METHOD FOR ASSESSING VALIDITY OF CELL THERAPY PRODUCT

Abstract

A composition according to an embodiment includes a first population of transformed mammalian cells with a transforming growth factor beta (TGF-β), the first population having a TGF-β expression level of 0.65 ng/10.sup.5 cells/24 hours or more, and a second population of mammalian cells which are not transformed with the transforming growth factor beta, the second population having an expression level of a thrombospondin 1 (TSP-1) expression level of 31 ng/10.sup.5 cells/24 hours or more.

Claims

1. A composition comprising: a first population of transformed mammalian cells with a transforming growth factor beta (TGF-β), the first population having a TGF-β expression level of not less than 0.65 ng/10.sup.5 cells/24 hours; and a second population of mammalian cells which are not transformed with the transforming growth factor beta.

2. The composition of claim 1, wherein the first population of the transformed mammalian cells are inactivated cells in replication incompetent state.

3. The composition of claim 1, wherein the number of the untransformed mammalian cells of the second population and the number of the transformed mammalian cells of the first population are mixed with a mixing ratio of 3:1.

4. The composition of claim 1, wherein the first population has the TGF-β expression level of not less than 1 ng/10.sup.5 cells/24 hours.

5. The composition of claim 1, wherein the first population has the TGF-β expression level of not less than 1.7 ng/10.sup.5 cells/24 hours.

6. The composition of claim 1, wherein the second population express thrombospondin 1 (TSP-1).

7. The composition of claim 1, wherein the second population has the thrombospondin 1 (TSP-1) expression level of not less than 31 ng/10.sup.5 cells/24 hours.

8. The composition of claim 1, wherein the second population has the thrombospondin 1 (TSP-1) expression level of not less than 50 ng/10.sup.5 cells/24 hours.

9. The composition of claim 1, wherein the second population has the thrombospondin 1 (TSP-1) expression level of not less than 90 ng/10.sup.5 cells/24 hours.

10. An osteoarthritis therapeutic agent comprising the composition according to claim 1.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1 is a graph illustrating an expression level of TGF-β1 protein in TC cells.

[0059] FIG. 2 is a graph illustrating the confirmed results of the expression level of TGF-β1 in each Manufacturing batch.

[0060] FIG. 3A is a graph illustrating von Frey filament test results when an animal model with MIA-induced osteoarthritis was treated with mixed cells and/or anti-TGF-β1 neutralizing antibody; and FIG. 3B is a graph illustrating the above von Frey filament test results by an area under the curve (AUC).

[0061] FIG. 4 is a graph illustrating the expression level of TSP-1 protein in HC cells and TC cells, respectively.

[0062] FIG. 5 is a graph and a table illustrating the confirmed results of the expression level of TSP-1 in each cell production batch.

[0063] FIG. 6A is a graph illustrating von Frey filament test results when the MIA-induced osteoarthritis animal model was treated with mixed cells and/or anti-TSP-1 neutralizing antibody; and FIG. 6B is a graph illustrating the above von Frey filament test results by an area under the curve (AUC).

[0064] FIG. 7A is a graph illustrating von Frey filament test results when an animal model with MIA osteoarthritis was treated with mixed cells having different TGF-β1 expression levels; FIG. 7B is a graph illustrating the above von Frey filament test results by an area under the curve (AUC); and FIG. 7C are photographs illustrating results of H & E staining tissue analysis.

[0065] FIG. 8A a graph illustrating 1, 2 VFF test results of 120 Gy mixed cells in order to determine minimum effective value of TGF-β1; and FIG. 8B a graph illustrating the counted results by AUC.

[0066] FIG. 9A a graph illustrating 1, 2 VFF test results of 120 Gy mixed cells in order to determine minimum effective value of TSP-1; FIG. 9B a graph illustrating counted results by AUC; and FIG. 9C are photographs illustrating results of H & E staining tissue analysis.

DETAILED DESCRIPTION

[0067] Hereinafter, the following embodiments are provided to describe the present invention in more detail. It will be apparent to those skilled in the art that the scope of the present invention in regard to the objects of invention is not limited by these embodiments.

Example 1. Preparation of Cell Therapeutic Agent

[0068] The cell therapeutic agent used in this example of the present invention is a transformed cell population so as to express TGF-β1 (NCBI Reference Sequence: NM 000660.6) (first population; hereinafter referred to as TC) and a normal cell population without transformation using the above gene (second population; hereinafter referred to as HC).

[0069] TC could be prepared by injecting cDNA of TGF-β1 into cells according to a known method. For instance, the cDNA of TGF-β1 is inserted into a known vector having a resistant gene such as ampicillin or neomycin [for example, pCI (containing ampicillin resistant gene) from Promega Co.] to construct a vector containing cDNA of TGF-β1, followed by injecting the vector into chondrocytes according to a known method such as a calcium phosphate method or a lipofectin method, thus to prepare TC.

[0070] The HC and TC are human-derived chondrocytes, wherein HC is a normal chondrocyte while TC is a transformed chondrocyte to secrete TGF-β1. A method for construction of HC and TC has been disclosed in known documents [Cytotherapy, 2012 February; 14 (2): 247-256) and U.S. Pat. Nos. 7,005,127 and 7,282,200.

[0071] A mixing ratio of HC and TC was 3:1 based on the number of cells and was applied to the following examples.

[0072] The prepared TC and HC were filled into a vial, respectively, and frozen and prepared/stored for use as a mixed cell-based therapeutic agent. At this time, the TC was inactivated by irradiation before or after freezing.

Example 2. Identification of TGF-β1 Secretion in Cell Therapeutic Agent

[0073] The present inventors have confirmed whether TC cells prepared according to the manufacturing process express TGF-β1 in order to establish a criterion for determining effectiveness of mixed cells as a cell therapeutic agent as in Example 1.

[0074] The TC cells were filled into a vial and inactivated in the frozen state. After inactivation, the frozen vial was taken out and thawed in a water bath at 37° C., and the cells were taken out of the vial and added in a conical tube containing a medium. The cells were centrifuged at 210×g for 5 minutes to remove supernatant. The cells were suspended in a culture medium and then inoculated into 3 wells to be 1.0×10.sup.5 cells/well in a 6-well plate. The cells were cultured in a 37° C. CO.sub.2 incubator for 24 hours, respectively.

[0075] After 24 hours, the medium was changed and the cells were cultured in a 37° C. CO.sub.2 incubator for 24 hours. After incubation, the spent medium was sampled by 1 mL At this time, 2 mL of medium was used as a negative control group in 3 wells. The amount of TGF-β1 in the sampling medium was measured by ELISA method and the negative control group was also measured in the same method. TGF-β1 secretion level was calculated as “average amount of TGF-β1 secretion in the sample—average amount of TGF-β1 secretion in the negative control group.”

[0076] According to the result, as shown in FIG. 1 and Table 1 below, it could be confirmed that TGF-β1 was expressed at an average of 5.07 ng/1×10.sup.5 cells/24 hours in TC cells.

TABLE-US-00001 TABLE 1 TGF-β1 (ng/1 × 10.sup.5 cells/24 hours) TC Average 5.07

Example 3. Determination of TGF-β1 Expression Level by Batch

[0077] The expression level of TGF-β1 was examined in each manufacturing batch in order to establish TGF-β1 expression criterion which exhibits effectiveness as a cell therapeutic agent in TC cells.

[0078] According to the result, as shown in FIG. 2 and Table 2 below, it could be confirmed that there was a difference in expression levels of TGF-β1 protein by batch.

TABLE-US-00002 TABLE 2 TC Batch 1 Batch 2 Batch 3 TGF-β1 (ng/1 × 10.sup.5 cells/24 hours) 9.86 9.87 12.46

[0079] As described above, since TGF-β1 protein expression level is different depending on the production batch, a greater difference may possibly occur when there is a change such as a process change. Therefore, it could be understood that a reference concentration for TGF-β1 expression should be established for quality control in regard to cell therapeutic agents capable of reproducing the same therapeutic effects.

Example 4. Verification of Relationship Between TGF-β1 Expression and Therapeutic Efficacy in Cell Therapeutic Agent

[0080] In order to verify whether there is substantially a close relationship between TGF-β1 expression level and therapeutic effects in TC cells, the present inventors have prepared an MIA osteoarthritis animal model, and then treated the model with the mixed cells and/or anti-TGF-β1 neutralizing antibody, followed by observing a change of pain.

[0081] 2 weeks after the MIA injection, CS-10 administration group (vehicle) as a control group and the mixed cells (1.2×10.sup.6) prepared by mixing HC and TC in a ratio of 3:1 were administered, respectively, into a joint cavity of a left knee of the test animal. For neutralization antibody test, a control antibody (IgG, 500 ng/30 μL) and TGF-β1 neutralizing antibody (anti-TGF-β1, 500 ng/30 μL) were administered into the joint cavity of the left knee on the day of the mixed cell administration and on 3.sup.rd day.

[0082] Thereafter, von Frey filament test was performed. This test was conducted using 50% up & down threshold method which was established in 1980 by Dixon (Chaplan S R et al., Quantitative assessment of tactile allodynia in the rat paw, Journal of Neuroscience Methods, 1994, 53: 55-63; and Dixon W. J., Efficient analysis of experimental observations, Annual Reviews Pharmacology Toxicology, 1980, 20: 441-62). Using a total of nine (9) von Frey filaments with N values of 0.4, 0.6, 1, 2, 4, 6, 8, and 15 grams (g), respectively, pain response was examined and a threshold value was calculated according to predetermined patterns.

[0083] According to the result, as shown in FIGS. 3A and 3B, the measured results were 1.46±0.54 in the CS-10 administration group (vehicle) and 4.87±0.8 in the mixed cell administration group, respectively, on 7th day after the administration. Such analgesic effects showed a similar tendency till 42th day after the administration of cells, and as compared to the CS-10 administration group, statistically significant analgesic efficacy was observed at all measured values (P<0.05).

[0084] Further, in order to identify how TGF-β1 secreted by TC influences on analgesic effects in a case of the mixed cell administration, when anti-TGF-β1 neutralizing antibody which neutralizes and inhibits activity of TGF-β1 protein thus to block therapeutic effects thereof was also added, it was observed that pain therapeutic effects on the mixed cell administration group correspond to the threshold value of 1.43±0.38 in TGF-β1 neutralizing antibody administration group (mixed cell+anti-TGF-β1) on 7th day after the administration, which exhibits pain similar to that of the CS-10 administration group. Further, no difference in pain therapeutic effects was detected till 42nd day. On the other hand, a group using the control antibody (IgG) (that is, mixed cells+IgG) retained pain therapeutic effects similar to the mixed cell administration group.

[0085] From the above result, it could be understood that TGF-β1 secreted from TC shows significant effects on osteoarthritis treatment.

Example 5. Identification of TSP-1 Expression in Cell Therapeutic Agent

[0086] In order to investigate whether or not TSP-1 (NCBI Reference Sequence: NM 003246.3) protein is expressed in HC and TC cells, respectively, as well as an expression level thereof, each cell vial being stored in the same method as described in Example 2 above was thawed and TSP-1 amount was measured by ELISA method.

[0087] According to the result, as shown in FIG. 4 and Table 3 below, TSP-1 expression levels of 180.37 ng/1×10.sup.5 cells/24 hours in HC cells and 0.29 ng/1×10.sup.5 cells/24 hours were expressed TC cells, respectively, thereby demonstrating inhibition of TSP-1 expression.

TABLE-US-00003 TABLE 3 TSP-1 (ng/1 × 10.sup.5 cells/24 hours) HC TC Average 180.37 0.29

Example 6. Determination of TSP-1 Expression Level by Batch

[0088] The expression level of TSP-1 was examined in each manufacturing batches as described in Example 2 above in order to establish TSP-1 expression standard showing effectiveness as a cell therapeutic agent in HC cells.

[0089] According to the result, as shown in FIG. 5 and Table 4 below, it could be confirmed that there was a difference in expression levels of TSP-1 by batch.

TABLE-US-00004 TABLE 4 HC Batch 1 Batch 2 Batch 3 TSP-1 (ng/1 × 10.sup.5 cells/24 hours) 76.41 162.30 116.14

[0090] As described above, since TSP-1 protein expression level is different depending on the production batch, a greater difference may possibly occur when there is a change such as a process change. Therefore, it could be understood that a reference concentration for TSP-1 expression should be established for quality control in regard to cell therapeutic agents capable of reproducing the same therapeutic effects.

Example 7. Verification of Relationship Between TSP-1 Expression and Therapeutic Efficacy in Cell Therapeutic Agent

[0091] In order to verify therapeutic effects of the mixed cells, the present inventors have prepared an MIA osteoarthritis animal model in the same manner as described in Example 4 above, and then treated the model with the mixed cells and/or anti-TSP-1 neutralizing antibody, followed by observing a change of pain.

[0092] According to the result, as shown in FIGS. 6A and 6B, the measured results were 1.19±0.23 in the CS-10 administration group (vehicle) and 6.79±1.03 in the mixed cell administration group, respectively, on 7th day after the administration. Such pain therapeutic effects showed a similar tendency till 42nd day after the mixed cell administration, and as compared to the CS-10 administration group, statistically significant analgesic efficacy was observed at all measured values (P<0.05).

[0093] Further, in order to identify how TSP-1 secreted by HC influences on analgesic efficacy of mixed cells in a case of the mixed cell administration, when anti-TSP-1 neutralizing antibody which neutralizes and inhibits activity of TSP-1 protein thus to block therapeutic effects thereof was also added, it was observed that pain therapeutic effects on the mixed cell administration group correspond to the threshold value of 2.28±0.54 in TSP-1 neutralizing antibody administration group (mixed cell+anti-TSP-1) on 7th day after the administration, which exhibits pain similar to that of the CS-10 administration group. Further, no difference in pain therapeutic effects was detected till 42 days. On the other hand, a group using the control antibody (IgM) (that is, mixed cells+IgM) exhibited pain therapeutic effects similar to the mixed cell administration group (p<0.05).

[0094] From the above result, it could be understood that TSP-1 secreted from HC shows significant effects on osteoarthritis treatment.

Example 8. Verification of minimum dosage of TGF-β1 in cell therapeutic agent

[0095] In order to determine a minimum value of TGF-β1 in TC which is a constitutive cell of mixed cells, the present inventors have used TCs showing different TGF-β1 values for osteoarthritis-induced subjects 2 weeks after MIA injection and treated the subjects with mixed cells (2.8×10.sup.5 cells) which were prepared by mixing HCs and TCs in a ratio of cells of 3:1, followed by observing a change of pain.

[0096] At this time, in order to embody a situation of TCs having different TGF-β1 expression levels under influence of different conditions, TCs having different TGF-β1 values were prepared. Such TCs were prepared by shRNA treatment of TGF-β1 and 150 Gy gamma irradiation, and TGF-β1 average (ng/1×10.sup.5 cells/24 hours) were determined as shown in Table 5 below.

TABLE-US-00005 TABLE 5 shRNA TC control shRNA 150 Gy TGF-β1 (ng/1 × 10.sup.5 cells/24 hours) 21.71 7.00 0.35 0.63

[0097] According to the result, as shown in FIGS. 7A and 7B, the measured results of CS-10 administration group (vehicle) and the mixed cell administration group were 1.17±0.53 and 6.06±1.91, respectively, on 14th day after the administration. Such pain therapeutic effects showed a similar tendency till 42nd day after the administration of cells, and as compared to the CS-10 administration group, statistically significant analgesic efficacy was observed at all measured values (P<0.05).

[0098] Further, on the 14th day after the administration of cells, the measured results were obtained as follows: 6.21±1.59 for the shRNA control (shCON) treatment administration group; 1.39±0.23 for the mixed cell administration group using 150 Gy gamma-irradiated TC; and 0.86±0.49 for the mixed cell administration group along with shRNA treatment to TGF-β1, respectively. These effects were similar till 42nd day after the administration of cells.

[0099] When von Frey filament measurement results were expressed as AUC values, statistically significant results were also demonstrated in the mixed cell administration group and the shRNA control (shCON) treatment administration group, as compared to the control group, i.e., the CS-10 administration group (p<0.05).

[0100] According to the result of H & E staining analysis for tissues isolated from the same animal model, improvement in the cartilage structure was observed in the mixed cell administration group and the shCON treatment administration group, as shown in FIG. 7C.

[0101] On the other hand, no improvement in the cartilage structure was observed in the CS-10 administration group (vehicle) as the control group, the mixed cell administration group along with shRNA treatment to TGF-β1 and the mixed cell administration group using 150 Gy gamma-irradiated TC, respectively.

[0102] In addition, in order to determine a minimum effective value for pain relief by TGF-β1 secreted from TC as well as cartilage structure improvement, analgesic effects and improvement in the cartilage structure by mixed cells using TC produced in another batch were examined.

[0103] TGF-β1 values of the prepared TC were 9 ng/1×10.sup.5 cells/24 hours and 1.7 ng/1×10.sup.5 cells/24 hours, respectively. These TCs were used in preparation of the mixed cells. The prepared mixed cells were indicated as 9 ng_mixed cells and 1.7 ng_mixed cells, respectively.

[0104] According to the result, as shown in FIGS. 8A and 8B, on 14th day after the administration of 9 ng_mixed cells, 1.7 ng_mixed cells and CS-10 administration group (vehicle) as the control group, respectively, the measured results were as follows: 1.48±0.26 for the CS-10 administration group; 5.57±1.13 for 9 ng_mixed cells; and 7.41±1.21 for 1.7 ng_mixed cells. There was a similar tendency till 42nd day after the administration of cells. In comparison with the CS-10 administration group, both of 9 ng_mixed cell and 1.7 ng_mixed cell administration groups showed statistical significance (P<0.05).

[0105] When von Frey filament measurement results were expressed as AUC values, statistically significant results were also demonstrated in the 9 ng mixed cell and 1.7 ng_mixed cell administration groups, as compared to the control group, i.e., the CS-10 administration group (p<0.05).

[0106] Therefore, in the results obtained after the administration of mixed cells using different TGF-β1s secreted from TC, the minimum effective values of TGF-β1 for pain relief and improvement in the cartilage structure were measured to be higher than 0.63 ng/1×10.sup.5 cells/24 hours determined in the 150 Gy gamma-irradiated mixed cell administration group, i.e., 0.65 ng/1×10.sup.5 cells/24 hours or more.

Example 9. Verification of Minimum Dosage of TSP-1 in Cell Therapeutic Agent

[0107] In order to determine a minimum value of TSP-1 in HC which is a constituent cell of mixed cells, the present inventors have treated osteoarthritis-induced subjects 2 weeks after MIA injection with mixed cells (2.8×10.sup.5), which were prepared by mixing HCs and TCs in a ratio of 3:1, wherein HCs exhibit different TSP-1 values for the subjects, followed by observing a change of pain.

[0108] At this time, in order to embody HC situation different in TSP-1 expression levels due to different conditions, HCs exhibiting different expression levels of TSP-1 were prepared.

[0109] Such HCs with different TSP-1 values were prepared using siRNA to TSP-1, and average TSP-1 values (ng/1×10.sup.5 cells/24 hours) were determined as shown in Table 6 below.

TABLE-US-00006 TABLE 6 siRNA siRNA siRNA HC control 1 2 TSP-1 (ng/1 × 10.sup.5 cells/24 hours) 349.03 200.09 92.13 30.53

[0110] According to the result, as shown in FIGS. 9A and 9B, the measured results were as follows: 0.6±0.19 for the CS-10 administration group (vehicle); 4.7±1.03 for the mixed cell administration group; and 4.7±1.67 for the mixed cell+siRNA control administration group, respectively, on 14th day after the administration. Such effects showed a similar tendency till 42nd day after the administration of cells, and as compared to the CS-10 administration group, statistically significant analgesic efficacy was observed at all measured values (P<0.05).

[0111] Further, on 14th day after the administration, the measured results for the mixed cell+siRNA 1 administration group and the mixed cell+siRNA 2 administration group were 3.95±0.94 and 1.89±0.9, respectively. As compared to the CS-10 administration group, statistically significant analgesic efficacy was observed in the mixed cell+siRNA 1 administration group. These pain therapeutic effects have a similar tendency till 42nd day after the administration of cells.

[0112] When von Frey filament measurement results were expressed as AUC values, statistically significant results were also obtained in the mixed cell administration group, the siRNA control treatment administration group and the mixed cell+siRNA 1 administration group, respectively, as compared to the control group, i.e., CS-10 administration group (vehicle) (P<0.05).

[0113] In addition, according to the result of H & E staining analysis for tissues isolated from the same animal model, as shown in FIG. 9C, improvements in the cartilage structure were observed in the mixed cell administration group, the siRNA control treatment administration group and the mixed cell+siRNA 1 administration group. On the other hand, for the CS-10 administration group (vehicle) as the control group and the mixed cell+siRNA 2 administration group, no improvement in the cartilage structure was observed.

[0114] Therefore, it was determined that the minimum effective value for pain relief and improvement in the cartilage structure of TSP-1 secreted from HC was higher than 30.53 ng/1×10.sup.5 cells/24 hours, which was determined in the mixed cell+siRNA 2 administration group, specifically, 31 ng/1×10.sup.5 cells/24 hours or more.

[0115] In conclusion, when using the method for evaluating effectiveness of an osteoarthritis therapeutic agent established according to the present invention, based on: (a) a specific TGF-β expression level in transformed mammalian cells with TGF-β as a first population; and (b) a specific TSP-1 expression level in untransformed mammalian cells with the same gene as a second population, effectiveness (i.e., therapeutic efficacy) of individual cell therapeutic agents can be reliably determined prior to initiation of treatment, thereby accomplishing uniform therapeutic effects.