Method for preparing and using cell ghost with active factors as synergist of lymphocyte in vitro culture

Abstract

Provided is a method for preparing and using cell ghosts with active factors as a synergist of a lymphocyte in vitro culture. The method for preparing cell ghosts comprises: washing a cell to obtain a washed cell; and cleaving the washed cell to obtain cell ghosts, wherein the cell has cytokines capable of promoting the proliferation and differentiation of lymphocytes on their surface.

Claims

1. A method for preparing cell ghosts, comprising: a) washing cells to obtain washed cells; and b) lysing the washed cells to obtain the cell ghosts, wherein said cells express on their surface cytokines capable of promoting lymphocyte proliferation and/or activation.

2. The method of claim 1, wherein washing the cells further comprises: a) suspending the cells in an isotonic solution to obtain a cell suspension; and b) centrifuging the cell suspension to obtain the washed cells.

3. The method of claim 2, wherein prior to suspending the cells in the isotonic solution, the isotonic solution is cooled to 4 C.

4. The method of claim 3, wherein the isotonic solution is an isotonic phosphate buffer at pH 7.4.

5. The method of claim 1, wherein lysing the washed cells further comprises: a) suspending the washed cells in a hypotonic solution at a predetermined volume ratio, followed by b) incubating the preparation of step (a) for 2 hours without mixing, to obtain cell lysates; and c) centrifuging the cell lysates to obtain the cell ghosts.

6. The method of claim 5, wherein the predetermined volume ratio of cells to total volume is 1:40.

7. The method of claim 5, wherein prior to suspending the washed cells in the hypotonic solution, the hypotonic solution is cooled to 4 C.

8. The method of claim 7, wherein the hypotonic solution is a hypotonic Tris HCl buffer.

9. The method of claim 1, wherein the cytokines capable of promoting lymphocyte proliferation and/or activation are at least one selected from the group consisting of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86, and 4-1BBL.

10. The cell ghosts prepared according to the method of claim 1.

11. A method for culturing lymphocytes, comprising: a) mixing lymphocytes with a culture medium to obtain a lymphocyte suspension; b) adding the lymphocyte suspension and the cell ghosts of claim 10 into a culture container; and c) culturing the combination of the lymphocyte suspension and the cell ghosts obtained in step (b) in an incubator containing water-saturated air and 5% CO.sub.2 at 37 C.

12. The method of claim 11, wherein the lymphocytes are at least one member selected from the group consisting of NK cells, CTL cells and Treg cells.

13. The method of claim 12, wherein the lymphocyte suspension has a volume of 40 ml and comprises 510.sup.6 of the lymphocytes, and the ratio of the cell ghosts to the lymphocytes is 1:1.

14. A method for amplifying and/or activating lymphocytes, comprising: a) isolating monocytes from the peripheral blood of a human subject; b) mixing the monocytes with 40 ml of culture medium for culturing lymphocytes to obtain a lymphocyte suspension, wherein the complete culture medium for culturing the lymphocytes contains a RPMI 1640 medium supplemented with 200 IU/ml IL-2, 10% of plasma from the human subject and 80 U/ml gentamicin, wherein 40 ml of the lymphocyte suspension contains 510.sup.6 of the monocytes; c) adding the lymphocyte suspension and 510.sup.6 of the cell ghosts of claim 10 into a T175 culture flask, and d) culturing the preparation of step (c) in an incubator containing water-saturated air and 5% CO.sub.2 at 37 C.; e) adding another 40 ml of complete RPMI 1640 culture medium on Day 4; f) transferring cells in the T175 culture flask into a culture bag, adding the complete RPMI 1640 culture medium up to a volume of 400 ml; g) adding 810.sup.7 cell ghosts of claim 10; h) transferring the culture obtained in step (g) into two culture bags each containing 640 ml of the complete RPMI 1640 culture medium on Day 10; and i) collecting the culture product from each bag to obtain the lymphocytes on Day 12, wherein the lymphocytes are at least one member selected from the group consisting of NK cells, CTL cells and Treg cells.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing comparison of in vitro amplification of NK cells in PBMCs stimulated by RPMI 8866-ghost and RPMI 8866-gamma, respectively, according to embodiment 1 of the present disclosure;

(2) FIG. 2 is a graph showing comparison of cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by RPMI 8866-gamma and RPMI 8866-ghost, respectively, according to embodiment 1 of the present disclosure;

(3) FIG. 3 is a graph showing comparison of in vitro amplification of NK cells in PBMCs stimulated by HFWT-gamma and HFWT-ghost, respectively, according to embodiment 2 of the present disclosure;

(4) FIG. 4 is a graph showing comparison of cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by HFWT-gamma and HFWT-ghost, respectively, according to embodiment 2 of the present disclosure;

(5) FIG. 5 is a graph showing comparison of in vitro amplification of NK cells in PBMCs stimulated by EBV-CLC-gamma and EBV-CLC-ghost, respectively, according to embodiment 3 of the present disclosure;

(6) FIG. 6 is a graph showing comparison of cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by EBV-CLC-gamma and EBV-CLC-ghost, respectively, according to embodiment 3 of the present disclosure;

(7) FIG. 7 is a graph showing comparison of in vitro amplification of NK cells in PBMCs stimulated by K15-41BBL-gamma and K15-41BBL-ghost, respectively, according to embodiment 4 of the present disclosure;

(8) FIG. 8 is a graph showing comparison of cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by K15-41BBL-gamma and K15-41BBL-ghost, respectively, according to embodiment 4 of the present disclosure;

(9) FIG. 9 is a graph showing comparison of in vitro amplification of NK cells in PBMCs stimulated by K21-41BBL-gamma and K21-41BBL-ghost, respectively, according to embodiment 5 of the present disclosure;

(10) FIG. 10 is a graph showing comparison of cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by K21-41BBL-gamma and K21-41BBL-ghost, respectively, according to embodiment 5 of the present disclosure;

(11) FIG. 11 is a graph showing comparison of in vitro amplification of NK cells in PBMCs stimulated by TK21-41BBL-gamma and TK21-41BBL-ghost, respectively, according to embodiment 6 of the present disclosure;

(12) FIG. 12 is a graph showing comparison of cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by TK21-41BBL-gamma and TK21-41BBL-ghost, respectively, according to embodiment 6 of the present disclosure;

(13) FIG. 13 is a graph showing comparison of in vitro amplification of NK cells in PBMCs stimulated by two formulations (fresh liquid and lyophilized) of PK21-41BBL-ghost and K21-41BBL-ghost, respectively, according to embodiment 7 of the present disclosure;

(14) FIG. 14 is a graph showing comparison of cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by two formulations (fresh liquid and lyophilized) of PK21-41BBL-ghost and K21-41BBL-ghost, respectively, according to embodiment 7 of the present disclosure;

(15) FIG. 15 is a graph showing comparison of in vitro amplification of NK cells in PBMCs stimulated by PBMC-ghost cIL21-41BBL and PBMC-ghost aIL21-41BBL, respectively, according to embodiment 8 of the present disclosure; and

(16) FIG. 16 is a graph showing comparison of cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by PBMC-ghost cIL21-41BBL and PBMC-ghost aIL21-41BBL, respectively, according to embodiment 8 of the present disclosure.

DETAILED DESCRIPTION

(17) The present disclosure provides a method of preparing cell ghosts bearing active cytokines as a booster for culturing lymphocytes in vitro and use thereof. Reference will be made in detail to embodiments of the present disclosure. The embodiments are explanatory, illustrative, and used to generally understand the present disclosure. The embodiments shall not be construed to limit the present disclosure. Specific techniques or conditions that are not indicated in the embodiments shall be in accordance with techniques or conditions as described in literatures of the present field or product specifications. The reagents and instruments without specified manufacturers are conventional products that can be purchased.

Embodiment 1

(18) 1. Preparation of RPMI 8866-Cell Ghost

(19) In accordance with the following steps, cell ghosts were prepared using a cell line, RPMI 8866, in which the PRMI 8866 is human B lymphoblastoid cell line. The details are as follows:

(20) The RPMI 8866 cells were suspended in 3-time volume of pH 7.4 isotonic PBS solution (pre-cooled to 4 C.), followed by centrifugation at 1500 rpm and 4 C. for 10 minutes. Remove the supernatant, and repeatedly washed 1-3 times to obtain the washed RPMI 8866 cells; then added the washed RPMI 8866 into 10 mmol/L hypotonic Tris HCl buffer solution (pre-cooled to 4 C.) at a volume ratio of 1:40, with slow stirring during addition. The obtained mixture was then put into 4 C. refrigerator for 2 hours to allow complete lysis of cells. Afterwards, the mixture was centrifuged at 9000 rpm, 4 C. for 10 minutes to precipitate the cell ghosts, followed by further repeated washing, and centrifugal precipitation for another 3-5 times to obtain RPMI 8866 cell ghosts, i.e. RPMI 8866-ghost. Then, aliquoted at a concentration of 210.sup.7 cell ghosts/ml in freeze solution, and stored at 80 C.; or preserved at 4 C. after lyophilization.

(21) 2. Preparation of RPMI 8866-Gamma Feeder Cell

(22) RPMI 8866-gamma feeder cells were prepared according to the following steps:

(23) The RPMI 8866 cells were suspended in pH 7.4 isotonic PBS solution at a concentration of 110.sup.7 cells/mL. The obtained cell suspension was subjected to 10 thousand rad gamma ray irradiation, and then washed with pH 7.4 isotonic PBS solution for three times to obtain the feeder cells, namely RPMI8866-gamma. The obtained feeder cells were then suspended in complete PRMI 1640 medium (supplemented with 10% fetal bovine serum, gentamycin 80 U/ml) at a concentration of 210.sup.7 feeder cells/ml, and stored at 80 C. in freeze solution.

(24) 3. Amplification of NK Cells in PBMCs Using RPMI 8866-Ghost and RPMI 8866-Gamma Feeder Cells.

(25) The NK cell proliferation in PBMCs was stimulated by RPMI 8866-ghost or RPMI 8866-gamma feeder cells. Peripheral blood mononuclear cells from 3 normal human subjects were tested as follows:

(26) 1) Preparation of autologous plasma: collecting 50 ml anticoagulant peripheral blood, followed by 700 g centrifugation for 20 min at room temperature; the plasma was collected and placed in 56 C. water bath for 30 min, then stood at 4 C. for another 15 min; lastly, the plasma was centrifuged again at 900 g, 4 C. for 30 min to obtain autologous plasma and stored at 4 C.

(27) 2) D-PBS was added into the cell layer after plasma extraction followed by well mixing. PBMCs were isolated with a lymphocyte separation solution via centrifugation at 800 g for 20 min;

(28) 3) Cell suspension (approximately 510.sup.6 lymphocytes) was prepared by adding isolated PBMCs, 40 ml complete culture medium for culturing lymphocytes (RPMI 1640 supplemented with about 200 IU/ml IL-2, autologous plasma 1-10%, gentamicin 80 U/ml) and 110.sup.7 feeder cells or cell ghosts in a T175 culture flask, followed by incubating in an incubator with saturated humidity, 37 C., and 5.0% CO.sub.2;

(29) 4) approximately 40 mL complete culture medium for culturing lymphocytes was added around Day 4;

(30) 5) All cells in the T175 culture flask were transferred into a culture bag followed by adding about 400 mL complete culture medium for culturing lymphocytes and adding about 810.sup.7 cell ghosts mentioned above around Day 7;

(31) 6) All cells in the culture bag were passaged to two new culture bags each containing about 640 mL complete culture medium for culturing lymphocytes around Day 10; and

(32) 7) collecting culturing products around Day 12, which were subjected to evaluation with amplification and activation on NK cells in PBSCs via different methods. The NK cells in PBSCs were cultured for different time period, such as for 18 to 20 continuous days, in accordance with the number of cells needed.

(33) 4. Comparison of In Vitro Amplification of NK Cells in PBMCs Stimulated by RPMI 8866-Gamma and RPMI 8866-Ghost

(34) Comparison of in vitro amplification of NK cells in PBMCs stimulated by RPMI 8866-ghost and RPMI 8866-gamma respectively was shown in FIG. 1. It indicates that the amplification effects of the NK cells respectively by the cell ghosts of the present disclosure and the feeder cells are comparable.

(35) 5. Comparison of Cytotoxicity of NK Cells in PBMCs Amplified and Activated by RPMI 8866-Gamma and RPMI 8866-Ghost Against Tumor Cells

(36) The NK cells obtained through in vitro amplification described above were taken as effector cells, and K562 leukemia cells (K562 cells) were taken as target cells. The cytotoxicity on K562 target cells after NK cells in PBMCs were stimulated and amplified by RPMI 8866-gamma and RPMI 8866-ghost were determined by Calcein-AM fluorescence scanning assay. Specific steps are as follows:

(37) A CAM solution was prepared by diluting fluorescent dye Calcein-AM with the complete culture medium (RPMI1640 supplemented with 10% fetal bovine serum, 80 U/ml gentamicin). 110.sup.6 target cells were suspended in 1 ml of the CAM solution, and incubated in an incubator with saturated humidity, 37 C., and 5.0% CO.sub.2 for one hour, with timely shaking during the culture. Then the target cells in culture were washed twice with NKEM medium, with 1200 rpm centrifugation for 5 minutes after every wash. The target cells were then counted for and then adjusted to be a concentration of 110.sup.5 cells/mL. The effector cells were diluted to be a concentration of 110.sup.6 cells/mL.

(38) The target cells were added into 3 wells of a U-shaped bottom 96-well cell culture plate each containing 200 L, corresponding to a ratio of effector cells to target cells (E:T) of 10:1. One of such three wells was added with 100 L of 2% Triton X-100 such that the target cells were lysed completely to be as a positive control with maximum release. Other two wells were added with 100 L of the complete culture medium to be as a negative control for natural release.

(39) The effector cells were double-diluted 5 times, with a ratio of the effector cells to the target cells (E:T) of 0.3125:1 in the last dilution.

(40) The number of sample wells on the 96-well plate is determined according to the total number of samples with different concentrations of effector cells, with 100 l target cells added in each well and centrifuged at 100 g for 1 min to induce cell attachment. After cultured in the incubator with saturated humidity, 37 C. and 5.0% CO.sub.2 for 4 hours, cells were gently pipetted up-and-down with a 100 l pipette to suspend released Calcein fluorescence dye, followed by 5 min centrifugation at 100 g to precipitate cells. 100 L supernatant was transferred to a new culture plate to be measured with a fluorescence plate-reader (excitation light at 485 nm, emission light at 530 nm). The cytotoxicity of NK cells in PBMCs after stimulated and amplified by RPMI 8866-gamma and RPMI 8866-ghost, respectively, on k562 cells, which is calculated in accordance with a formula of: killer rate=[(absorbance of the test groupabsorbance of the natural release group)/(absorbance of the maximum release groupabsorbance of the natural release group)]100. Results are shown in FIG. 2.

(41) As shown in FIG. 2, NK cells in PBMCs exhibits strong toxicity on K562 cells after amplified and stimulated by RPMI8866-gamma and RPMI8866-ghost.

Embodiment 2

(42) HFWT-gamma and HFWT-ghost were prepared with a cell line, HFWT cells (HFWT Wilms tumor cell line), respectively; and experiments on amplification and cytotoxicity of NK cells were performed via methods the same as above description in Embodiment 1.

(43) Comparison of in vitro amplification of NK cells in PBMCs stimulated by HFWT-gamma and HFWT-ghost, respectively is shown in FIG. 3. It can be seen that the NK cells are amplified by about 100 times averagely either by HFWT-gamma and HFWT-ghost on Day 12, indicating that the amplification of the NK cells respectively by the cell ghosts of the present disclosure and the feeder cells are comparable.

(44) Cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by HFWT-gamma and HFWT-ghost are shown in FIG. 4. It can be seen that the NK cells in PBMCs after amplified and stimulated respectively by HFWT-gamma and HFWT-ghost exhibit strong toxicity on the K562 cells.

Embodiment 3

(45) EBV-CLC-gamma feeder cells and EBV-CLC-ghost were prepared with EBV-CLC cells respectively, and experiments on amplification and cytotoxicity of NK cells were performed via methods the same as above description in Embodiment 1, in which EBV-CLC is a B lymphoblastoid cell line formed by EBV transformation of human B lymphocytes. The in vitro amplification of NK cells in PBMCs stimulated by EBV-CLC-gamma and

(46) EBV-CLC-ghost are shown in FIG. 5. It can be seen that average amplification rates by EBV-CLC-gamma and EBV-CLC-ghost are about 150 times on Day 12 respectively, indicating that the amplification of the NK cells by the cell ghosts of the present disclosure and the feeder cells are comparable.

(47) Cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by EBV-CLC-gamma and EBV-CLC-ghost are shown in FIG. 6. It can be seen that the NK cells in PBMCs after amplified and stimulated by EBV-CLC-gamma and EBV-CLC-ghost exhibit strong toxicity on the K562 cells.

Embodiment 4

(48) K15-41BBL-gamma feeder cells and K15-41BBL-ghosts were prepared with a genetic recombinant cell line K15-41BBL; and experiments on amplification and cytotoxicity of NK cells were performed via methods the same as above description in Embodiment 1, with a difference that the amplification of NK cells was detected on Day 20 during cell amplification process. K15-41BBL is a cell line prepared by expressing IL-15 and 4-1BBL on the surface of K562 cells.

(49) The in vitro amplification of the NK cells in PBMCs stimulated by K15-41BBL-gamma feeder cells and K15-41BBL-ghosts are shown in FIG. 7. It can be seen that average amplification rates by K15-41BBL-gamma feeder cells and K15-41BBL-ghosts are about 2500 times without significant differences on Day 20.

(50) Cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by K15-41BBL-gamma feeder cells and K15-41BBL-ghosts are shown in FIG. 8. It can be seen that the NK cells in PBMCs after amplified and stimulated by K15-41BBL-gamma feeder cells and K15-41BBL-ghosts exhibit strong toxicity on the K562 cells without significant difference.

Embodiment 5

(51) K21-41BBL-gamma feeder cells and K21-41BBL-ghosts were prepared with a genetic recombinant cell line K21-41BBL; and experiments on amplification and cytotoxicity of NK cells were performed via methods the same as above description in Embodiment 1, with a difference that the amplification of NK cells was detected on Day 20 during cell amplification process. K21-41BBL is a cell line prepared by expressing IL-21, IL-15, CD86, CD64, CD19, and 4-1BBL on the surface of K562 cells.

(52) The in vitro amplification of the NK cells in PBMCs stimulated by K21-41BBL-gamma feeder cells and K21-41BBL-ghosts are shown in FIG. 9. It can be seen that average amplification rates by K21-41BBL-gamma feeder cells and K15-41BBL-ghosts are about 40,000 times without significant difference on Day 20.

(53) Cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by K21-41BBL-gamma feeder cells and K21-41BBL-ghosts are shown in FIG. 10. It can be seen that the NK cells in PBMCs after amplified and stimulated respectively by K21-41BBL-gamma feeder cells and K21-41BBL-ghosts exhibit strong toxicity on the K562 cells without significant difference.

Embodiment 6

(54) TK21-41BBL-gamma feeder cells and TK21-41BBL-ghosts were prepared with a TK21-41BBL cell line, respectively; and experiments on amplification and cytotoxicity of NK cells were performed via methods the same as above description in Embodiment 1, with a difference that the amplification of NK cells was detected on Day 20 during cell amplification process. TK21-41BBL is a cell line prepared by transient transfecting with genes encoding IL-21, IL-15, CD64, CD19, CD86, and 4-1BBL proteins into K562 leukemia cells.

(55) The in vitro amplification of the NK cells in PBMCs stimulated by TK21-41BBL-gamma feeder cells and TK21-41BBL-ghosts are shown in FIG. 11. It can be seen that average amplification rates by K21-41BBL-gamma feeder cells and K15-41BBL-ghosts are about 20,000 times without significant difference on Day 20.

(56) Cytotoxicity on K562 target cells after NK cells in PBMCs are stimulated and amplified by TK21-41BBL-gamma feeder cells and TK21-41BBL-ghosts are shown in FIG. 12. It can be seen that the NK cells in PBMCs after amplified and stimulated by TK21-41BBL-gamma feeder cells and TK21-41BBL-ghosts exhibit strong toxicity on the K562 cells without difference.

Embodiment 7

(57) 1. Preparation of Cells Ghost

(58) Cells ghosts were prepared with K562 cells according to the method for preparing the cell ghosts in Embodiment 1, thereby obtaining K562-ghost. The K562-ghosts were crosslinked with a cytokine mixture of IL-21, IL-15, CD86, CD64, CD19 and 4-1BBL via a glutaraldehyde method, thereby obtaining PK21-41BBL-ghosts.

(59) 2. NK Cells Amplification

(60) NK cells in PBMCs were stimulated and amplified respectively by two formulations (freshly prepared (directly obtained without preservation) and lyophilized (prepared, lyophilized and preserved in 4 C. refrigerator)) of the PK21-41BBL-ghosts and the K21-41BBL-ghost prepared in Embodiment 6 via methods the same as above description in Embodiment 1. The in vitro amplification of NK cells in PBMCs stimulated by the two formulations of K21-41BBL-ghosts and PK21-41BBL-ghosts, respectively, were detected in on Day 20, of which result is shown in FIG. 13. It can be seen that average amplification rates by PK21-41BBL-ghosts and K21-41BBL-ghosts are about 40,000 times without significant difference on Day 20.

(61) 3. Cytotoxicity Test

(62) Cytotoxicity on K562 target cells after the effector NK cells in PBMCs are stimulated and amplified by two formulations (freshly prepared (directly obtained without preservation) and lyophilized (prepared, lyophilized and preserved in 4 C. refrigerator)) of K21-41BBL-ghosts and the PK21-41BBL-ghosts, respectively, are shown in FIG. 14. It can be seen that the NK cells in PBMCs after stimulated and amplified by the two formulations (freshly prepared and lyophilized) of PK21-41BBL-ghost and K21-41BBL-ghost exhibit strong cytotoxicity on the K562 cells.

Embodiment 8

(63) 1. Preparation of PBMC Ghosts, PBMC Ghosts Crosslinked with Cytokines, and PBMC Ghosts Attached with Cytokines

(64) PBMC-ghost was prepared with PBMCs via the method in Embodiment 1; the PBMC-ghosts were crosslinked with IL-21, IL-15, CD86, CD64, CD19 and 4-1BBL, thereby obtaining PBMC-ghost-cIL21-41BB.

(65) Besides, PBMC-ghosts were mixed with soluble cytokines, such as IL-21, IL-15, CD86, CD64, CD19 and 4-1BBL, such that the cytokines were attached on the PBMC-ghost, i.e., PBMC-ghost-aIL21-41BB.

(66) 2. NK Cell Amplification

(67) NK cells in allogeneic PBMCs (i.e., the PBMC used for preparing cell ghosts and feeder cells is different from the PBMC used for amplification) were stimulated and amplified by PBMC-ghost-cIL21-41BBL and PBMC-ghost-aIL21-41BBL mentioned above via the same method in Embodiment 1.

(68) The in vitro amplification of NK cells in PBMCs stimulated by PBMC-ghost-cIL21-41BBL and PBMC-ghost-aIL21-41BBL were measured in on Day 20, of which result is shown in FIG. 15. It can be seen that average amplification rates by PBMC-ghost-cIL21-41BBL and PBMC-ghost-aIL21-41BBL are about 40,000 times without significant difference on Day 20.

(69) 3. Cytotoxicity Test

(70) Cytotoxicity on K562 target cells after the effector NK cells in PBMCs are stimulated and amplified by PBMC-ghost-cIL21-41BBL and PBMC-ghost-aIL21-41BBL, respectively, are shown in FIG. 16. It can be seen that the NK cells in PBMCs after stimulated and amplified respectively by PBMC-ghost-cIL21-41BBL and PBMC-ghost-aIL21-41BBL exhibit strong cytotoxicity on the K562 cells without significant difference.

(71) Reference throughout this specification to an embodiment, some embodiments, one embodiment, another example, an example, a specific examples, or some examples, means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as in some embodiments, in one embodiment, in an embodiment, in another example, in an example, in a specific examples, or in some examples, in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

(72) Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.