agaccctgag cgtgacctgc accgtgagcg gcgtgagcct gccggattat 540

ggcgtgagct ggattcgcca gccgccgcgc aaaggcctgg aatggctggg cgtgatttgg 600

ggcagcgaaa ccacctatta taacagcgcg ctgaaaagcc gcctgaccat tagcaaagat 660

aacagcaaaa gccaggtgag cctgaaaatg agcagcgtga ccgcggcgga taccgcgatt 720

tattattgcg cgaaacatta ttattatggc ggcagctatg cgatggatta ttggggccag 780

ggcaccagcg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1500

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1560

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1620

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1740

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1800

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1860

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1920

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 1980

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

<210> 22

<211> 706

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-10

<400> 22

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Met Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 23

<211> 2150

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223> DNA NK19H-no Flag-11

<400> 23

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga tattcagatg acccagagcc cgagcagcct gagcgcgagc 120

gtgggcgatc gcgtgaccat tacctgccgc gcgagccagg atattagcaa atatctgaac 180

tggtatcagc agaaaccggg cggcaccgtg aaactgctga tttatcatac cagccgcctg 240

catagcggcg tgccgagccg ctttagcggc agcggcagcg gcaccgattt taccctgacc 300

attagcagcc tgcagccgga agatattgcg acctattttt gccagcaggg caacaccctg 360

ccgtatacct ttggcggcgg caccaaactg gaaattaccg gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg aaagcggccc gggcctggtg 480

aaaccgagcc agaccctgag cgtgacctgc accgtgagcg gcgtgagcct gccggattat 540

ggcgtgagct ggattcgcca gccgccgcgc aaaggcctgg aatggctggg cgtgatttgg 600

ggcagcgaaa ccacctatta taacagcgcg ctgaaaagcc gcctgaccat tagcaaagat 660

aacagcaaaa gccaggtgag cctgaaaatg agcagcgtga ccgcggcgga taccgcgatt 720

tattattgcg cgaaacatta ttattatggc ggcagctatg cgatggatta ttggggccag 780

ggcaccagcg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1500

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1560

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1620

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1740

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1800

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1860

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1920

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 1980

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

<210> 24

<211> 706

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-11

<400> 24

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Met Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 25

<211> 2150

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223> DNA NK19H-no Flag-12

<400> 25

gaattcgccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccgga tattcagatg acccagagcc cgagcagcct gagcgcgagc 120

gtgggcgatc gcgtgaccat tacctgccgc gcgagccagg atattagcaa atatctgaac 180

tggtatcagc agaaaccgga tggcaccgtg aaactgctga tttatcatac cagccgcctg 240

catagcggcg tgccgagccg ctttagcggc agcggcagcg gcaccgatta taccctgacc 300

attagcagcc tgcagccgga agatattgcg acctattttt gccagcaggg caacaccctg 360

ccgtatacct ttggcggcgg caccaaactg gaaattaccg gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctcaggtg cagctgcagg aaagcggccc gggcctggtg 480

aaaccgagcc agaccctgag cgtgacctgc accgtgagcg gcgtgagcct gccggattat 540

ggcgtgagct ggattcgcca gccgccgcgc aaaggcctgg aatggctggg cgtgatttgg 600

ggcagcgaaa ccacctatta taacagcgcg ctgaaaagcc gcctgaccat tagcaaagat 660

aacagcaaaa gccaggtgag cctgaaaatg agcagcgtga ccgcggcgga taccgcgatt 720

tattattgcg cgaaacatta ttattatggc ggcagctatg cgatggatta ttggggccag 780

ggcaccagcg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1020

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1080

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1500

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1560

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1620

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1680

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1740

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1800

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1860

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1920

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 1980

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2040

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2100

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2150

<210> 26

<211> 706

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19H-without Flag-12

<400> 26

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Met Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Arg Asp Gln

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu

500 505 510

Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Tyr Cys

705

<210> 27

<211> 2210

<212> DNA

<213> Homo sapiens

<220>

<221> other_sign

<223> NK19-1 DNA

<400> 27

ggatccgaat tcgccgccac catggcctta ccagtgaccg ccttgctcct gccgctggcc 60

ttgctgctcc acgccgccag gccggactac aaagacgatg acgataaagg cggtggtggc 120

tctggtggtg gcggcagcga catccagatg acacagacta catcctccct gtctgcctct 180

ctgggagaca gagtcaccat cagttgcagg gcaagtcagg acattagtaa atatttaaat 240

tggtatcagc agaaaccaga tggaactgtt aaactcctga tctaccatac atcaagatta 300

cactcaggag tcccatcaag gttcagtggc agtgggtctg gaacagatta ttctctcacc 360

attagcaacc tggagcaaga agatattgcc acttactttt gccaacaggg taatacgctt 420

ccgtacacgt tcggaggggg gaccaagctg gagatcacag gtggcggtgg ctcgggcggt 480

ggtgggtcgg gtggcggcgg atctgaggtg aaactgcagg agtcaggacc tggcctggtg 540

gcgccctcac agagcctgtc cgtcacatgc actngtctcag gggtctcatt acccgactat 600

ggtgtaagct ggattcgcca gcctccacga aagggtctgg agtggctggg agtaatatgg 660

ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat catcaaggac 720

aactccaaga gccaagtttt cttaaaaatg aacagtctgc aaactgatga cacagccatt 780

tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggccaa 840

ggaacctcag tcaccgtctc ctcaaccacg acgccagcgc cgcgaccacc aacaccggcg 900

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 960

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 1020

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg ccggagggac 1080

cagaggctgc cccccgatgc ccacaagccc cctgggggag gcagtttccg gacccccatc 1140

caagaggagc aggccgacgc ccactccacc ctggccaaga tcagagtgaa gttcagcagg 1200

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1260

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1320

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1380

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1440

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1500

caggccctgc cccctcgcgg ctctggcgag ggaaggggtt ccctgcttac ttgcggcgac 1560

gtcgaagaga atcccggtcc gatggccctc ccagtaactg ccctcctttt gcccctcgca 1620

ctccttcttc atgccgctcg ccccaactgg gtcaacgtga ttagcgattt gaagaaaatc 1680

gaggacctta tacagtctat gcatattgac gctacactgt atactgagag tgatgtacac 1740

ccgtcctgta aggtaacggc catgaaatgc tttcttctgg agctccaggt catcagcttg 1800

gagtctgggg acgcaagcat ccacgatacg gttgaaaacc tcatcatcct tgcgaacaac 1860

tctctctcat ctaatggaaa cgttacagag agtgggtgta aggagtgcga agagttggaa 1920

gaaaaaaaca tcaaagaatt tcttcaatcc ttcgttcaca tagtgcaaat gttcattaac 1980

acgtccacta ccacacccgc cccgaggcca cctacgccgg caccgactat cgccagtcaa 2040

cccctctctc tgcgccccga ggcttgccgg cctgcggctg gtggggcggt ccacacccgg 2100

ggcctggatt ttgcgtgcga tatatacatc tgggcacctc ttgccggcac ctgcggagtg 2160

ctgcttctct cactcgttat tacgctgtac tgctaagcgg ccgcgtcgac 2210

<210> 28

<211> 724

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_sign

<223> AK sequence NK19-1

<400> 28

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 Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Ile Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser

355 360 365

Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu

370 375 380

Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

465 470 475 480

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

485 490 495

Pro Pro Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

500 505 510

Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu

515 520 525

Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asn Trp Val

530 535 540

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

Thr Leu Tyr Cys

<---

Claims (39)

1. A method for enhancing the proliferation of natural killer (NK) cells, comprising: (a) co-cultivation in a culture medium of a population of NK cells with a population of feeder cells, wherein the feeder cell population contains cells engineered to express 4-1BBL and membrane-bound interleukin-15 (mbIL15); (b) enrichment of the culture medium with interleukin-2 (IL2) and (c) enriching the culture medium with at least one soluble stimulatory agent, wherein the at least one soluble stimulatory agent comprises a combination of (i) soluble interleukin-12 (IL12) at a concentration of from 0.01 ng/ml to 8 ng/ml and (ii) soluble interleukin-18 (IL18) at a concentration of from 0.01 ng/ml to 30 ng/ml. 2. The method according to claim 1, wherein the medium is enriched for the first time with IL2 at a concentration of less than 500 IU/ml. 3. The method according to claim 1 or 2, wherein the medium is enriched for the first time with IL2 at a concentration of less than 50 IU/ml. 4. The method according to any one of claims 1 to 3, wherein the culture medium is enriched a second time with IL2 at a concentration that exceeds the concentration of IL2 in the culture medium enriched the first time. 5. The method according to item 4, wherein enrichment of the culture medium for the second time is performed in the interval from 12 hours to 120 hours from enrichment of the culture medium for the first time. 6. The method according to any one of claims 1-5, wherein the population of feeder cells comprises K562 cells. 7. The method of claim 6, wherein the K562 cells are irradiated prior to co-cultivation with the NK cell population. 8. The method according to any one of claims 1-7, further comprising contacting the NK cell population with a vector encoding a chimeric antigen receptor (CAR). 9. The method of claim 8, wherein the CAR is configured to target one or more of CD19, CD123, CD70, BCMA, or natural killer cell receptor group D (NKG2D) ligand. 10. The method according to any one of claims 1 to 9, wherein the method provides for an increase in the persistence of NK cells compared to the persistence of NK cells co-cultured with feeder cells in the absence of at least one soluble stimulating agent. 11. The method according to any one of claims 1-10, wherein the method provides for an increase in the cytotoxicity of NK cells compared to the cytotoxicity of NK cells co-cultured with feeder cells in the absence of at least one soluble stimulating agent. 12. The method according to any one of claims 4 to 11, wherein the medium is enriched a second time with IL2 to a concentration of less than 500 IU/ml. 13. A method for enhancing the proliferation of natural killer (NK) cells, comprising: (a) co-cultivating in a culture medium a population of NK cells with a population of feeder cells, wherein the population of feeder cells contains cells engineered to express 4-1BBL and membrane-bound interleukin-15 (mbIL15); (b) enrichment of the culture medium with interleukin-2 (IL2), soluble interleukin-12 (IL12) and soluble interleukin-18, wherein the culture medium is enriched with (i) soluble IL12 at a concentration of 0.01 ng/ml to 8 ng/ml and (ii) soluble IL18 at a concentration of 0.01 ng/ml to 30 ng/ml, and (c) bringing a population of NK cells into contact with a vector encoding a chimeric antigen receptor (CAR). 14. The method according to claim 13, wherein the culture medium is enriched with IL2 at a concentration of less than 500 IU/ml. 15. The method according to claim 12 or 14, wherein the culture medium is enriched with IL2 at a concentration of less than 50 IU/ml. 16. A method for enhancing the proliferation of natural killer (NK) cells, comprising: (a) co-cultivating in a culture medium a population of NK cells with a population of feeder cells, wherein the population of feeder cells comprises cells engineered to express 4-1BBL and membrane-bound interleukin-15 (mbIL15); (b) enrichment of the culture medium with interleukin-2 (IL2); (c) enriching the culture medium at a first time point with at least one soluble stimulatory agent, wherein, within 120 hours of said co-cultivation, the at least one soluble stimulatory agent comprises a combination of (i) interleukin-12 (IL12) at a concentration of from 0.01 ng/ml to 8 ng/ml and (ii) interleukin-18 (IL18) at a concentration of from 0.01 ng/ml to 30 ng/ml; (d) enriching the culture medium at a second time with an additional amount of at least one soluble stimulant; where the first and second moments of time are separated by more than 12 hours to less than 120 hours, and (e) co-cultivation of the NK cell population with feeder cells for a second period of time, wherein the method results in enhanced proliferation of NK cells compared to co-cultivation of NK cells with feeder cells in the absence of at least one soluble stimulating agent. 17. The method of claim 16, wherein the population of feeder cells comprises K562 cells. 18. The method of claim 16 or 17, further comprising contacting the NK cell population with a vector encoding a chimeric antigen receptor (CAR). 19. The method according to claim 17, wherein the K562 cells are irradiated prior to co-cultivation with the NK cell population. 20. The method of claim 18, wherein the CAR is configured to target one or more of CD19, CD123, CD70, BCMA, or natural killer cell receptor group D (NKG2D) ligand. 21. The method according to any one of claims 16-20, wherein the method provides for an increase in the persistence of NK cells compared to the persistence of NK cells co-cultured with feeder cells in the absence of at least one soluble stimulating agent. 22. The method according to any one of claims 16-20, wherein the method provides for an increase in the cytotoxicity of NK cells compared to the cytotoxicity of NK cells co-cultured with feeder cells in the absence of at least one soluble stimulating agent. 23. The method according to any one of claims 16-22, wherein the medium is first enriched with IL2 to a concentration of less than 500 IU/ml. 24. The method according to any one of claims 16-23, wherein the culture medium is enriched a second time with IL2 at a concentration that exceeds the concentration of IL2 in the culture medium enriched the first time.