Snippets from a recent patent covering GvHDRegPotency Assay
[0180] In an example, the present disclosure encompasses a method for determining the therapeutic efficacy of mesenchymal lineage precursor or stem cells comprising: [0181] (i) obtaining a population comprising mesenchymal lineage precursor or stem cells; [0182] (ii) culturing the cells in a culture medium; and [0183] (iii) determining the amount of TNF-R1 expressed by the cells into the culture medium under culture conditions.
[0184] In an example, an amount of at least about 200 pg/ml [23.5 pg/10.sup.6 cells] TNF-R1 is indicative of therapeutic efficacy. In an example, an amount of at least about 225 pg/ml [26.5 pg/10.sup.6 cells] TNF-R1 is indicative of therapeutic efficacy. In another example, at least about 230 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, at least about 250 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, at least about 260 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, the at least about 270 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, at least about 280 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, at least about 290 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, at least about 300 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, between 200 pg/ml and 500 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, between 220 pg/ml and 450 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, between 225 pg/ml and 450 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, a corresponding amount of TNF-R1 in pg/10.sup.6 cells is indicative of therapeutic efficacy. In another example, between 230 pg/ml and 450 pg/ml TNF-R1 is indicative of therapeutic efficacy. In another example, a corresponding amount of TNF-R1 in pg/10.sup.6 cells is indicative of therapeutic efficacy.
[0185] In another example, at least about 55% inhibition of IL-2Rα expression is indicative of therapeutic efficacy. In another example, at least about 58% inhibition of IL-2Rα expression is indicative of therapeutic efficacy. In another example, at least about 60% inhibition of IL-2Rα expression is indicative of therapeutic efficacy. In another example, between 55% and 75% inhibition of IL-2Rα expression is indicative of therapeutic efficacy. In another example, between 58% and 65% inhibition of IL-2Rα expression is indicative of therapeutic efficacy.
[0186] In an example, the present disclosure encompasses a method for determining the therapeutic efficacy of mesenchymal lineage precursor or stem cells comprising: [0187] (i) obtaining a population comprising mesenchymal lineage precursor or stem cells; [0188] (ii) culturing the cells in a culture medium; and [0189] (iii) determining the amount of IL-2Rα inhibition by the cells under culture conditions. In an example, at least about 55% inhibition of IL-2Rα expression is indicative of therapeutic efficacy. In another example, at least about 58% inhibition of IL-2Rα expression is indicative of therapeutic efficacy. In another example, at least about 60% inhibition of IL-2Rα expression is indicative of therapeutic efficacy. In another example, between 55% and 75% inhibition of IL-2Rα expression is indicative of therapeutic efficacy. In another example, between 58% and 65% inhibition of IL-2Rα expression is indicative of therapeutic efficacy.
[0190] In an example, the population of mesenchymal lineage precursor or stem cells is obtained from a 3D cell culture. For example, the population can be obtained from a bioreactor.
[0191] In an example, the population of mesenchymal lineage precursor or stem cells are culture expanded in 3D cell culture from a cryopreserved intermediate MLPSC preparation. In an example, the level of TNF-R1 is determined before the cells are cryopreserved.
Example 3: Improved Manufacturing Method
[0225] Mesenchymal stem cells (MSC)s were defrosted from a cryopreserved intermediate population of MSCs and established in culture before being culture expanded in cell factories and then a bioreactor using a streamlined manufacturing process that reduces cell handling. Cells were culture expanded without CO.sub.2 priming of cell factories and cytomate was eliminated from the culture process after passage 3. Removing CO.sub.2 priming from the culture process reduced handling of cell factories and elimination of cytomate from passage 3 onwards required fewer sterile washes of the cells to complete a passage step. Furthermore, cells were contacted with trypsin for a minimum of 15 minutes when passaged, minimising handling of culture vessels.
[0226] TNF-R1 expression typically decreases significantly following cryopreservation of cells. However, TNF-R1 expression analysis of MSCs culture expanded from a cryopreserved intermediate via the improved manufacturing process with reduced cell handling surprisingly revealed that the cells expressed high levels of TNF-R1. As shown inFIG.5, the cells express higher levels than previously produced cells (333 pg/ml v 218 pg/ml).
[0227] Next, clinical data was interrogated to examine directly whether there was a relationship between survival and whether patients received product made with cells culture expanded using previous techniques (“original manufacturing process”) or the improved manufacturing process discussed above. As shown below in Table 2, MSCs produced via the improved manufacturing process expressed significantly higher levels of TNF-R1 and significantly increased IL2-Rα inhibition. Furthermore, patients dosed with these MSCs had elevated 28 day overall response (OR) and significantly higher prospects of 100 day overall survival (OS).
TABLE-US-00003 TABLE 2 Higher levels of TNF-R1, IL2-Rα inhibition and day 100 survival in patients treated with MSCs produced using the improved manufacturing process. TNFR1 (SD) IL-2Ra (SD) Day 28 Day 100 All Patients (pg/mL) (% inhibition) OR OS Only Original 213 (32) 56 (25) 63% 58% process (N = 348) Only Improved 328 (39) 79 (6) 70% 75% process (n = 92) P-value <0.0001 <0.0001 0.2643 0.0026 *p-value for mean TNF-R1 from t-test; p-value for 100 day OS from Fisher's Exact test
[0228] Taken together with the above findings from Examples 1 and 2, the present inventors have identified a population of MLPSCs with improved therapeutic efficacy, in particular MLPSCs that express at least 200 pg/ml TNF-R1, preferably 225 pg/ml and have provided a method of producing such cells, in particular MLPSCs expressing levels of TNF-R1 exceeding 331 pg/ml.
Claims
1. A composition comprising a population of culture expanded mesenchymal lineage precursor or stem cells (MLPSC), wherein the population of MLPSCs are culture expanded from a cryopreserved intermediate MLPSC preparation and the culture expanded MLPSCs express a receptor which, upon activation via an inflammatory stimulus, phosphorylates NF-κB, wherein the MLPSCs express a level of the receptor that is sufficient to increase phosphorylation of NF-κB upon activation via the inflammatory stimulus at least 3.5 fold greater than unstimulated MLPSCs.
2. (canceled)
3. The composition according to claim 1, wherein the receptor is one or both of TNF-R1 or IL-1R.
4. The composition according to claim 1, wherein activation of the MLPSCs via the inflammatory stimulus increases secretion of one or more of MCP-1, M-CSF and PGE2 under culture conditions.
5. (canceled)
6. The composition according to claim 1, wherein the receptor which, upon activation via an inflammatory stimulus, phosphorylates NF-κB is TNF-R1 and the MLPSCs express at least about 100 pg/ml to about 225 pg/ml TNF-R1 under culture conditions.
7. The composition according to claim 1, wherein the composition comprises at least 25×10.sup.6 cells.
8-9. (canceled)
10. A composition comprising a population of culture expanded mesenchymal lineage precursor or stem cells, wherein the composition comprises at least 25×10.sup.6 cells and, wherein the population of culture expanded cells has been selected for use in treatment of an inflammatory disease by determining expression of least about 200 pg/ml TNF-R1 under culture conditions.
11. The composition according to claim 10, wherein the population of culture expanded cells express at least about 100 pg/ml TNF-R1 to about 270 pg/ml TNF-R1 under culture conditions.
12-13. (canceled)
14. The composition according to claim 10, wherein the inflammatory disease is graft versus host disease (GvHD).
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