Selection of cryopreserved umbilical cord blood units for the production of enhanced natural killer cells.

Abstract

Embodiments of this disclosure relate to methods and compositions relating to the optimization and selection of umbilical cord blood units for the production of immune cells, such as natural killer (NK) cells, for use in adoptive cell therapy. In certain embodiments, specific properties of an umbilical cord blood unit and / or the properties of cells obtained therefrom are analyzed. If a threshold measurement is met for one or more properties of the umbilical cord blood unit and / or the properties of cells obtained therefrom, the umbilical cord blood unit is used as a source for the production of immune cells. Specific properties to be measured include, for example, the viability of umbilical cord blood cells, the time from birth to cryopreservation of the umbilical cord blood unit, the total nucleated cell recovery rate, infant weight, infant sex, maternal age, gestational age, CD34-positive cell percentage, and / or nucleated red blood cell content. These properties may be determined before and / or after cryopreservation.

Claims

1. A method for selecting cryopreserved umbilical cord blood units (CBUs) for the production of improved immune cells, comprising selecting CBUs based on the time elapsed since birth of the infant from which the CBUs originated and the cryopreservation of the CBUs.

2. The method according to claim 1, further comprising selecting a CBU that has been cryogenically stored within approximately or exactly 32, 30, 28, 26, 24, 22, or 20 hours after the birth of the infant from which the CBU originated.

3. The method according to claim 1, wherein the CBU is cryogenically stored within exactly or approximately 24 hours after the birth of the infant.

4. The method according to claim 1, further comprising selecting a CBU having the following nucleated red blood cell (NRBC) content: a) If measured after reduction, 8.5 × 10 ７ , 8.0 x 10 ７ , or 7.5 x 10 ７ Less than one cell, or exactly or about 8.5 × 10⁻⁶ ７ , 8.0 x 10 ７ , or 7.5 x 10 ７ Individual cells, NRBC content, b) If measured before reduction, less than 9.9×10 ７ , 9.4×10 ７ , or less than 8.9×10 ７ cells, or exactly or approximately 9.9×10 ７ , 9.4×10 ７ , or 8.9×10 ７ cells, the NRBC content, and / or c) NRBC content, when measured after reduction, being less than 5%, 4%, or 3% of total nucleated cells (TNCs), or exactly or approximately 5%, 4%, or 3%.

5. If the NRBC content is measured after reduction, then 8.0 × 10 ７ Less than one cell, or exactly or about 8.0 x 10⁻⁶ ７ If measured before reduction, the individual cells are 9.4 × 10⁻⁶. ７ Less than one cell, or exactly or approximately 9.4 x 10⁻⁶ ７ The method according to claim 4, wherein the cells are individual cells and / or, when measured after reduction, are less than 4% of the total nucleated cells (TNCs), or exactly or about 4%.

6. The method according to claim 1, wherein the CBU is not selected based on the relative levels of one or more immune cells.

7. The method according to claim 6, wherein the CBU is not selected based on the proportion of NK cells, CD8+ T cells, CD4+ T cells, regulatory T cells, B cells, monocyte-derived dendritic cells (Mo-DCs), and / or plasmacytoid dendritic cells (pDCs).

8. The method according to claim 6, wherein the CBU does not have a significant difference in the proportion of NK cells, CD8+ T cells, CD4+ T cells, regulatory T cells, B cells, monocyte-derived dendritic cells (Mo-DCs), and / or plasmacytoid dendritic cells (pDCs).

9. The method according to claim 1, further comprising selecting a CBU based on the following: a) Total cell viability before low-temperature storage, b) Percentage of total CD34-positive cells, c) Infant weight, d) Race of the infant's parents, e) The age of the infant's mother, f) The gestation period of an infant, g) Method of collecting umbilical cord blood, h) Sex of the infant, and / or i) The volume of umbilical cord blood collected before processing.

10. The method according to claim 9, further comprising selecting a CBU based on the following: a) The total cell viability before low-temperature storage is greater than 95%, exactly 95%, or approximately 95%. b) The percentage of total CD34-positive cells is greater than 0.2%, exactly 0.2%, or approximately 0.2%. c) The infant's weight is greater than, exactly, or approximately 3,000 grams, d) The infant has at least one Caucasian parent, e) The mother's age is under 34 years, or exactly or approximately 34 years, f) The gestational age of the infant is less than 40 weeks, or exactly 40 weeks or approximately 40 weeks. g) The umbilical cord blood is collected intrauterine and / or extrauterine, h) The infant is male, and / or i) The volume of the collected umbilical cord blood before processing is less than 150 mL, or exactly or approximately 150 mL.

11. The method according to claim 9, further comprising selecting a CBU based on the following: a) The total cell viability before low-temperature storage is greater than 98.5%, or exactly or approximately 98.5%. b) The percentage of total CD34-positive cells is greater than 0.245%, or exactly or approximately 0.245%. c) The infant's weight is greater than, exactly, or approximately 3,650 grams, d) The infant has at least one Caucasian parent, e) The mother's age is under 32 years, or exactly or approximately 32 years, f) The gestational age of the infant is less than 38 weeks, or exactly or approximately 38 weeks, g) The umbilical cord blood is collected in the uterus. h) The infant is male, and / or i) The volume of the collected umbilical cord blood before processing is less than 120 mL, or exactly or approximately 120 mL.

12. The method according to claim 11, wherein at least three of the selection factors described above are used.

13. The method according to claim 1, wherein the phenotypic, transcriptional, and / or epigenetic signature of the immune cells is different from that of immune cells not selected based on the time from birth of the infant from which the CBU was derived and the cryopreservation of the CBU.

14. The method according to claim 13, wherein the immune cells have an increased multifunctional strength index (PSI).

15. The method according to claim 14, wherein the increased PSI includes increased effector PSI, increased irritant PSI, and / or increased chemoattractant PSI.

16. The method according to claim 13, wherein the immune cells have increased chromatin accessibility and / or transcription levels of genes encoding ZIC2, GLI3, TBX21, IRF2, IRF3, IRF4, IRF7, IRF8, IRF9, NKX2-3, NKX2-8, GLI2, EOMES, GZMA, CXCR6, CMKLR1, NKG2D, CD16, 2B4, T-BET, PFN, GZMA, and / or PRF1.

17. The method according to claim 13, wherein the immune cells have increased population doubling acceleration and / or increased protein secretion rate.

18. The method according to claim 13, wherein the immune cells have increased basal respiration and / or maximum respiratory rate.

19. The method according to claim 13, wherein the immune cells have reduced chromatin accessibility and / or transcription levels of genes encoding ATF1, ATF2, ATF3, ATF7, CREB1, CREB5, NFAT2, NFATC2, FOX, JUN, JUNB, SMAD2, SMAD3, HIF1A, MAFF, JMJD6, DDIT3, SIAH2, NR4A1, DNAJA1, BAK1, NFKB1, IL-10, LAG3, HASP90AB1, HSPA5, and / or HSPA13.

20. The method according to claim 13, wherein the immune cells have a reduced rate of trogocytosis and / or reduced transcription levels of NF-κβ-mediated TNFα signaling, UV response, hypoxia, IL2 STAT5 signaling, heme metabolism, apoptosis, inflammatory response, early estrogen response, G2M checkpoint, TGFβ signaling, p53 pathway, cholesterol homeostasis, KRAS signaling, and / or hallmark of Myc target V1.

21. The method according to claim 13, wherein the immune cells have reduced regulon activity of NR4A1, JUND, BCL3, MEF2D, HOXA5, FOXB, JUN, MAFF, ZNF281, KLF6, REL, CEBPG, KLF16, HIF1A, FOS, BCLAF1, GATA3, FOSL2, RARG, EGR2, and / or MAF.

22. The method according to claim 1, wherein the immune cells are natural killer (NK) cells.

23. The method according to claim 22, further comprising the step of expanding and proliferating the NK cells.

24. The method according to claim 23, wherein the CBU is reselected based on the following: a) Expansion and proliferation of NK cells from day 0 to day 15 of culture, and / or b) Expansion and proliferation of NK cells from day 6 to day 15 of culture.

25. In the above method, a) The expansion and proliferation of NK cells from day 0 to day 15 of culture is greater than 350 times, or exactly or approximately 350 times, and / or b) The expansion and proliferation of NK cells from day 6 to day 15 of culture is greater than 50 times, or exactly 50 times, The method according to claim 24.

26. In the above method, a) The expansion and proliferation of NK cells from day 0 to day 15 of culture is greater than 450 times, exactly or approximately 450 times, and / or b) The expansion and proliferation of NK cells from day 6 to day 15 of culture is greater than 70 times, exactly 70 times, or approximately 70 times. The method according to claim 24.

27. The method according to claim 22, wherein the method further includes a step of modifying the NK cells.

28. The method according to claim 27, wherein the NK cells are modified to express one or more non-endogenous gene products.

29. The method according to claim 28, wherein the non-endogenous gene product includes an antigen receptor, a cytokine, a homing receptor, a chemokine receptor, and combinations thereof.

30. The method according to claim 29, wherein the non-endogenous receptor is a chimeric receptor.

31. The method according to claim 30, wherein the chimeric receptor is a chimeric antigen receptor (CAR).

32. The method according to claim 31, wherein the CAR targets CD19, CD70, and / or TROP2.

33. The method according to claim 29, wherein the non-endogenous receptor is a T cell receptor (TCR).

34. The method according to claim 28, wherein the method further comprises the expression of one or more non-endogenous cytokines.

35. The method according to claim 34, wherein the cytokine is IL-15 and / or IL-21.

36. The method according to claim 22, wherein the NK cells are pre-activated by one or more cytokines.

37. The method according to claim 36, wherein the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

38. The method according to claim 22, wherein the NK cells include one or more manipulated mutations in an endogenous gene.

39. The method according to claim 38, wherein the endogenous gene is GR, TGFBR2, CISH, and / or CD38.

40. A composition comprising a CBU identified by the method described in any one of claims 1 to 39.

41. The composition according to claim 40, wherein the composition is contained in a pharmaceutically acceptable carrier.

42. The composition according to claim 40, wherein the composition is blended with one or more cryoprotective agents.

43. A composition comprising a population of immune cells derived from a CBU selected using the method described in any one of claims 1 to 39.

44. A method for treating a subject having cancer, comprising administering a population of immune cells as described in claim 43.

45. The method according to claim 44, wherein the subjects have a proportion of increased overall response rate (OR), complete response (CR), progression-free survival (PFS), and / or overall survival (OS) compared to subjects not treated with the population of immune cells.

46. A method for producing engineered immune cells, comprising manipulating a population of immune cells to express one or more non-endogenous gene products, wherein the immune cells are obtained from a population of umbilical cord blood cells from the birth of an infant, and the population of umbilical cord blood cells has the following characteristics before cryopreservation: (a) The umbilical cord blood cells were cryogenically stored within approximately or exactly 32, 30, 28, 26, 24, 22, or 20 hours after the birth of the infant from whom the umbilical cord blood cells were obtained; and (b) Contains the following nucleated red blood cell (NRBC) content: i) If measured after reduction, 8.5 × 10 ７ , 8.0 x 10 ７ , or 7.5 x 10 ７ Less than one cell, or exactly or about 8.5 × 10⁻⁶ ７ , 8.0 x 10 ７ , or 7.5 x 10 ７ Individual cells, NRBC content, ii) If measured before reduction, 9.9 × 10 ７ , 9.4 x 10 ７ , or 8.9 × 10 ７ Less than one cell, or exactly or approximately 9.9 × 10⁻⁶ ７ , 9.4 x 10 ７ , or 8.9 × 10 ７ Individual cells, NRBC content, and / or iii) NRBC content, when measured after reduction, being less than 5%, 4%, or 3% of total nucleated cells (TNCs), or exactly or approximately 5%, 4%, or 3%; In addition, optionally, (c) The total cell viability before cryopreservation is greater than 95%, exactly 95%, or approximately 95%. (d) The percentage of total CD34-positive cells is greater than 0.2%, exactly 0.2%, or approximately 0.2%. (e) The infant's weight is greater than, exactly, or approximately 3,000 grams, (f) The infant has at least one Caucasian parent, (g) The mother's age is under 34, or exactly or approximately 34 years old. (h) The infant's gestation period is less than 40 weeks, or exactly 40 weeks or approximately 40 weeks. (i) Umbilical cord blood is collected intrauterine and / or extrauterine, (j) The infant is male, and / or (k) The volume of the collected umbilical cord blood before processing is less than 150 mL, or exactly or approximately 150 mL.

47. In the above method, (a) The umbilical cord blood cells have been cryopreserved at precisely or within approximately 24 hours after the birth of the infant; and (b) Contains the following nucleated red blood cell (NRBC) content: i) If measured after reduction, 8.0 × 10 ７ Less than one cell, or exactly or about 8.0 x 10⁻⁶ ７ Individual cells, NRBC content, ii) If measured before reduction, 9.4 × 10 ７ Less than one cell, or exactly or approximately 9.4 × 10⁻⁶ ７ Individual cells, NRBC content, and / or iii) NRBC content, when measured after reduction, is less than 4% of total nucleated cells (TNCs), or exactly or approximately 4%; In addition, optionally, (c) The total cell viability before cryopreservation is greater than 98.5%, exactly 98.5%, or approximately 98.5%. (d) The percentage of total CD34-positive cells is greater than 0.245%, exactly 0.245%, (e) The infant's weight is greater than, exactly, or approximately 3,650 grams, (f) The infant has at least one Caucasian parent, (g) The mother's age is under 32, or exactly 32 years old or approximately 32 years old. (h) The infant's gestation period is less than 38 weeks, or exactly 38 weeks or approximately 38 weeks. (i) Umbilical cord blood is collected in the uterus. (j) The infant is male, and / or (k) The volume of the collected umbilical cord blood before processing is less than 120 mL, or exactly or approximately 120 mL. The method according to claim 46.

48. A method for producing raw materials for the production of a composition containing immune cells, comprising cryopreserving a cell population containing immune cells derived from a population of umbilical cord blood cells from the birth of an infant, Before low-temperature storage, the population of umbilical cord blood cells has the following characteristics: (a) The umbilical cord blood cells were cryogenically stored within approximately or exactly 32, 30, 28, 26, 24, 22, or 20 hours after the birth of the infant from whom the umbilical cord blood cells were obtained; and (b) Contains the following nucleated red blood cell (NRBC) content: i) If measured after reduction, 8.5 × 10 ７ , 8.0 x 10 ７ , or 7.5 x 10 ７ Less than one cell, or exactly or about 8.5 × 10⁻⁶ ７ , 8.0 x 10 ７ , or 7.5 x 10 ７ Individual cells, NRBC content, ii) If measured before reduction, 9.9 × 10 ７ , 9.4 x 10 ７ , or 8.9 × 10 ７ Less than one cell, or exactly or approximately 9.9 × 10⁻⁶ ７ , 9.4 x 10 ７ , or 8.9 × 10 ７ Individual cells, NRBC content, and / or iii) NRBC content, when measured after reduction, being less than 5%, 4%, or 3% of total nucleated cells (TNCs), or exactly or approximately 5%, 4%, or 3%; In addition, optionally, (c) The total cell viability before cryopreservation is greater than 95%, exactly 95%, or approximately 95%. (d) The percentage of total CD34-positive cells is greater than 0.2%, exactly 0.2%, or approximately 0.2%. (e) The infant's weight is greater than, exactly, or approximately 3,000 grams, (f) The infant has at least one Caucasian parent, (g) The mother's age is under 34, or exactly or approximately 34 years old. (h) The infant's gestation period is less than 40 weeks, or exactly 40 weeks or approximately 40 weeks. (i) Umbilical cord blood is collected intrauterine and / or extrauterine, (j) The infant is male, and / or (k) The volume of the collected umbilical cord blood before processing is less than 150 mL, or exactly or approximately 150 mL.

49. In the above method, (a) The umbilical cord blood cells have been cryopreserved at precisely or within approximately 24 hours after the birth of the infant; and (b) Contains the following nucleated red blood cell (NRBC) content: i) If measured after reduction, 8.0 × 10 ７ Less than one cell, or exactly or about 8.0 x 10⁻⁶ ７ Individual cells, NRBC content, ii) If measured before reduction, 9.4 × 10 ７ Less than one cell, or exactly or approximately 9.4 × 10⁻⁶ ７ Individual cells, NRBC content, and / or iii) NRBC content, when measured after reduction, is less than 4% of total nucleated cells (TNCs), or exactly or approximately 4%; In addition, optionally, (c) The total cell viability before cryopreservation is greater than 98.5%, exactly 98.5%, or approximately 98.5%. (d) The percentage of total CD34-positive cells is greater than 0.245%, exactly 0.245%, (e) The infant's weight is greater than, exactly, or approximately 3,650 grams, (f) The infant has at least one Caucasian parent, (g) The mother's age is under 32, or exactly 32 years old or approximately 32 years old. (h) The infant's gestation period is less than 38 weeks, or exactly 38 weeks or approximately 38 weeks. (i) Umbilical cord blood is collected in the uterus. (j) The infant is male, and / or (k) The volume of the collected umbilical cord blood before processing is less than 120 mL, or exactly or approximately 120 mL. The method according to claim 48.

50. A composition comprising a population of isolated umbilical cord blood-derived immune cells, wherein the immune cells are obtained from one or more umbilical cord blood units from the birth of an infant. The umbilical cord blood unit has the following characteristics: (a) The umbilical cord blood cells were cryogenically stored within approximately or exactly 32, 30, 28, 26, 24, 22, or 20 hours after the birth of the infant from whom the umbilical cord blood cells were obtained; and (b) Contains the following nucleated red blood cell (NRBC) content: i) If measured after reduction, 8.5 × 10 ７ , 8.0 x 10 ７ , or 7.5 x 10 ７ Less than one cell, or exactly or about 8.5 × 10⁻⁶ ７ , 8.0 x 10 ７ , or 7.5 x 10 ７ Individual cells, NRBC content, ii) If measured before reduction, 9.9 × 10 ７ , 9.4 x 10 ７ , or 8.9 × 10 ７ Less than one cell, or exactly or approximately 9.9 × 10⁻⁶ ７ , 9.4 x 10 ７ , or 8.9 × 10 ７ Individual cells, NRBC content, and / or iii) NRBC content, when measured after reduction, being less than 5%, 4%, or 3% of total nucleated cells (TNCs), or exactly or approximately 5%, 4%, or 3%; In addition, optionally, (c) The total cell viability before cryopreservation is greater than 95%, exactly 95%, or approximately 95%. (d) The percentage of total CD34-positive cells is greater than 0.2%, exactly 0.2%, or approximately 0.2%. (e) The infant's weight is greater than, exactly, or approximately 3,000 grams, (f) The infant has at least one Caucasian parent, (g) The mother's age is under 34, or exactly or approximately 34 years old. (h) The infant's gestation period is less than 40 weeks, or exactly 40 weeks or approximately 40 weeks. (i) Umbilical cord blood is collected intrauterine and / or extrauterine, (j) The infant is male, and / or (k) The volume of the collected umbilical cord blood before processing is less than 150 mL, or exactly or approximately 150 mL.

51. In the above composition, (a) The umbilical cord blood cells have been cryopreserved at or within approximately 24 hours after the birth of the infant; and (b) Contains the following nucleated red blood cell (NRBC) content: i) If measured after reduction, 8.0 × 10 ７ Less than one cell, or exactly or about 8.0 x 10⁻⁶ ７ Individual cells, NRBC content, ii) If measured before reduction, 9.4 × 10 ７ Less than one cell, or exactly or approximately 9.4 × 10⁻⁶ ７ Individual cells, NRBC content, and / or iii) NRBC content, when measured after reduction, is less than 4% of total nucleated cells (TNCs), or exactly or approximately 4%; In addition, optionally, (c) The total cell viability before cryopreservation is greater than 98.5%, exactly 98.5%, or approximately 98.5%. (d) The percentage of total CD34-positive cells is greater than 0.245%, exactly 0.245%, (e) The infant's weight is greater than, exactly, or approximately 3,650 grams, (f) The infant has at least one Caucasian parent, (g) The mother's age is under 32, or exactly 32 years old or approximately 32 years old. (h) The infant's gestation period is less than 38 weeks, or exactly 38 weeks or approximately 38 weeks. (i) Umbilical cord blood is collected in the uterus. (j) The infant is male, and / or (k) The volume of the collected umbilical cord blood before processing is less than 120 mL, or exactly or approximately 120 mL. The composition according to claim 50.

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