ABCB5 ligand and substrate

文档序号：913544 发布日期：2021-02-26 浏览：10次中文

阅读说明：本技术 Abcb5配体和底物 (ABCB5 ligand and substrate ) 是由马库斯·H·弗兰克于 2019-04-25 设计创作，主要内容包括：本发明涉及用于调节ABCB5+干细胞活性的方法和组合物。本发明还涉及用于操纵和表征调节ABCB5+细胞信号转导之化合物的测定和试剂。(The present invention relates to methods and compositions for modulating the activity of ABCB5+ stem cells. The invention also relates to assays and reagents for the manipulation and characterization of compounds that modulate ABCB5+ cell signaling.)

1.A method of enhancing ABCB5 positive cell function comprising administering to a subject in need thereof an effective amount of a composition that enhances the ABCB5-PIP2 pathway.

2. The method of claim 1, further comprising assessing ABCB5-PIP2 binding after administration of the composition.

3. The method of claim 1, wherein the composition is a PIP2 or PIP2 agonist.

4. The method of claim 1, wherein the subject is a human or non-human animal, including a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, cat, dog, llama, or primate, such as a monkey.

5. The method of claim 1, wherein the composition comprises a phospholipid.

6. The method of claim 1, wherein the composition comprises [ PIP2 (6: 0/18: 0) -H]^-And a pharmaceutically acceptable carrier.

7. The method of claim 1, wherein the composition comprises a phospholipid comprising a compound having the structure:

wherein R1 and R2 are independent fatty acid chains; and is

Wherein the length of R1 and R2 is at least twice as long as the other of R1 and R2.

8. The method of claim 7, wherein the structure has a 22: 0-26: 0 total fatty acid chain.

9. The method of claim 7, wherein the structure has a 24: 0 total fatty acid chain.

10. The method of claim 1, wherein the subject is a healthy subject.

11. The method of claim 1, wherein the composition promotes wound healing.

12. The method of claim 1, wherein the composition promotes tissue regeneration.

13. The method of claim 1, wherein the composition promotes angiogenesis.

14. The method of claim 1, wherein the composition promotes cell survival.

15. The method of claim 1, wherein the composition inhibits cell death.

16. The method of claim 1, wherein the composition is administered by oral, intravenous, subcutaneous, topical, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular, or intrathecal routes.

17. A method of inhibiting ABCB5 positive cancer cell function comprising administering to a subject in need thereof an effective amount of a composition that inhibits the ABCB5-PIP2 pathway, and further comprising assessing ABCB5-PIP2 binding after administration of the composition.

18. A method of inhibiting ABCB5 positive cancer cell function comprising administering to a subject in need thereof an effective amount of a composition that inhibits ABCB5-PIP2 binding, wherein the composition is selected from the group comprising: small molecules, lipid analogs, anti-ABCB 5 antibodies or fragments specific for a cyclic or linear form of the extracellular polypeptide of the protein, enzymes, and anti-ABCB 5 antibodies or fragments thereof that alter the conformation of the ABCB5 PIP2 binding site.

19. The method of claim 18, wherein the anti-ABCB 5 antibody or fragment thereof that alters the conformation of the ABCB5 PIP2 binding site inhibits the production of PIP 3.

20. The method of claim 18, wherein the anti-ABCB 5 antibody or fragment thereof that alters the conformation of ABCB5 PIP2 binding site inhibits the PI3K pathway.

21. The method of claim 18, further comprising assessing ABCB5-PIP2 binding after administration of the composition.

22. The method of claim 17 or 18, wherein the composition is a PIP2 antagonist.

23. The method of claim 17, wherein the composition is selected from the group comprising: small molecules, lipid analogs, anti-ABCB 5 antibodies or fragments specific for a cyclic or linear form of the extracellular polypeptide of the protein, and enzymes.

24. The method of claim 17 or 18, wherein the composition is a small molecule.

25. The method of claim 17 or 18, wherein the composition is an anti-ABCB 5 antibody or fragment specific for a circular or linear form of an extracellular polypeptide of the protein.

26. The method of claim 17 or 18, wherein the composition is an ABCB5 antibody or fragment that alters the conformation of the ABCB5 PIP2 binding site.

27. The method of claim 17 or 18, wherein the composition is a lipid analog.

28. The method of claim 17 or 18, wherein the composition is an enzyme.

29. The method of claim 17 or 18, wherein the subject is a human or non-human animal, including a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, cat, dog, llama or primate, such as a monkey.

30. The method of claim 17 or 18, wherein the composition is administered by oral, intravenous, subcutaneous, topical, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular or intrathecal route.

31. A method for identifying an enhancer or inhibitor of the ABCB5-PIP2 pathway comprising:

contacting an ABCB5+ cell with a putative composition that modulates ABCB5-PIP2 binding; determining a level of a PIP2 pathway product compound and comparing said level to a baseline level of said PIP2 pathway product compound, wherein if said level is greater than said baseline level, then said putative composition is an ABCB5-PIP2 pathway enhancer, and if the level of the PIP2 pathway product compound is less than said baseline level, then said putative composition is an ABCB5-PIP2 inhibitor.

32. The method of claim 31, wherein the putative composition that modulates the ABCB5-PIP2 pathway is a PIP2 or PIP2 agonist.

33. The method of claim 31, wherein the putative composition that modulates the ABCB5-PIP2 pathway is a small molecule.

34. The method of claim 31, wherein the putative composition that modulates the ABCB5-PIP2 pathway is an anti-ABCB 5 antibody or fragment thereof.

35. The method of claim 31, wherein the PIP2 pathway compound is PIP 3.

36. The method of claim 31, wherein the PIP2 pathway compound is a member of the PI3K pathway.

37. The method of claim 31, wherein the ABCB5+ cells comprise ABCB5 isoform 1 in which amino acid 970 is lysine.

38. The method of claim 31, wherein the ABCB5+ cells comprise ABCB5 isoform 2 wherein amino acid 525 is lysine.

39. A composition comprising a synthetic phospholipid comprising a compound having the structure:

wherein R1 and R2 are independent fatty acid chains; and is

Wherein the length of R1 and R2 is at least twice as long as the other of R1 and R2.

40. The composition of claim 39, wherein the phospholipid has a pH of 22: 0-26: 0 total fatty acid chain.

41. The composition of claim 40, wherein the phospholipid has a pH of 24: 0 total fatty acid chain.

42. The composition of claim 39, wherein the phospholipid has the formula: C33H65O19P 3.

43. The composition of claim 39, wherein the phospholipid comprises [ PIP2 (6: 0/18: 0) -H]^-And a pharmaceutically acceptable carrier.

44. The composition of claim 39, wherein the composition comprises a PIP2 analog.

45. The composition of claim 39, wherein the composition enhances the ABCB5-PIP2 pathway.

46. The composition of claim 45, wherein the composition promotes wound healing.

47. The composition of claim 45, wherein the composition promotes tissue regeneration.

48. The composition of claim 45, wherein the composition promotes angiogenesis.

49. The composition of claim 45, wherein the composition promotes cell survival.

50. The composition of claim 45, wherein the composition inhibits cell death.

51. The composition of claim 39, wherein the phospholipid comprises phosphorylated PIP3 (6: 0/18: 0) -H^-(C33H65O19P4) and a pharmaceutically acceptable carrier.

52. A human anti-ABCB 5 antibody or an ABCB5 binding fragment thereof that inhibits the ABCB5-PIP2 pathway, wherein the anti-ABCB 5 antibody or an ABCB5 binding fragment thereof binds to the three-dimensionally configured extracellular loop of ABCB 5.

53. The human anti-ABCB 5 antibody or ABCB5 binding fragment of claim 52, wherein the antibody can be prepared by a method comprising affinity maturation to specifically bind to the extracellular loop of the nonlinear form of ABCB 5.

54. The human anti-ABCB 5 antibody or ABCB5 binding fragment of claim 52, wherein the antibody has a sequence corresponding to an antibody that can be made by a method comprising affinity maturation to specifically bind to the extracellular loop of the nonlinear form of ABCB 5.

55. A method of making a human anti-ABCB 5 antibody or ABCB5 binding fragment that inhibits the ABCB5-PIP2 pathway of claim 52, wherein said anti-ABCB 5 antibody or ABCB5 binding fragment is subjected to affinity maturation to specifically bind to the extracellular loops of the non-linear form of the protein.

56. A method for identifying an antibody or fragment that inhibits the ABCB5-PIP2 pathway comprising:

contacting an ABCB5+ cell with a putative antibody or fragment that binds to ABCB 5; assessing ABCB5-PIP2 binding after treatment with the antibody or fragment; determining a level of a PIP2 pathway product compound and comparing said level to a baseline level of said PIP2 pathway product compound, wherein if the level of said PIP2 pathway product compound is below said baseline level, then said putative antibody or fragment is an inhibitor of the ABCB5-PIP2 pathway.

57. The method of claim 56, wherein the PIP2 pathway compound is PIP 3.

58. The method of claim 56, wherein the PIP2 pathway compound is a member of the PI3K pathway.

ABCB5 isoform 1 comprising two transmembrane domains (TMD) and 12 transmembrane helices (TM 1-12), wherein the glutamic acid at position 970 of TM12 has been mutated to lysine, or wherein the glutamic acid at position 970 of TM12 is glutamic acid.

ABCB5 isoform 2 comprising one transmembrane domain (TMD) and 6 transmembrane helices (TM 1-6), wherein the glutamic acid at position 525 of TM6 has been mutated to lysine, or wherein the glutamic acid at position 525 of TM 12.

61. A human anti-ABCB 5 isotype antibody or binding fragment thereof that inhibits the ABCB5-PIP2 pathway, wherein the anti-ABCB 5 antibody or ABCB5 binding fragment thereof specifically binds to ABCB5 isotype 1 of claim G1 or ABCB5 isotype 2 of claim 60.

62. A method for identifying an enhancer or inhibitor of the ABCB5-PIP2 pathway comprising:

contacting ABCB5 isoform 1 of claim 59 or ABCB5 isoform 2 of claim 60 with a putative composition that modulates ABCB5-PIP2 binding; determining a level of a PIP2 pathway product compound and comparing said level to a baseline level of said PIP2 pathway product compound, wherein if said level is greater than said baseline level, then said putative composition is an ABCB5-PIP2 pathway enhancer, and if the level of a PIP2 pathway compound is less than said baseline level, then said putative composition is an ABCB5-PIP2 inhibitor.

63. The method of claim 62, wherein the putative composition that modulates the ABCB5-PIP2 pathway is a PIP2 or PIP2 agonist.

64. The method of claim 62, wherein the putative composition that modulates the ABCB5-PIP2 pathway is a small molecule.

65. The method of claim 62, wherein the putative composition that modulates the ABCB5-PIP2 pathway is an anti-ABCB 5 antibody or fragment thereof.

66. The method of claim 62, wherein the PIP2 pathway compound is PIP 3.

67. The method of claim 62, wherein the PIP2 pathway compound is a member of the PI3K pathway.

68. The method of any one of claims 62-67, wherein the ABCB5 isoform is recombinantly expressed.

69. A method for treating cancer in a subject, comprising:

disrupting an endogenous ABCB5 gene in a cell using gene editing; treating the cancer in the subject by contacting the cell with a Cas protein, CRISPR RNA that hybridizes to the endogenous ABCB5 gene, and a tracrRNA, wherein after contacting the Cas protein, CRISPR RNA, and tracrRNA, the endogenous ABCB5 gene is modified such that an AAA sequence in a region of the gene encoding a terminal transmembrane helix of the ABCB5 gene is replaced with GAA, and wherein the gene editing treats the cancer in the subject.

70. The method of claim 69, wherein the subject has ABCB + stem cells associated with the cancer prior to gene editing that are ABCB5 homozygous isoform 2K 525/K525.

71. The method of claim 69 or 70, wherein the cancer is melanoma or glioblastoma.

72. A method for treating cancer in a subject, comprising:

administering to the subject an ABCB1 inhibitor in an amount effective to inhibit the function of the ABCB5-PIP2 pathway to treat the cancer in the subject, wherein:

i) the cancer comprises cancer cells, and the cancer cells express negligible ABCB1 or do not express ABCB 1; or

ii) the method further comprises detecting the presence of ABCB5+ stem cells prior to the administering step; or

iii) wherein the ABCB1 inhibitor is a pump inhibitor and the cancer is not being treated concurrently with a chemotherapeutic agent; or

iv) the method further comprises assessing ABCB5-PIP2 binding after administration of the composition.

73. The method of claim 72, wherein the subject has ABCB + stem cells associated with the cancer prior to gene editing that are ABCB5 homozygous isoform 2K 525/K525.

74. The method of claim 72 or 73, wherein the cancer is melanoma or glioblastoma.

75. A method for characterizing cancer, comprising:

isolating a cancer cell from a subject, and determining whether the cancer cell is an ABCB5 homozygous isoform 2K525/K525, an ABCB5 homozygous isoform 2E525/E525, or an ABCB5 heterozygous isoform 2K525/E525 to characterize the cancer.

Technical Field

The present invention relates to methods and compositions and related assays and reagents for modulating stem cell activity to treat disease. The invention also relates to methods and compositions for wound healing and tissue engineering involving ABCB5 positive cells.

Background

Tumor development and progression are associated at the DNA level with cumulative changes in oncogenes, tumor suppressor genes and repair/stability genes. At the cellular level, human cancers have been recognized to consist of phenotypically heterogeneous populations of cells with different self-renewal and tumor spreading capabilities. This observation has led to the development of a tumor stem cell (CSC) model of tumor initiation and growth, which has been widely demonstrated in a variety of malignancies, including melanoma and colorectal cancer. CSCs have been shown to result in failure of existing treatments to consistently eradicate malignancies through a variety of molecular mechanisms, including epithelial-mesenchymal transition (EMT) associated with the ability of human cancers to invade the vascular system (vacuoluture) and spread to new anatomic sites, which leads to tumor progression and treatment resistance.

ABCB5 is a multidrug resistance (MDR) mediator expressed in diverse human malignancies, where it was specifically overexpressed in a subpopulation of treatment-resistant CD133(+) tumors previously found to represent CSCs. ABCB5 confers resistance to cancer cells to chemotherapeutic agents, such as 5-fluorouracil (5-FU).

ABCB5+ stem cells are also present in normal tissues and play a role in tissue regeneration and senescence. Regenerative medicine involves the use of foreign materials (e.g., scaffolds) to repair, regenerate, maintain and replace tissues and organs. The scaffold may be seeded with cells, such as primary or stem cells, and various factors that promote tissue growth. However, many challenges remain in the design of suitable materials for regenerative medicine and tissue engineering.

Summary of The Invention

In some aspects, the invention relates to methods and compositions for modulating the activity of ABCB5+ stem cells. The invention also relates to assays and reagents for the manipulation and characterization of compounds that modulate ABCB5+ cell signaling.

Some aspects of the invention relate to a method of enhancing ABCB5 positive cell function comprising administering to a subject in need thereof an effective amount of a composition that enhances the ABCB5-PIP2 pathway.

In some embodiments, the invention further comprises assessing ABCB5-PIP2 binding after administration of the composition.

In some embodiments, the composition is a PIP2 or PIP2 agonist.

In some embodiments, the subject is a human or non-human animal, including a goat, sheep, bison, camel, cow, pig, rabbit, buffalo (buffalo), horse, rat, mouse, cat, dog, llama (llama), or primate, e.g., monkey.

In some embodiments, the composition comprises a phospholipid.

In some embodiments, the composition comprises [ PIP2 (6: 0/18: 0) -H ] -and a pharmaceutically acceptable carrier.

In some embodiments, the composition comprises a phospholipid comprising a compound having a structure described herein. In some embodiments, the structures comprise R1 and R2 groups. In some embodiments, R1 and R2 are separate fatty acid chains. In some embodiments, the structure comprises R1 and R2, R1 and R2 being at least twice as long as the other of R1 and R2. In some embodiments, the structures have a total fatty acid chain of 22: 0 to 26: 0. In some embodiments, the structure has 24: 0 total fatty acid chains.

In some embodiments, the subject is a healthy subject. In some embodiments, the composition promotes wound healing. In some embodiments, the composition promotes tissue regeneration. In some embodiments, the composition promotes angiogenesis. In some embodiments, the composition promotes cell survival. In some embodiments, the composition inhibits cell death. In some embodiments, the composition is administered by oral, intravenous, subcutaneous, topical (topic), parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, intracameral (ocular), or intrathecal routes.

Aspects of some of the invention are methods of inhibiting ABCB5 positive cancer cell function comprising administering to a subject in need thereof an effective amount of a composition that inhibits the ABCB5-PIP2 pathway, and further comprising assessing ABCB5-PIP2 binding after administration of the composition.

Other aspects of the invention relate to a method of inhibiting ABCB5 positive cancer cell function comprising administering to a subject in need thereof an effective amount of a composition that inhibits ABCB5-PIP2 binding, wherein the composition is selected from the group comprising: small molecules, lipid analogs, anti-ABCB 5 antibodies or fragments specific for a cyclic or linear form of the extracellular polypeptide of the protein, enzymes, and anti-ABCB 5 antibodies or fragments thereof that alter the conformation of the ABCB5 PIP2 binding site.

In some embodiments, an anti-ABCB 5 antibody or fragment thereof that alters the conformation of the ABCB5 PIP2 binding site inhibits the production of PIP 3. In some embodiments, an anti-ABCB 5 antibody or fragment thereof that alters the conformation of the ABCB5 PIP2 binding site inhibits the PI3K pathway.

In some embodiments, ABCB5-PIP2 binding is assessed after administration of the composition.

In some embodiments, the composition is a PIP2 antagonist. In some embodiments, the composition is selected from the group comprising: small molecules, lipid analogs, anti-ABCB 5 antibodies or fragments specific for a cyclic or linear form of the extracellular polypeptide of the protein, and enzymes. In some embodiments, the composition is a small molecule. In some embodiments, the composition is an anti-ABCB 5 antibody or fragment specific for a circular or linear form of an extracellular polypeptide of the protein. In some embodiments, the composition is an ABCB5 antibody or fragment that alters the conformation of the ABCB5 PIP2 binding site. In some embodiments, the composition is a lipid analog. In some embodiments, the composition is an enzyme.

In some embodiments, the subject is a human or non-human animal, including a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, cat, dog, llama, or primate, e.g., monkey.

In some embodiments, the composition is administered by oral, intravenous, subcutaneous, topical, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular, or intrathecal routes.

Some aspects of the invention relate to methods for identifying an enhancer or inhibitor of the ABCB5-PIP2 pathway comprising. In some embodiments, the invention comprises contacting an ABCB5+ cell with a putative composition that modulates ABCB5-PIP2 binding; the level of the PIP2 pathway product compound is determined and compared to a baseline level of the PIP2 pathway product compound. In some embodiments, if the level is greater than the baseline level, the putative composition is identified as an ABCB5-PIP2 pathway enhancer. In some embodiments, if the level of PIP2 pathway product compound is less than the baseline level, the putative composition is identified as an ABCB5-PIP2 pathway inhibitor.

In some embodiments, a putative composition that modulates the ABCB5-PIP2 pathway is a PIP2 or PIP2 agonist. In some embodiments, the putative composition that modulates the ABCB5-PIP2 pathway is a small molecule. In some embodiments, the putative composition that modulates the ABCB5-PIP2 pathway is an anti-ABCB 5 antibody or fragment thereof. In some embodiments, the PIP2 pathway compound is PIP 3. In some embodiments, the PIP2 pathway compound is a member of the PI3K pathway. In some embodiments, the ABCB5+ cells comprise ABCB5 isoform (isoform)1 in which amino acid 970 is lysine. In some embodiments, the ABCB5+ cell comprises ABCB5 isoform 2 wherein amino acid 525 is lysine.

In some embodiments, the assay involves determining the number of ABCB5 alleles and then testing how many positive for K and how many positive for E, i.e., an allele-specific quantification program that extracts both copy number and allelic type information.

Some aspects of the invention relate to compositions comprising a synthetic phospholipid comprising a compound having a structure described herein. In some embodiments, the structures comprise R1 and R1 groups. In some embodiments, R1 and R2 are separate fatty acid chains. In some embodiments, the length of R1 and R2 is at least twice as long as the other of R1 and R2. In some embodiments, the phospholipids have total fatty acid chains ranging from 22: 0 to 26: 0. In some embodiments, the phospholipids have a total fatty acid chain of 24: 0. In some embodiments, the phospholipid has the formula: C33H65O19P 3. In some embodiments, the phospholipid comprises [ PIP2 (6: 0/18: 0) -H ] -and a pharmaceutically acceptable carrier.

In some embodiments, the composition comprises a PIP2 analog. In some embodiments, the composition enhances the ABCB5-PIP2 pathway. In some embodiments, the composition promotes wound healing. In some embodiments, the composition promotes tissue regeneration. In some embodiments, the composition promotes angiogenesis. In some embodiments, the composition promotes cell survival. In some embodiments, the composition inhibits cell death.

In some embodiments, the phospholipid comprises phosphorylated PIP3 (6: 0/18: 0) -H^-(C33H65O19P4) and a pharmaceutically acceptable carrier.

Some aspects of the invention relate to a human anti-ABCB 5 antibody or ABCB5 binding fragment thereof that inhibits the ABCB5-PIP2 pathway, wherein the anti-ABCB 5 antibody or ABCB5 binding fragment thereof binds to the three-dimensional configured extracellular loop of ABCB 5.

In some embodiments, a human anti-ABCB 5 antibody or ABCB5 binding fragment can be prepared by a method that includes affinity maturation to specifically bind to the extracellular loop of the nonlinear form of ABCB 5. In some embodiments, the human anti-ABCB 5 antibody or ABCB5 binding fragment has a sequence corresponding to an antibody that can be prepared by a method that includes affinity maturation to specifically bind to the extracellular loop of the nonlinear form of ABCB 5.

Some aspects of the invention relate to methods of making a human anti-ABCB 5 antibody or ABCB5 binding fragment that inhibits the ABCB5-PIP2 pathway described herein. In some embodiments, the anti-ABCB 5 antibody or ABCB5 binding fragment is subjected to affinity maturation to specifically bind to the extracellular loop of the nonlinear form of the protein.

Some aspects of the invention relate to methods for identifying antibodies or fragments that inhibit the ABCB5-PIP2 pathway. In some embodiments, an antibody or fragment that inhibits the ABCB5-PIP2 pathway is identified by: contacting an ABCB5+ cell with a putative antibody or fragment that binds to ABCB 5; assessing ABCB5-PIP2 binding after treatment with the antibody or fragment; the level of the PIP2 pathway product compound is determined and compared to a baseline level of the PIP2 pathway product compound.

In some embodiments, if the level of PIP2 pathway product compound is below a baseline level, the antibody or fragment is assumed to be an inhibitor of the ABCB5-PIP2 pathway. In some embodiments, the PIP2 pathway compound is PIP 3. In some embodiments, the PIP2 pathway compound is a member of the PI3K pathway.

Some aspects of the invention relate to ABCB5 isoform 1 comprising two transmembrane domains (TMDs) and 12 transmembrane helices (TM 1-12). In some embodiments, the glutamic acid at position 970 of TM12 has been mutated to a lysine, or TM12 is a glutamic acid at position 970.

Some aspects of the invention relate to ABCB5 isoform 2 comprising one transmembrane domain (TMD) and 6 transmembrane helices (TM 1-6). In some embodiments, the glutamic acid at position 525 of TM6 has been mutated to a lysine, or the glutamic acid at position 525 of TM 12.

Some aspects of the invention relate to human anti-ABCB 5 isotype antibodies or binding fragments thereof that inhibit the ABCB5-PIP2 pathway. In some embodiments, the anti-ABCB 5 antibody or ABCB5 binding fragment thereof specifically binds to ABCB5 isoform 1 described herein or ABCB5 isoform 2 described herein.

Some aspects of the invention relate to methods for identifying an enhancer or inhibitor of the ABCB5-PIP2 pathway. In some embodiments, the methods comprise contacting ABCB5 isoform 1 or ABCB5 isoform 2 described herein with a putative composition that modulates ABCB5-PIP2 binding; the level of the PIP2 pathway product compound is determined and compared to a baseline level of the PIP2 pathway product compound. In some embodiments, if the level is greater than the baseline level, then the composition is assumed to be an ABCB5-PIP2 pathway enhancer. In some embodiments, if the level of PIP2 pathway compound is below a baseline level, the composition is assumed to be an ABCB5-PIP2 inhibitor.

Some aspects of the invention relate to methods for treating cancer in a subject. In some embodiments, the method comprises disrupting an endogenous ABCB5 gene in the cell using gene editing. In some embodiments, editing comprises contacting the cell with a Cas protein, CRISPR RNA that hybridizes to an endogenous ABCB5 gene, and a tracrRNA. In some embodiments, after contact with the Cas protein, CRISPR RNA, and tracrRNA, the endogenous ABCB5 gene is modified such that the AAA sequence in the region of the gene encoding the terminal transmembrane helix of the ABCB5 gene is replaced with GAA. In some embodiments, the gene editing treats cancer in the subject.

In some embodiments, the subject has cancer-associated ABCB + stem cells prior to gene editing that are ABCB5 homozygous isoform 2K 525/K525. In some embodiments, the cancer is melanoma or glioblastoma.

Some aspects of the invention relate to methods for treating cancer in a subject. In some embodiments, the method comprises administering to the subject an ABCB1 inhibitor in an amount effective to inhibit ABCB5-PIP2 pathway function to treat cancer in the subject. In some embodiments. In some embodiments, the cancer comprises cancer cells, and the cancer cells express negligible ABCB1 or do not express ABCB 1. In some embodiments, the method further comprises detecting the presence of ABCB5+ stem cells prior to the administering step. In some embodiments, the ABCB1 inhibitor is a pump inhibitor and the cancer is not simultaneously treated with a chemotherapeutic agent. In some embodiments, the method further comprises assessing ABCB5-PIP2 binding after administration of the composition.

In some embodiments, the subject has ABCB + stem cells associated with the cancer prior to gene editing that are ABCB5 homozygous isoform 2K 525/K525. In some embodiments, the cancer is melanoma or glioblastoma.

Some aspects of the invention relate to methods for characterizing cancer. In some embodiments, the method comprises isolating a cancer cell from a subject, determining whether the cancer cell is ABCB5 homozygous isoform 2K525/K525, ABCB5 homozygous isoform 2E525/E525, or ABCB5 heterozygous isoform 2K525/E525, to characterize the cancer.

Each limitation of the invention may encompass various embodiments of the invention. It is therefore contemplated that each limitation of the invention relating to any one element or combination of elements may be incorporated into every aspect of the invention. The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Brief Description of Drawings

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

pipp 2 and PIP3 are biological ligands of ABCB5, ABCB5 acting as receptors for PIP2 and PIP 3. Immunoblotting using the ABCB5 monoclonal antibody (fig. 1A, top panel) showed that immunoprecipitation of PIP2 from human ABCB 5-expressing melanoma cells using anti-PIP 2 antibody pull-down revealed co-precipitation of ABCB5 protein. Immunoblotting using PIP2 antibody confirmed PIP2 pull-down (fig. 1A, bottom panel). Immunoblotting with the ABCB5 monoclonal antibody (fig. 1B) showed that immunoprecipitation of PIP3 from human ABCB 5-expressing melanoma cells using anti-PIP 3 antibody pull-down revealed co-precipitation of ABCB5 protein. Figure 1C shows that PIP1, PIP2, and PIP3 all bound to recombinant human ABCB5 and murine ABCB5, whereas there was no detection of binding in the tissues of the ABCB5 knockout mouse. Therefore, PIP2 and PIP3 bind ABCB5 more efficiently than PIP1 binds ABCB 5.

Figure 2 binding of PIP1, PIP2 and PIP3 to ABCB5 can be inhibited by competitive pharmacological ligands. The data show that PtdIns- (1, 2-dicaprylyl) competitively inhibits binding of PIP1, PIP2, or PIP3 to ABCB5 with significant saturation of action at concentrations as low as 0.1 mM.

Fig. 3 ABCB5 monoclonal antibody can block binding of PIP1, PIP2 or PIP3 to ABCB 5. Surface Plasmon Resonance (SPR) analysis demonstrated binding of PIP1, PIP2 and PIP3 to ABCB 5. Competition with the anti-ABCB 5 monoclonal antibody resulted in a concentration-dependent decrease in signal on all three surfaces (PIP 3> PIP2 > PIP1) by as much as 50% on the PIP3 surface.

Fig. 4 ABCB5 is functionally needed to more efficiently convert PIP2 to PIP 3.ABCB5 monoclonal antibody, but not isotype control antibody, significantly reduced the PIP3/PIP2 ratio in human melanoma cells (left panel). In addition, examination of murine ABCB5 gene knockout skin tissue also showed a significantly reduced PIP3/PIP2 ratio compared to ABCB5 wild type skin (right panel).

Figure 5 ABCB5 is required to maintain the PIP2/PIP3 dependent PI3K/AKT signaling axis in malignancies, and ABCB5 inhibition results in inhibition of the PI3K/AKT signaling axis and associated tumor growth and treatment resistance. 4-HT treatment inducible Tyr in the context of ABCB5 WT or ABCB5 KO: : CreER; brafca; analysis of the Ptenlox/lox genetic mouse melanoma model showed significant inhibition of the PI3K/AKT signaling axis in ABCB5 KO compared to ABCB5 WT tumor, with reduced expression of p-AKT, p-mTOR and p-S6 in other dysregulated molecules.

Figure 6 ABCB5 KO status resulted in decreased tumor cell proliferation compared to ABCB5 WT status, as determined by determining the percentage of tumor cells positively stained for the proliferation marker Ki-67.

7A-7B. ABCB5 KO status resulted in down-regulation of pro-angiogenic molecules (FIG. 7A, left panel) compared to ABCB5 WT status, and as a result, a reduction in CD 31-positive microvascular density (FIG. 7A right panel and FIG. 7B).

Figure 8 ABCB5(+) CRC cells express the receptor tyrosine kinase AXL that maintains EMT, which acts as a mediator of ABCB 5-dependent cancer invasion. A set of scatter plots depicting representative flow cytometry analysis of AXL protein expression in ABCB5KD compared to control transfected cell lines is shown. Also shown is a bar graph showing AXL mRNA expression in ABCB5KD compared to control transfected human CRC cells. Western blot analysis of AXL, AKT and phosphorylated AKT protein expression in anti-ABCB 5 mAb-treated or isotype control-treated CRC cells is also shown. Data were analyzed using unpaired t-test. Error bars indicate s.e.m. P < 0.05, P < 0.01, P < 0.001.

Fig. 9 ABCB5 plays a key role in tumor vemurafenib resistance through its function in the intact PIP2/PIP 3-dependent PI3K/pAKT signal transduction axis required to maintain vemurafenib resistance. 4-HT treatment inducible Tyr in the context of ABCB5 WT or ABCB5 KO: : CreER; brafca; the Ptenlox/lox genetic mouse melanoma model, compared to the ABCB5 WT status, ABCB5 KO status resulted in complete sensitivity to the effects of the BRAF inhibitor vemurafenib, which resistance is driven in part by the functional PI3K/pAKT signaling axis. No tumor formation was observed after genetic induction in vemurafenib treated ABCB5 KO mice, in contrast ABCB5 WT mice exhibited 100% formation of vemurafenib resistant tumors (left panel), and survival was significantly prolonged in ABCB5 KO mice compared to ABCB5 WT mice (right panel).

Figure 10 psoriasis exacerbations were found in an ABCB5 knockout imiquimod-induced psoriasis mouse model compared to ABCB5 wild-type mice.

Figure 11 amino acid residue 525 of TM6 of ABCB5 isoform 2 is an important molecular switch in the physiological ligand/substrate binding quality of ABCB 5. Experimental induction of molecular switch from K (lysine, AAA codon) to E (glutamate, GAA codon) in one allele by criprpr/Cas 9 mediated gene editing in wild type K525/K525 human melanoma cells expressing only ABCB5 isoform 2 at baseline resulted in a clone hybrid ABCB5K 525/E525 melanoma cell variant with impaired ABCB5 signaling function and resulted in significant inhibition of ABCB5 driven tumor growth (P < 0.05).

Detailed Description

The present invention in some aspects relates to the following findings: ATP-binding cassette subfamily B (MDR/TAP) member 5(ABCB5) [ Frank, n.y.et al.regulation of promoter cell fusion by ABCB5P-glycoprotein, a novel human ATP-binding cassette transporter.j Biol Chem 278, 47156-65(2003) and Schatton, t.et al.identification of ls inducing human melatomas. nature 451, 345-9(2008) ], preferentially expressed at high levels in the plasma membranes of cancer stem cells and normal tissue-specific stem cells, and to a lesser extent, as receptors for phosphatidylinositol 4, 5-bisphosphonates (PtdIns (4, 5) P2, also PIP2), abbreviated as PIP1 and PIP 3. As used herein, "ABCB 5(+) stem cell" refers to a cell that has the ability to self-renew and differentiate into mature cells of various adult cell lineages and is characterized by expression of ABCB5 on the cell surface. PIP2 is the minor phosphoinositide (phosphoinositide) phospholipid component of the cell membrane that is abundant in the plasma membrane, where it is a substrate for many important signal transduction proteins, regulating signal transduction, for example, by Receptor Tyrosine Kinases (RTKs), either through the PI3K pathway or the IP3/DAG pathway of G protein-coupled receptors. Inhibition of the ABCB5-PIP2 pathway by inhibition of ABCB5 blocks binding of PIP2 to ABCB5 and subsequent phosphorylation of PIP2 to produce PIP 3. Thus, disruption of this pathway results in inhibition of downstream PI3K signaling by tyrosine kinase receptors (e.g., VEGFR1, EGFR, and AXL) and abrogation of their stem cell specific functions. ABCB5 PIP2 binding can be assessed using a variety of methods known in the art. For example, ABCB5 PIP2 binding can be assessed by methods including immunoprecipitation, western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, microscopy and spectroscopy (see fig. 1-3).

Some aspects of the invention relate to methods for enhancing the function of ABCB5 positive cells. As used herein, "ABCB 5 positive cell function" refers to the activity of ABCB5 in healthy subjects and has a positive effect on the subject. For example, ABCB5 positive cell functions include promoting wound healing, tissue regeneration, angiogenesis, cell survival, and inhibiting cell death. It is understood that wound healing, tissue regeneration, angiogenesis, cell survival and cell death are determined by comparing the level or rate of each with the level or rate in a control sample. Herein, "control sample" refers to a sample lacking the function of ABCB 5. As used herein, a "healthy subject" is a subject that is otherwise free of disease.

As used herein, the phrase "stem cell specific function" relates to ABCB5 activity associated with stem cells. For example, skin-related healthy ABCB5+ stem cells use ABCB5 enhanced PI3K signaling and downstream AKT phosphorylation and mTOR signaling for angiogenesis and anti-apoptotic signaling, leading to stem cell survival and vascular differentiation, as well as other downstream functions required for normal wound healing. ABCB5+ limbal stem cells use this pathway for anti-apoptotic signaling required for stem cell maintenance. ABCB5+ cancer stem cells (e.g. in melanoma or colorectal cancer) use this pathway for cell survival, angiogenic mimicry, drug resistance and EMT and metastatic invasion (as shown in figure 1, i.e. inhibition of pAKT phosphorylation and EMT and invasion by ABCB5 blocking).

Among other functions, ABCB5 binding of PIP2 can be used to increase its rate of phosphorylation to PIP3, and thus represents a stem cell specific interaction to enhance signal transduction of PIP2 in cells that do not express ABCB 5. ABCB5-PIP2 binding may also be inhibited by small molecule ABCB5 competitive ligands or substrates or compositions comprising them, which also inhibit downstream signaling of critical ABCB 5-dependent biological stem cell function. Thus, the present invention has several important uses.

Thus, the invention described herein can be used to promote regeneration in healthy subjects.

The invention may also be used to treat a subject having or at risk of having a disease, such as a subject having or at risk of having cancer.

Subject shall refer to a human or vertebrate mammal, including but not limited to goats, sheep, bison, camels, cows, pigs, rabbits, buffalos, horses, rats, mice, cats, dogs, llamas and primates, such as monkeys. Thus, the invention may also be used to treat a disease or condition in a non-human subject. For example, cancer is one of the leading causes of death in companion animals (i.e., cats and dogs). Preferably, the subject is a human.

A subject at risk for developing cancer is a subject with a high likelihood of developing cancer. These include, for example, subjects with genetic abnormalities whose presence has been shown to correlate with a higher likelihood of developing cancer, as well as subjects exposed to carcinogens (e.g., tobacco, asbestos, or other chemical toxins), or subjects who have previously been treated for cancer and significantly remitted. Subjects at risk of having cancer also include subjects with pre-cancerous lesions. Precancerous lesions are areas of tissue with altered properties and with a risk of becoming skin cancer. Precancerous lesions can be caused by, for example, UV radiation, genetics, exposure to carcinogens (e.g., arsenic, tar, or X-rays).

A subject with cancer is a subject with detectable cancer cells. The cancer may be a malignant or non-malignant cancer. Cancers or tumors include, but are not limited to, biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial tumors; lymphoma; liver cancer; lung cancer (e.g., small cell and non-small cell); melanoma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; a sarcoma; skin cancer; testicular cancer; thyroid cancer; and kidney cancer, as well as other cancers and sarcomas. Preferably, the cancer comprises cancer stem cells expressing ABCB 5.

Optionally, prior to treatment, the binding molecules described herein can be used to detect the presence of ABCB5 positive stem cells. The detection or diagnostic methods provided herein generally comprise contacting one or more molecules of the invention with a sample in or from a subject. Preferably, the sample is first harvested from the subject, however in vivo detection methods are also contemplated. The sample may comprise any body tissue or fluid suspected of containing cancer stem cells. For example, stem cells are often present in or around tumor masses.

ABCB5 or ATP binding cassette B subfamily 5

As the name implies, ABCB5 is a member of ATP-binding cassette transporter subfamily B. It is a transmembrane protein encoded by the ABCB5 gene. ATP-binding cassette (ABC) transporters play a key role in physiology and pathology. It is involved in the transport of structurally diverse molecules from small ions, sugars and peptides to more complex organic molecules (Chen et al 2005).

As used herein, "ABCB 5+ stem cells" or "ABCB 5+ cells" refers to cells that have the ability to self-renew and differentiate into mature cells of multiple adult cell lineages. In some embodiments, the cells are characterized by expressing ABCB 5. In some embodiments of the invention, the ABCB5+ cells are cancer stem cells. In some embodiments of the invention, the ABCB5+ cells are healthy stem cells.

Some aspects of the invention relate to the identification of ABCB5 isoforms involved in cancer. In some embodiments, the ABCB5 isoform is involved in melanoma or glioblastoma. As used herein, the "ABCB 5 isoform" is an ABCB5 protein having a variant of the ABCB5 structure. In some embodiments, the ABCB5 isoform is ABCB5 isoform 1(1257 amino acids). ABCB5 isoform 1 contains two transmembrane domains (TMDs), each with 6 Transmembrane (TM) helices, i.e., it contains a total of 12 transmembrane helices (TM 1-12). In some embodiments, the ABCB5 isoform is isoform 2(812 amino acids). ABCB5 isoform 2 contains one TMD with 6 Transmembrane (TM) helices (TM 1-6). TM 1-6 of ABCB5 isoform 2 corresponds to TM 7-12 of ABCB5 isoform 1. In other embodiments, the presence of a lysine residue at a particular position in both isoforms is correlated with cancer prevalence. In other embodiments, the residue is 970 in TM12 of ABCB5 isoform 1 and 525 in TM6 of ABCB5 isoform 2. It is shown herein that non-synonymous Single Nucleotide Polymorphisms (SNPs) in ABCB5 coding regions that provide AA 970E > K in TM12 of ABCB5 isoform 1 and correspond to AA525E > K in TM6 of ABCB5 isoform 2 are important for ABCB5 function in cancer cells. For example, 970 in TM12 of ABCB5 isoform 1 and 525 in TM6 of ABCB5 isoform 2 are important for ABCB5 positive stem cell function. In other embodiments, these residues are required for ABCB 5-positive cancer stem cell function.

Further residues involved in substrate binding of ABCB5 are N702 and H706 in TM7 of ABCB5 isoform 1, N257 and H261 in TM1 corresponding to ABCB5 isoform 2, and 857A > T in TM10 of ABCB5 isoform 1 (rs80123476), and 412A > T in TM4 of ABCB5 isoform 2 (rs 80123476).

Genes encoding higher functional ABCB5 isoform 2-K525 protein sequences or ABCB5 isoform 2-K525 protein itself and genes encoding lower functional ABCB5 isoform 2-E525 protein sequences or ABCB5 isoform 2-E525 protein itself are useful compositions. These compositions are useful, for example, in: 1. recombinantly expressing ABCB5 isoform 2-K525 or ABCB5 isoform 2-E525; 2. ABCB5 isoform 2-K525 and ABCB5 isoform 2-E525 were used in docking and binding experiments to identify new sequence specific ABCB5 ligands and substrates; the use of ABCB5 isoform 2-K525 or ABCB5 isoform 2-E525 in molecular screening to identify synthetic compounds and naturally occurring substances that competitively inhibit the binding of PIP1, PIP2 or PIP3 to ABCB5 and thus also inhibit ABCB 5-dependent receptor tyrosine kinase and G protein-coupled receptor signal transduction; ABCB5 isoform 2-K525 or ABCB5 isoform 2-E525 were used in molecular screening to identify novel ABCB5 monoclonal antibodies that inhibit the binding of PIP1, PIP2 or PIP3 to ABCB5 and thus also inhibit ABCB 5-dependent receptor tyrosine kinase and G protein-coupled receptor signaling.

Compounds that competitively inhibit PIP1, PIP2, or PIP3 binding to ABCB5 and thus inhibit ABCB5 dependent signal transduction are useful according to the present invention. In some embodiments, these compounds include, but are not limited to, PtdIns- (1, 2-dioctanoyl), a synthetic analog of natural phosphatidylinositol (PtdIns) comprising C8: 0 fatty acids at the n-1 and sn-2 positions (CAS accession No. 899827-36-2). These compounds are useful for treating cancers associated with ABCB5+ stem cells.

In some aspects, the invention is a method of treating cancer by administering an ABCB1 inhibitor to a subject having cancer. It is found herein that ABCB1 inhibitors may also be useful in the treatment of ABCB5+ cancers. These compounds competitively inhibit PIP1, PIP2, or PIP3 binding to ABCB5 and, therefore, ABCB 5-dependent signal transduction.

As used herein, an ABCB1 inhibitor is a compound that reduces or eliminates ABCB1 function in a cell. ABCB1 inhibitors are known in the art and include anti-ABCB 1 antibodies and functional fragments thereof, as well as small molecules. Some ABCB1 inhibitors are ABCB1 agents for the treatment of heart disease or vascular disease, ABCB1 agents for the treatment of ABCB1+ cancer, ABCB1 agents for the treatment of infectious diseases, ABCB1 agents for the treatment of gastric diseases, and ABCB1 agents for the treatment of other diseases. In some embodiments, ABCB1 inhibitors include, for example, PSC833 (vasporder), Zosuquidar, Tariquidar, and laniquodar, i.e., inhibitors of the substrate and/or associated substrate binding site of the highly homologous ABCB1 molecule.

ABCB1 substrates or inhibitors are known for the treatment of a variety of diseases. Based on the discovery of new ABCB5 isoform 2-AA525 substrate binding sites for PIP1, PIP2, or PIP3, these compounds are therefore useful as small molecule inhibitors of ABCB 5-dependent PIP1, PIP2, or PIP3 binding, as well as PIP-dependent signal transduction and pAKT phosphorylation, for example, to block therapeutic inhibition of ABCB 5-driven human cancer growth and progression in ABCB 5-expressing cancers through functional ABCB 5.

In some embodiments, an ABCB1 inhibitor useful in a method of treating cancer is an ABCB1 agent for treating cardiac/vascular disease. Non-limiting examples of these compounds are shown in the following list.

ABCB1 agent for treating heart disease/blood vessel disease

In some embodiments, an ABCB1 inhibitor useful in a method of treating cancer is an ABCB1 agent for treating an infectious disease. Non-limiting examples of these compounds are shown in the following list.

ABCB1 agent for treating infectious diseases

In some embodiments, an ABCB1 inhibitor useful in a method of treating cancer is an ABCB1 agent for treating cancer. In some embodiments, the cancer is ABCB5+ cancer, and the cancer has no ABCB1 or has negligible ABCB 1. Non-limiting examples of these compounds are shown in the following list.

ABCB1 inhibitors for the treatment of ABCB5+ cancer

In some embodiments, an ABCB1 inhibitor useful in a method of treating cancer is an ABCB1 agent useful in treating gastric disorders. Non-limiting examples of these compounds are shown in the following list.

ABCB1 preparation for treating gastropathy

Omeprazole (Omepazole)

Nizatidine (Nizatidine)

Domperidone (Domperidone)

Lansoprazole (Lansoprazole)

Ranitidine (Ranitidine)

Pantoprazole (Pantoprazole)

Other ABCB1 inhibitors useful in the methods of the invention include, but are not limited to: cyclosporin (Ciclosporin), Cimetidine (Cimetidine), Aldosterone (Aldosterone), Tacrolimus (Tarrolimus), Phenobarbital (Phenobarbital), Dexamethasone (Dexamethane), Carbamazepine (Carbamazepine), Colchicine (Colchicine), Loperamide (Loperamide), Imipramine (Imipramine), Hydrocortisone (Hydrocortisone), Citalopram (Citalopram), taurocholine (Taurocholic Acid), Fexofenadine (Fexofenadine), Prednisone (Prednisonone), Estrone (statin), Diazepam (Diazepam), Digitoxin (Digitoxin), Methylprednisolone (Metalnisolone), Quetiapine (Qutiaoxine), Olanzapine (Oxazapine), Prednisolone (Epalvine), levofloxacin (Lebetamethasone), Prednisolone (Levone), levofloxacin (Levodione), Prednisolone (Levone (Levodione), Prednisolone (Levoxil), Prednisolone (Levoxil), levofloxacin (Levoxiletine), Prednisone (Levone), levofloxacin (Levoxiletine), Prednisolone (Levoxiletine), levosultaine (Levone), levosultaine (Levoxil), levosultaine (Levoxil (E), levosultaine (Levoxil), levosultam, Lamotrigine (Lamotrigine), Sitagliptin (Sitagliptin), katazoam (Ketazolam), Silodosin (Silodosin), Rivaroxaban (Rivaroxaban), Dabigatran etexilate (Dabigatran etexilate), Fesoterodine (Fesoterodine), Indacaterol (Indacaterol), Clobazam (Clobazam), Linagliptin (Linagliptin), Mirabegron (Mirabegron), Bosutinib (Bosutinib), Fluticasone furoate (Fluticasone furoate), Mycophenolate (Mycophenolate mofetil), lagranolozin (Dapagliflozin), Umeclidinium bromide (Umeclidinium), Edoxaban (edonbacin), oryzanol (nidulan), oryzanol (nitine), oryzanol (oryzanol), oryzanol (ritol), oryzanol (lovastatin), oryzanol (oryzanol), oryzanol (clavulanol (clavulanate), oryzanol (oryzanol), oryzanol (ketozanol), oryzanol (ketozanol), vinpocetine, vinpoc, Estradiol diheptanoate (Estradiol dienanthrate), Estradiol valerate (Estradiol valerate), Testosterone propionate (Testosterone propionate), Ashretavir (Asunaporvir), Somatostatin (Somatostatin), Alvatripopa (Avatrombopag), Venlafaxine (Venlafaxine), Trimipramine (Trimipramine), Tacrine (Tarcine), Eletriptan (Eletriptan), Sumatriptan (Sumatriptan), rapamycin (Sirolimus), palivir (Paritaporivir), Dasabrevivir (Dasabur), Erythromycin (Erythromycin), gramicin D (Gramicidin D), Itraconazole (Itraconazole), Tetracycline (Tetracycline), validamycin (Vatrimipramine), triamcinolone (Validavir), hydramine (Aminovir), Triflupirtine (Aminovir), Validazine (Aminovir), Validarubine (Aminovir), Valtamsulosin (Validamol), Validamol (Validamol G), Validamol (Validamol), Validamol (Valtamiprole), Valtamsultamsulbactam), Valtamsulbactam (Valtamiprole), Valtamsultamsulbactam), Valtamsultamsultamsultamarine (Valtamarine), Valtamarine (Valtamarine), Valtamarin, Ascorbic acid (Ascorbic acid), Chlorpromazine (Chlorpromazine), Phenothiazine (Phenothiazine), Atorvastatin (Atorvastatin), Bromperidol (Bromperidol), Morphine (Morphine), Pentazocine (Pentazocine), Propranolol (Propranolol), Neostigmine (Neostigmine), Moxidectin (Moxidectin), Mefloquine (Mefloquine), Fluticasone (Fluticasone), Fluticasone propionate (Fluticasone propionate), Elagolix, Chloroquine (Chloroquine), Paliperidone (Paliperidone), Lusutropoap (Lusumolopag), Posaconazole (Posaconazole), Dipyridamole (Dipyridamole), Quinine (Quincin), indomethacin (Acetafenoxan), Acetaminophen (Paracetamol), Paracetamol (Benzofine), Paracetamol (Paracetamol), Paracetamol (Benzimine (Paracetamol), Paracetamol (Paracetamol), and L (Paracetamol), Paracetamol (Paracetamol) (Benzimine (Paracetamol), Paracetamol) (Benzimine (Paracetamol) (Paraben (Acidol), and L) (Parabenomyl) (Paraben (Acidol) (Octamole (Acidol) (Acroline), and L) (Acbenomyl) (Acbenomy, Tenofovir (Tenofovir), Ledipasvir (Ledipasvir), Sildenafil (Sildenafil), Vardenafil (Vardenafil), Cabergoline (Cabergoline), Prucalopride (Prucalopride), Risperidone (Risperidone), Tramadol (Tramadol), Azithromycin (Azithromycin), Fluconazole (Flubonazole), Ranolazine (Ranolazine), Cetirizine (Cetirizine), Tegaserod (Tegaserod) and Doxepin (Doxepin)

PIP2 or phosphatidylinositol 4, 5-bisphosphate

PIP2 is a phospholipid that is present at low levels in cells but is involved in a variety of important cellular processes. Some cellular functions of PIP2 include the regulation of endocytosis, exocytosis, phagocytosis, and cell signaling (Czech et al, 2000).

As used herein, "PIP 2" refers to a phospholipid that binds to ABCB 5.

Some aspects of the invention are methods for enhancing ABCB5/PIP 2-dependent signaling in normal stem cells by an ABCB5-PIP2 binding enhancer to enhance the function of ABCB5 normal stem cells. Such methods comprise administering to a subject in need thereof an effective amount of a composition that enhances the ABCB5-PIP2 pathway, and assessing ABCB5-PIP2 binding after administration of the composition. In some embodiments, the composition comprises PIP2, a PIP2 agonist, a phospholipid, and [ PIP2 (6: 0/18: 0) -H ] -. In some embodiments, the composition comprises a phospholipid comprising a compound having the structure:

in some embodiments, R1 and R2 are separate fatty acid chains. In some embodiments, the length of R1 and R2 is at least twice as long as the other of R1 and R2. In some embodiments, the structure has a total fatty acid chain of 22: 0 to 26: 0. In some embodiments, the structure has 22: 0, 23: 0, 24: 0, 25: 0, or 26: 0 total fatty acid chains. In some embodiments of the invention, the composition is administered to a healthy subject. The subject may be a human or non-human animal including a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, cat, dog, llama or primate, e.g. monkey. In some embodiments, the compositions are used in a subject to promote wound healing, tissue regeneration, angiogenesis, and cell survival, reduce aging, and inhibit cell death. In some embodiments of the invention, the composition is administered by oral, intravenous, subcutaneous, topical, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular or intrathecal routes. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

In some embodiments, the composition comprises a PIP2 agonist. In some embodiments, the composition comprises:

one advantage of the method of the invention is that it allows for selective targeting of tissues having stem cells expressing ABCB 5. While existing therapies targeting the tyrosine kinase pathway have potential for a wide range of side effects, methods targeting ABCB5/PIP2 binding (enhancers or inhibitors) that also modulate PI3K signaling limit their effects to subpopulations of cells that also express ABCB 5. Therefore, the method should provide a lower potential side effect rate. ABCB5 targeting can be used as a stand-alone treatment for disseminated disease or as an adjunct treatment for sensitizing cancer cells to chemotherapeutic agents, especially in those patients currently suffering from refractory metastatic disease.

Some aspects of the invention relate to methods for inhibiting ABCB5-PIP2 binding by an inhibitory molecule contained in a composition to inhibit ABCB 5-dependent cancer stem cell function. Such methods represent functional blockade of ABCB5, and further comprise assessing ABCB5-PIP2 binding after administration of the composition. In some embodiments, the compositions inhibit the PI3K pathway and inhibit tumorigenesis, metastasis, and/or resistance to drugs that modulate PI3K signaling, e.g., melanoma resistance to vemurafenib mediated through PI3K signaling, or cancer resistance to EGFR inhibitors mediated through increased PI3K signaling by ABCB 5.

In some embodiments, the composition comprises a PIP2 antagonist. In some embodiments, the composition is selected from the group comprising: small molecules, lipid analogs, anti-ABCB 5 antibodies or ABCB5 binding fragments specific for a circular or linear form of the extracellular polypeptide of the protein, and enzymes. In some embodiments, the composition comprises an anti-ABCB 5 antibody or ABCB5 binding fragment specific for a circular or linear form of an extracellular polypeptide of ABCB 5. In some embodiments, the composition comprises an ABCB5 antibody or an ABCB5 binding fragment that alters the conformation of the ABCB5 PIP2 binding site. In some embodiments, the ABCB5 antibody is selected from, for example, a list comprising: a monoclonal antibody, a polyclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, a single chain antibody, a F (ab') 2, a Fab, a Fd, a Fv, or a single chain Fv fragment. In some embodiments, the ABCB5 antibody is a human anti-ABCB 5 antibody or ABCB5 binding fragment that binds to the three-dimensional configured extracellular loop of ABCB 5. In some embodiments, the human anti-ABCB 5 antibody is subjected to affinity maturation to recognize and specifically bind the non-linear form of the extracellular loop of ABCB 5. The human anti-ABCB 5 antibody or ABCB5 binding fragment described herein has a sequence corresponding to an antibody that can be prepared by a method that includes affinity maturation to specifically bind to the extracellular loop of ABCB5 in a non-linear form.

Some aspects of the invention relate to the production (e.g., preparation) of human anti-ABCB 5 antibodies or ABCB5 binding fragments that inhibit the ABCB5-PIP2 pathway. In some embodiments, the anti-ABCB 5 antibody or ABCB5 binding fragment is subjected to affinity maturation to specifically bind to the extracellular loop of the nonlinear form of the protein. The affinity maturation process can occur by: a. phage display, yeast display, or ribosome display; panning techniques. For example, once antibodies have been raised against linear extracellular loop peptides, the resulting antibodies can be matured using a display method by presenting and allowing the peptide proteins to be processed by antigen presenting cells.

In some embodiments of the invention, the composition is administered to a healthy subject.

In some embodiments, the subject may be a human or non-human animal, including a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, cat, dog, llama, or primate, e.g., monkey. In some embodiments of the invention, the composition inhibits drug resistance, cell survival, Epithelial Mesenchymal Transition (EMT) and metastasis, and promotes cell death. In some embodiments of the invention, the composition is administered by oral, intravenous, subcutaneous, topical, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular or intrathecal routes.

Some aspects of the invention relate to methods for inhibiting ABCB 5-dependent cancer stem cell function by administering to a subject in need thereof an effective amount of a composition that inhibits ABCB5-PIP2 binding. Such methods represent functional blockade of ABCB5, and further comprise assessing ABCB5-PIP2 binding after administration of the composition. In some embodiments, the compositions inhibit the PI3K pathway and inhibit tumorigenesis, metastasis, and/or resistance to drugs that modulate PI3K signaling, e.g., melanoma resistance to vemurafenib mediated through PI3K signaling, or cancer resistance to EGFR inhibitors mediated through increased PI3K signaling by ABCB 5.

In one aspect, the invention is useful as a screening tool in methods of developing molecular compounds that inhibit or enhance ABCB5-PIP2 binding and thus represent functional ABCB5 blockers or enhancers. Such compounds include lipid analogs, PIP2 or PIP2 agonists, small molecule drugs (e.g., PSC833), and a novel ABCB5 inhibitory monoclonal antibody subset that binds to ABCB5 and blocks PIP2 binding and inhibits pAKT phosphorylation and downstream signaling/effector pathways by inducing spatial changes in the molecule. The methods of the invention further comprise contacting an ABCB5+ cell with a putative composition comprising a compound that modulates ABCB5-PIP2 binding and determining the level of a PIP2 pathway product compound. If the level is greater than the baseline level, the composition is assumed to be an ABCB5-PIP2 pathway enhancer, and if the level of the PIP2 pathway compound is below the baseline level, the composition is assumed to be an ABCB5-PIP2 inhibitor. In some embodiments, the PIP2 pathway product compound is a compound member of the PIP3, or PI3K pathway. As used herein, "baseline level" refers to the level of PIP2 pathway product compound in a sample that has not been exposed to a hypothetical composition comprising a compound that modulates ABCB5-PIP2 binding.

Some aspects of the invention disclose novel phospholipid analogs of PIP2 that have been characterized by mass spectrometry. The fatty acid chain composition of PIP2 analogs represents a novel endogenously existing PIP2 variant compound (formula C)₃₃H₆₅O₁₉P₃PIP2 with 24: 0 total fatty acid chains, identified as [ PIP2 (6: 0/18: 0) -H]). This analog was particularly enriched in ABCB5 knockout cells, indicating that ABCB5 is functionally required for high efficiency PIP2 switching.

PIP2 has formula C₄₇H₈₀O₁₉P₃And the following structure:

in other aspects, the invention is a novel compound which is a functional analog of PIP2 and has the structure [ PIP2 (6: 0/18: 0) -H]And formula C₃₃H₆₅O₁₉P₃And 24: 0 total fatty acid chains. In some embodiments, the compound inhibits the ABCB5-PIP2 pathway. In some embodiments of the invention, the compounds inhibit drug resistance, cell survival, Epithelial Mesenchymal Transition (EMT) and metastasis, and promote cell death.

In some aspects, the invention is a compound having the structure:

wherein R is₁And R₂Independently fatty acid chains such that the structure has a total fatty acid chain of 22: 0 to 26: 0, and wherein R is₁And R₂One of which has a length R₁And R₂At least twice as long as the other. In some embodiments, the structures have 22: 0, 23: 0, 24: 0, 25: 0, or 26: 0 total fatty acid chains. In some embodiments, the compound inhibits the ABCB5-PIP2 pathway. In some embodiments of the invention, the compounds inhibit drug resistance, cell survival, Epithelial Mesenchymal Transition (EMT) and metastasis, and promote cell death.

In some aspects, the invention is a method for screening ABCB5 antagonists and enhancers using the novel compositions of the invention or PIP2 or other PIP2 analogs. The methods of the invention further comprise contacting an ABCB5+ cell with a putative composition that modulates ABCB5-PIP2 binding and determining the level of a PIP2 pathway product compound. If the level is greater than the baseline level, the composition is assumed to be an ABCB5-PIP2 pathway enhancer, and if the level of the PIP2 pathway product compound is below the baseline level, the composition is assumed to be an ABCB5-PIP2 inhibitor. In some embodiments, the PIP2 pathway compound is a member of the PIP3, or PI3K pathway.

In some embodiments, the ABCB5+ cells comprise ABCB5 isoform 1 with a lysine at amino acid 970. In some embodiments, the ABCB5+ cells comprise ABCB5 isoform 2 with a lysine at amino acid 525. ABCB5 expressing this SNP is most common in human cancers.

In others, the compositions are tools for such screening assays.

In other aspects, the invention is a method of using the novel composition or PIP2 or other PIP2 analog as a therapeutic compound to enhance ABCB 5-dependent stem cell function when administered exogenously.

An effective amount

In the methods described herein, the term "effective amount" refers to an amount of a composition that can achieve a desired therapeutic effect (e.g., enhance or inhibit the ABCB5-PIP2 pathway).

In some embodiments, the composition comprises PIP2, a PIP2 agonist, a PIP2 antagonist, a phospholipid, or [ PIP2 (6: 0/18: 0) -H ] -. In some embodiments, the composition comprises PIP 2. In some embodiments, the amount of PIP2 in the composition is from 1% to 100%. In some embodiments, the amount of PIP2 in the composition is at least 1%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more. In some embodiments, the composition comprises a PIP2 agonist. In some embodiments, the amount of PP2 agonist in the composition is from 1% to 100%. In some embodiments, the amount of PIP2 agonist in the composition is at least 1%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more.

In some embodiments, the composition comprises a PIP2 antagonist. In some embodiments, the amount of PIP2 antagonist in the composition is 1% to 100%. In some embodiments, the PIP2 antagonistic dose in the composition is at least 1%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more.

In some embodiments, the composition comprises a phospholipid. In some embodiments, the amount of phospholipid in the composition is 1% to 100%. In some embodiments, the amount of phospholipid in the composition is at least 1%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more. In some embodiments, the compositions comprise [ PIP2 (6: 0/18: 0) -H ] -. In some embodiments, the amount of [ PIP2 (6: 0/18: 0) -H ] -in the composition is 1% to 100%. In some embodiments, the amount of [ PIP2 (6: 0/18: 0) -H ] -in the composition is at least 1%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more.

Pharmaceutical composition

The compounds, antibodies, and encoding nucleic acids or nucleic acid sets, vectors comprising these, or host cells comprising the vectors described herein can be mixed with pharmaceutically acceptable carriers (excipients) to form pharmaceutical compositions for treating a target disease. By "acceptable" is meant that the carrier must be compatible with the active ingredients of the composition (and preferably capable of stabilizing the active ingredients) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) include buffers well known in the art. See, e.g., Remington: the Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed.K.E.Hoover.

The pharmaceutical compositions used in the present methods may comprise pharmaceutically acceptable carriers, excipients or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed.K.E.Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and alphaA methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexa-hydrocarbonic quaternary ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, for example methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, e.g. TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

In some embodiments, the pharmaceutical compositions described herein comprise liposomes containing the compound or antibody (or encoding nucleic acid) which can be prepared by methods known in the art, for example, as described in Epstein, et al, proc.natl.acad.sci.usa 82: 3688 (1985); hwang, et al, proc.natl.acad.sci.usa 77: 4030 (1980); and U.S. patent nos. 4,485,045 and 4,544,545. U.S. Pat. No.5,013,556 discloses liposomes with extended circulation time. Particularly useful liposomes can be produced by reverse phase evaporation methods with lipid compositions comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters that define the pore size to produce liposomes with the desired diameter.

The compounds or antibodies or encoding nucleic acids may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively; encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or encapsulated in macroemulsion (macroemulsion). Such techniques are known in The art, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In other embodiments, the pharmaceutical compositions described herein may be formulated in a sustained release form. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the compound or antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Some examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D- (-) -3-hydroxybutyric acid.

In other embodiments, the pharmaceutical compositions described herein may be formulated in sustained release form by implementing certain protease biology techniques to affect selective binding to a tissue or tumor, e.g., peptide masking of antibody antigen binding sites to allow for passage of one or more proteases, e.g., probodies, in the tumor microenvironment^TMOr Conditionationary Active Biologics^TMSelective protease cleavage of (3). In a normal microenvironment, activation may be stated as reversible.

Pharmaceutical compositions for in vivo administration must be sterile. This is readily accomplished by filtration, for example, through sterile filtration membranes. The therapeutic compound or antibody composition is typically placed into a container having a sterile access port, e.g., an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein may be in unit dosage form for oral, parenteral or rectal administration, or administration by inhalation or insufflation, for example, as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories.

For preparing solid compositions (e.g., tablets), the principal active ingredient may be mixed with a pharmaceutically acceptable carrier (e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums) and other pharmaceutically acceptable diluents (e.g., water) to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equivalent unit dosage forms such as tablets, pills and capsules. The solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500mg of the active ingredient of the present invention. Tablets or pills of the novel compositions may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, a tablet or pill may contain an inner dose and an outer dose component, the latter being in the form of an over-the-former capsule. The two components may be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials may be used for such enteric layers or coatings, such materials including a variety of polymeric acids, and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

Suitable surfactants include in particular nonionic agents, such as polyoxyethylene sorbitan (e.g. Tween @)^TM20. 40, 60, 80 or 85) and other sorbitan (e.g. Span)^TM20. 40, 60, 80, or 85). Compositions with surfactants will conveniently comprise 0.05% to 5% surfactant, and may be 0.1% to 2.5%. It will be appreciated that other ingredients, such as mannitol or other pharmaceutically acceptable carriers, may be added if desired.

Suitable emulsions may be prepared using commercially available fat emulsions, for example, Intralipid^TM、Liposyn^TM、Infonutrol^TM、Lipofundin^TMAnd Lipiphysan^TM. The active ingredient may be dissolved in a pre-mixed emulsion composition, orAlternatively, it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and mixed with a phospholipid (e.g., lecithin, soybean phospholipid, or soybean lecithin) and water to form an emulsion. It will be appreciated that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions will typically comprise up to 20% oil, for example 5% to 20%. The fat emulsion may comprise fat droplets of 0.1 to 1.0.im., in particular 0.1 to 0.5.im., and a pH of 5.5 to 8.0.

The emulsion composition may be prepared by combining the compound or antibody with an Intralipid^TMOr their components (soybean oil, lecithin, glycerin and water).

Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions, and powders in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof. The liquid or solid composition may comprise suitable pharmaceutically acceptable excipients as described above. In some embodiments, the composition is administered by the oral or nasal respiratory route for local or systemic effect.

The composition in a preferably sterile pharmaceutically acceptable solvent can be nebulized by the use of a gas. The nebulized solution may be breathed directly from the nebulizing device, or the nebulizing device may be connected to a mask, tent, or intermittent positive pressure ventilator. Solution, suspension or powder compositions may be administered from a device that delivers the formulation in a suitable manner, preferably orally or nasally.

Therapeutic applications

Any of the compounds or antibodies described herein, as well as encoding nucleic acids or nucleic acid sets, vectors comprising these, or host cells comprising the vectors, can be used to treat cancer, inflammation, infectious disease, or other malignancies in which stimulation of an immune response is desired.

To practice the methods disclosed herein, an effective amount of a pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment by a suitable route, e.g., intravenous administration, e.g., as a bolus injection (bolus) or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or topical routes. Commercially available nebulizers for liquid formulations (including jet nebulizers and ultrasonic nebulizers) can be used for administration. The liquid formulation can be directly nebulized, and the lyophilized powder can be nebulized after reconstitution. Alternatively, fluorocarbon (fluorocarbon) formulations and metered dose inhalers can be used to aerosolize the compounds or antibodies described herein, or to inhale as lyophilized and milled powders.

The subject to be treated by the methods described herein can be a human patient having, at risk of having, or suspected of having cancer or other malignancy requiring stimulation of an immune response. Subjects with the target disease or disorder can be identified by routine medical examination, such as laboratory tests, organ function tests, CT scans, or ultrasound. A subject suspected of having any such target disease/disorder may exhibit one or more symptoms of the disease/disorder. A subject at risk for a disease/disorder may be a subject with one or more risk factors for the disease/disorder.

The methods and compositions described herein can be used to treat cancer. Some examples of cancers that can be treated with the methods and compositions described herein include, but are not limited to: lung cancer, melanoma, kidney cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, stomach cancer, pancreatic cancer, thyroid cancer, hematological cancer, lymphoma, leukemia, skin cancer, ovarian cancer, bladder cancer, urothelial cancer, head and neck cancer, metastatic lesions of the cancer, and all types of cancer diagnosed as being at a high mutational burden. In a specific embodiment, the cancer has a high mutation burden. Subjects suffering from or at risk for a variety of cancers can be identified by routine medical procedures.

In some examples, human patients have high microsatellite instability-high (MSI-H) or mismatch repair defects (dMMR) found in soft tissue cancers, glioblastoma, esophageal and EGJ cancers, breast cancer, non-small cell lung cancer, epithelial cancers of the ovarian surface, unknown primary cancers, small cell lung cancer, non-epithelial ovarian cancer, pancreatic cancer, other female genital tract malignancies, uveal melanoma, retroperitoneal or peritoneal sarcoma, thyroid cancer, uterine sarcoma, biliary tract cancer, prostate adenocarcinoma (prostata adenocarcinomas), hepatocellular carcinoma, neuroendocrine tumors, cervical cancer, colorectal adenocarcinoma, small intestine malignancy, gastric adenocarcinoma, and endometrial cancer.

As recognized by one skilled in the art, effective amounts vary according to: the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, height, sex and weight, duration of treatment, the nature of concurrent therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of a health practitioner. Empirical considerations, such as half-life, will generally aid in the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to prolong the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The frequency of administration can be determined and adjusted during the course of treatment and is typically (but not necessarily) based on the treatment and/or inhibition and/or amelioration and/or delay of the target disease/disorder. Alternatively, a sustained continuous release formulation of the antibody may be suitable. Various formulations and devices for achieving sustained release are known in the art

In one example, the dosage for a compound or antibody described herein can be determined empirically in an individual who has been administered one or more administrations of the compound or antibody. The compound is administered to the individual at increasing doses. To assess the efficacy of a compound, indicators of disease/disorder can be followed.

Generally, for administration of any of the compounds or antibodies described herein, the initial candidate dose may be about 2 mg/kg. For purposes of this disclosure, a typical daily dose, weekly dose, biweekly dose, or triweekly dose may be any of about 0.1 to 3 to 30 to 100 to 300 to 0.6mg/kg, lmg/kg, 3 to 10mg/kg, 30 to 100mg/kg, or more depending on the factors described above. For repeated administrations over several days, weeks, months or longer, depending on the condition, the treatment is continued until the desired suppression of symptoms occurs, or until a sufficient therapeutic level is reached to alleviate the target disease or disorder or symptoms thereof. An exemplary dosing regimen includes administering an initial dose of about 3mg/kg every 3 weeks, followed by a maintenance dose of about 1mg/kg of compound or antibody once within 6 weeks, or followed by a maintenance dose of about 1mg/kg every 3 weeks. However, other dosage regimens may be useful depending on the pharmacokinetic decay pattern that the practitioner wishes to achieve. For example, a combination therapy of 1mg/kg administered once every 3 weeks with at least one additional immunotherapeutic agent is contemplated. In some embodiments, about 3 μ g/mg to about 3mg/kg (e.g., about 3 μ g/mg, about 10 μ g/mg, about 30 μ g/mg, about 100 μ g/mg, about 300 μ g/mg, about 1mg/kg and about 3mg/kg) can be used for administration. In some embodiments, the frequency of administration is once per week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once per month, every 2 months, or every 3 months or longer. The progress of such treatment is readily monitored by conventional techniques and assays. The dosage regimen, including the compound or antibody used, may vary over time.

In some embodiments, for adult patients of normal weight, a dose of about 0.1 to 5.0mg/kg may be administered. In some examples, the dose described herein may be 10 mg/kg. The particular dosing regimen, i.e., dose, time and repetition, will depend on the particular individual and the individual's medical history, as well as the nature of the individual agent (e.g., the half-life of the agent, and other considerations known in the art).

For the purposes of this disclosure, the appropriate dosage of a compound or antibody described herein will depend on the particular compound or antibody, antibody and/or non-antibody peptide (or composition thereof) used, the type and severity of the disease/disorder, whether the compound or antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antagonist, and the discretion of the attending physician. Typically, the clinician will administer the compound or antibody until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a reduction in tumor size, an extension of progression-free survival, and/or an improvement in overall survival. Methods of determining whether a dosage produces the desired result will be apparent to those skilled in the art. Administration of one or more compounds or antibodies may be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the skilled practitioner. Administration of the compound or antibody may be substantially continuous over a preselected period of time, or may be carried out in a series of spaced doses, for example, before, during, or after the onset of the disease or disorder of interest.

As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having, having symptoms of, or predisposed to a disease/disorder of interest, with the aim of curing, treating, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease, symptoms of the disease, or predisposition to the disease or disorder. Alleviating the target disease/disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease. Treatment reduces the likelihood that the subject will develop the disease and that treatment will follow the subject to combat the disease, prevent the disease from worsening or slow the progression of the disease, as compared to no treatment.

Alleviation of the disease does not necessarily require a curative effect. As used herein, "delaying" the onset of a target disease or disorder means delaying, impeding, slowing, arresting, stabilizing and/or delaying the progression of the disease. Such delays may have varying durations, depending on the history of the disease and/or the individual being treated. A method of "delaying" or alleviating the onset of disease or delaying the onset of disease is a method that: reducing the likelihood of one or more symptoms of a disease occurring within a given time frame and/or reducing the extent of symptoms within a given time frame when compared to not using the method. Such comparisons are typically based on clinical studies using a number of subjects sufficient to give statistically significant results.

"onset" or "progression" of a disease means the initial manifestation and/or subsequent progression of the disease. The occurrence of disease can be detected and assessed using standard clinical techniques well known in the art. However, occurrence also refers to a progression that may not be detected. For the purposes of this disclosure, onset or progression refers to the biological process of a symptom. "onset" includes occurrence, recurrence and seizure. As used herein, "onset" or "occurrence" of a target disease or disorder includes initial onset and/or recurrence.

In some embodiments, a compound or antibody described herein is administered to a subject in need of treatment in an amount sufficient to inhibit the activity of ABCB5 or other products in the ABCB5-PIP2 pathway by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) in vivo. In other embodiments, the compound or antibody is administered in an amount effective to reduce the level of activity of ABCB5 or other products in the ABCB5-PIP2 pathway by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

Depending on the type of disease to be treated or the site of the disease, the pharmaceutical composition may be administered to the subject using conventional methods known to those of ordinary skill in the medical arts. The composition may also be administered by other conventional routes, e.g., parenterally, topically, orally, by inhalation spray, rectally, nasally, buccally, vaginally, or by implanted reservoirs. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intraperitoneal, intratumoral, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. In addition, administration to a subject can be by an injectable depot (injectable depot) route of administration, e.g., using 1 month, 3 months, or 6 months injectable depot or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

Injectable compositions may contain various carriers such as vegetable oils, dimethyl lactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycols, and the like). For intravenous injection, the water-soluble compound or antibody may be administered by the instillation method by which a pharmaceutical formulation comprising the compound or antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, e.g., sterile preparations of the compound or antibody in the form of a suitable soluble salt, can be dissolved and administered in a pharmaceutically acceptable excipient such as water for injection, 0.9% saline, or 5% dextrose solution.

In one embodiment, the compound or antibody is administered by site-specific or targeted local delivery techniques. Some examples of site-specific or targeted local delivery techniques include various implantable depot sources of compounds or antibodies or local delivery catheters, such as infusion catheters, indwelling catheters, or needle catheters; synthesizing a graft; adventitial wrap (advanced wrap), shunt and stent or other implantable device; a site-specific vector; direct injection or direct application. See, for example, PCT publication No. wo 00/53211 and U.S. patent No.5,981,568.

Targeted delivery of therapeutic compositions comprising antisense polynucleotides, expression vectors, or subgenomic polynucleotides may also be used. Receptor-mediated DNA delivery techniques are described in the following: for example, Findeis et al, Trends Biotechnol. (1993) 11: 202; chiou et al, Gene Therapeutics: methods and Applications of Direct Gene Transter (J.A. Wolff, ed.) (1994); wu et al, j.biol.chem. (1988) 263: 621 of the first and second substrates; wu et al, j.biol.chem. (1994) 269: 542; zenke et al, proc.natl.acad.sci.usa (1990) 87: 3655; wu et al, j.biol.chem. (1991) 266: 338.

therapeutic compositions comprising polynucleotides (e.g., those encoding the antibodies or other proteins described herein) are administered at about 100ng to about 200mg of DNA for topical administration in a gene therapy regimen. In some embodiments, concentration ranges of about 500ng to about 50mg, about 1 μ g to about 2mg, about 5 μ g to about 500 μ g, and about 20 μ g to about 100 μ g DNA or higher may also be used during a gene therapy regimen.

Therapeutic polynucleotides and polypeptides described herein can be delivered using a gene delivery vehicle. Gene delivery vehicles can be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy (1994) 1: 51; Kimura, Human Gene Therapy (1994) 5: 845; Connelly, Human Gene Therapy (1995) 1: 185; and Kaplitt, Nature Genetics (1994) 6: 148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of a coding sequence may be constitutive or regulated.

Viral-based vectors for delivering a desired polynucleotide and expressing in a desired cell are well known in the art. Exemplary virus-based carriers include, but are not limited to, recombinant retroviruses (see, e.g., PCT publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; British patent No.2,200,651; and European patent No. 0345242); alphavirus-based vectors (e.g., Sindbis virus vector (Sindbis virus vector), Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (Ross River virus) (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-1250; ATCC 124923 VR 9; ATCC VR-532) as well as adeno-associated virus (AAV) vectors (see, e.g., PCT publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655.) administration of DNA linked to killed adenovirus as described in Curiel, hum. Gene Ther (1992) 3: 147 can also be used.

Non-viral delivery vehicles and methods can also be used, including but not limited to polycationic condensed DNA linked or not to a killed adenovirus alone (see, e.g., Curiel, hum. gene Ther. (1992) 3: 147); ligand-linked DNA (see, e.g., Wu, j.biol.chem. (1989) 264: 16985); eukaryotic cell delivery vehicle cells (see, e.g., U.S. Pat. No.5,814,482; PCT publication No. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nuclear charge (nuclear charge) neutralization or fusion with the cell membrane. Naked DNA (naked DNA) may also be used. Exemplary naked DNA introduction methods are described in PCT publication No. WO 90/11092 and U.S. Pat. No.5,580,859. Liposomes useful as carriers for gene delivery are described in the following: U.S. Pat. Nos. 5,422,120; PCT publication nos. wo 95/13796; WO 94/23697; WO 91/14445; and european patent No. 0524968. Additional methods are described in Philip, mol.cell.biol. (1994) 14: 2411 and methods described in Woffendin, proc.natl.acad.sci. (1994) 91: 1581.

The particular dosage regimen, i.e., dose, time, and repetition, used in the methods described herein will depend upon the particular subject and the subject's medical history.

In some embodiments, more than one compound or antibody, or a combination of a compound or antibody and another suitable therapeutic agent, may be administered to a subject in need of treatment. The compound or antibody may also be used in combination with other agents for enhancing and/or supplementing the effectiveness of the agent.

The efficacy of treatment of the target disease/disorder can be assessed by methods well known in the art.

The treatment methods described in relation to, for example, the present disclosure may be used in conjunction with other types of treatments for the target diseases or disorders disclosed herein. Some examples include chemotherapy, immunotherapy (e.g., therapies involving therapeutic antibodies, CAR T cells, or cancer vaccines), surgery, radiation, gene therapy, etc., or anti-infectious therapy. Such treatment may be administered simultaneously or sequentially (in any order) with treatment according to the present disclosure. In some cases, the target disease is a cancer (e.g., those disclosed herein) and the combination therapy includes an immune checkpoint (e.g., inhibition checkpoint) antagonist. Some examples include PD-1/PD-L1 antagonists (e.g., nivolumab, pembrolizumab, avilumab, dervolumab, and atezolizumab), LAG3 antagonists, TIM-3 antagonists, VISTA antagonists, TIGIT antagonists, CSF1R antagonists, CD112R (PVRIG) antagonists, PVR (CD155) antagonists, PD-L2 antagonists, A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, or BTLA antagonists. Additional examples include activators that enhance the activity of the stimulated checkpoint, such as CD122(IL2) agonists, 4-1BB, ICOS ligand, GITR, and OX 40.

Additional useful agents are also found in the physicians' Desk Reference, 59.sup.th edition, (2005), Thomson P D R, Montvale n.j.; gennaro et al, eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; braunwald et al, eds. Harrison's Principles of Internal Medicine, 15.sup.th edition, (2001), McGraw Hill, NY; berkow et al, eds. the Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

When co-administered with additional therapeutic agents, the appropriate therapeutically effective dose of each agent may be reduced by additive or synergistic effects.

The efficacy of the methods described herein can be assessed by any method known in the art and will be apparent to the skilled medical professional. For example, the efficacy of antibody-based immunotherapy can be assessed by survival of the subject or the burden of cancer in the subject or a tissue or sample thereof. In some embodiments, the methods are assessed based on the safety or toxicity of the treatment in the subject, e.g., by the overall health of the subject and/or the presence of an adverse event or severe adverse event.

This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Unless defined otherwise herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, a noun without a quantitative modification would include one or more. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, the nomenclature and the techniques used in connection with, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.

The invention is further illustrated by the following examples, which should in no way be construed as further limiting. All references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference in their entirety.

Examples

Example 1: ABCB5 promotes tumor invasion by modulating AXL.

Recent studies have shown that the receptor tyrosine kinase AXL, which is associated with a poor prognosis of colorectal cancer (CRC), is responsible for EMT induction in other malignancies. It is shown herein that AXL mRNA expression was reduced by > 90% in COLO741 ABCB5KD CRC cell cultures and AXL protein expression was reduced by > 50% in these cells compared to control transfected cells (fig. 8). Furthermore, mAb-mediated ABCB5 blockade consistently inhibited AXL expression in all four CRC cell lines tested (COLO741, SW620, HT29, and HCT116, with ABCB5(+) tumor cell frequencies from 9% to 27%), as determined by Western blot analysis (fig. 8), and also inhibited its downstream target phospho-AKT to a lesser extent, demonstrating an important signaling pathway for ABCB5+ cell use. The functional relationship between ABCB5 and AXL was supported by AXL expression that was significantly up-regulated at both mRNA and protein levels in untreated ABCB5(+) cells sorted from all four cell lines via flow cytometry (fig. 8).

Furthermore, AXL expression (as determined at mRNA and protein levels) and downstream signaling (p-AKT/AKT ratio) were enhanced in the transfer-derived COLO741MET compared to parental COLO741 cells (fig. 8).

Example 2: PIP2 and PIP3 are natural in vivo binding ligands of ABCB5 as determined by immunoprecipitation from human tissue.

Immunoprecipitation of human ABCB5 expressing melanoma cells from PIP2 pulled down using anti-PIP 2 antibody revealed co-precipitation of ABCB5 protein as shown in fig. 1A by immunoblotting using ABCB5 monoclonal antibody (upper panel). The PIP2 pull-down was confirmed by immunoblotting using the PIP2 antibody in fig. 1A (lower panel). Similarly, immunoprecipitation of human ABCB5 expressing melanoma cells from PIP3 pulled down using anti-PIP 3 antibody revealed co-precipitation of ABCB5 protein as shown in fig. 1B by immunoblotting using ABCB5 monoclonal antibody. These data indicate that PIP2 and PIP3 are biological ligands of ABCB5, and ABCB5 acts as a receptor for PIP2 and PIP 3.

Example 3: PIP1, PIP2 and PIP3 bound to ABCB5 as determined by ELISA.

ELISA plates coated with PIP1, PIP2 or PIP3 were incubated with purified recombinant human ABCB5 isoform 2(812aa, NCBI reference sequence: NP-848654.3), wild type mouse Abcb5 expressing skin tissue or Abcb5 non-expressing skin tissue derived from ABCB5 knock-out mice (as a specific control), followed by binding to ABCB5 detection using a mouse ABCB5 specific monoclonal antibody (Kscan et al Nature 2014). As shown in figure 1C, all PIP1, PIP2, and PIP3 bound to recombinant human ABCB5 and murine ABCB5, with no binding detection in ABCB5 knockout mouse tissues. Therefore, PIP2 and PIP3 bind ABCB5 more efficiently than PIP1 binds ABCB 5. These data confirm that ABCB5 binds to PIP2 and PIP3 and also reveal the ability of ABCB5 to PIP1, albeit with significantly lower affinity. Similar results were obtained using murine ABCB5 mAb clone 3C2-1D12(Frank NY et al J Biol chem.2003) as the detection antibody.

Example 4: binding of PIP1, PIP2 and PIP3 to ABCB5 can be inhibited by competitive pharmacological ligands.

ELISA plates coated with PIP1, PIP2 or PIP3 were incubated with purified recombinant human ABCB5 (P-glycoprotein ABCB5[ homo sapiens ] GenBank: AAO73470.1) for binding in the absence or presence of increasing doses of PtdIns- (1, 2-dioctanoyl), a synthetic analogue of natural phosphatidylinositol (PTDIns) containing C8: 0 fatty acids at the sn-1 and sn-2 positions (CAS registry No. 899827-36-2). The data illustrated in figure 2 show that the molecule competitively inhibits binding of PIP1, PIP2 or PIP3 to ABCB5 with significant saturation at concentrations as low as 0.1 mM.

These results provide proof of principle as follows: PIP analogs (including PIP2 analogs and chemical PIP2 variants that cannot be phosphorylated to biologically active PIP 3), or additional synthetic chemical or biological agents that compete for PIP1, PIP2, or PIP3 binding of ABCB5, but differ from PIP1, PIP2, or PIP3 that do not play a role in signal transduction of different receptor tyrosine kinases (listed in the table below) or multiple G protein-coupled receptors, may be used as therapeutic agents to modulate ABCB5/PIP1, ABCB5/PIP2, or ABCB5/PIP3 receptor/ligand interactions that are associated with PIP-dependent signal transduction mechanisms of such receptors in a variety of disease states, where ABCB5 is functional, particularly but not limited to initiation and progression of human cancer.

Example 5: the binding of PIP1, PIP2 and PIP3 to ABCB5 was inhibited by ABCB5 monoclonal antibody.

Surface Plasmon Resonance (SPR) analysis also demonstrated binding of PIP1, PIP2 and PIP3 to ABCB 5. Competition with the anti-ABCB 5 monoclonal antibody resulted in a decrease in signal in a concentration-dependent manner on all three surfaces (PIP 3> PIP2 > PIP1) by up to 50% on the PIP3 surface (see figure 3). Isotype control monoclonal antibodies showed no significant effect (not exemplified). These results indicate that ABCB5 monoclonal antibody can block PIP1, PIP2 or PIP3 from binding to ABCB 5.

Example 6: ABCB5 is needed to more efficiently convert PIP2 to PIP 3.

Functional experiments involving ABCB5 blockade using ABCB5 monoclonal antibody in human melanoma cells, or ABCB5 functional ablation in ABCB5 knock-out mouse derived tissues revealed that ABCB5 is functionally required for a more efficient conversion of PIP2 to PIP3, possibly through its function as a PIP2 docking receptor. As illustrated in figure 4, ABCB5 monoclonal antibody, but not isotype control antibody, significantly reduced the PIP3/PIP2 ratio (left) in human melanoma cells. Furthermore, examination of murine ABCB5 knockout skin tissue also revealed a significantly reduced PIP3/PIP2 ratio (right) compared to ABCB5 wild-type skin.

Also, the results of SPR analysis indicate that ABCB5 monoclonal antibody can block ABCB5/PIP2 receptor/ligand interactions, which indicate that ABCB5/PIP2 binding interactions are functionally required for more efficient phosphorylation of PIP2 to PIP3, suggesting that ABCB5 plays a key role in PIP 2-dependent signal transduction regarding receptor tyrosine kinase and G protein-coupled receptor signaling in ABCB 5-expressing cells, including cancer stem cells involved in tumor formation, cancer progression and resistance to treatment in various malignancies, including melanoma, colorectal cancer, glioblastoma multiforme, Merkel cell carcinoma (Merkel cell carcinoma), SCC and hepatocellular carcinoma, among others. In addition, physiological tissue-specific stem cells, including skin, eye and intestinal stem cells express ABCB5 at high levels and rely on receptor tyrosine kinases and G protein-coupled receptor signaling to perform their tissue regeneration functions. Thus, the present findings provide a means to block ABCB 5-dependent PIP2 binding and phosphorylation to PIP3 in receptor tyrosine kinase signaling, or to block processing to IP3 and DAG in G-protein coupled receptor signaling, by ABCB5 monoclonal antibody or small molecule/chemical inhibitors of ABCB5/PIP2 binding, resulting in a G-protein coupled receptor signaling-dependent mechanism that inhibits cancer initiation/progression/treatment resistance-associated receptor tyrosine kinase signaling (e.g., AXL (see Guo et al.j Biol chem.2018), EGFR), or inhibits cancer initiation/progression/treatment resistance. Furthermore, enhancing ABCB5/PIP2 binding interactions, such as increased expression/binding by ABCB5 or by exogenous PIP2 addition, would enhance biological stem cell function to treat stem cell deficiency-related disorders.

Example 7: ABCB5 is required to maintain the PIP2/PIP3 dependent PI3K/AKT signaling axis in malignancies, and ABCB5 inhibition results in inhibition of the PI3K/AKT signaling axis as well as dependent tumor growth and treatment resistance.

Treatment of 4-HT in the context of ABCB5 WT or ABCB5 KO induced Tyr: CreER; brafca; analysis of the Ptenlox/lox genetic mouse melanoma model revealed significant inhibition of the PI3K/AKT signaling axis in ABCB5 KO compared to ABCB5 WT tumors, with reduced expression of p-AKT, p-mTOR and p-S6 in additional dysregulated molecules (see figure 5).

Furthermore, in this model, ABCB5 KO status resulted in decreased tumor cell proliferation compared to ABCB5 WT status, as determined by determining the percentage of tumor cells that stained positive for the proliferation marker Ki-67 (see fig. 6):

in addition, in this model, ABCB5 KO status resulted in down-regulation of pro-angiogenic molecules compared to ABCB5 WT status (see figure 7, left panel) and as a result, decreased CD 31-positive microvascular density (see figure 7, right panel).

It is also shown that a particular ABCB5 monoclonal antibody disrupts the PIP2/PIP 3-dependent PI3K/pAKT signaling axis in colorectal cancer (see Guo et al j biol chem 2018), where treatment resulted in inhibition of the pAKT/AKT ratio, and similar results were obtained when examining the effect of ABCB5 monoclonal antibodies on human melanoma cells, where treatment with those ABCB5 antibodies that also inhibited PIP2/PIP3 binding to ABCB5 and/or PIP2 transformation to PIP3 resulted in significant inhibition of the pAKT/AKT ratio. These data indicate that functional blockade of ABCB5 disrupts the PIP2/PIP 3-dependent PI3K/pAKT signaling axis important for tumor growth and tumor angiogenesis, providing clear evidence for the anticancer therapeutic utility of functional blockade of ABCB5/PIP2/PIP3 receptor/ligand interactions.

Furthermore, in the context of ABCB5 WT or ABCB5 KO, treatment at 4-HT induced Tyr: creer; brafca; in the Ptenlox/lox genetic mouse melanoma model, ABCB5 KO status resulted in complete sensitivity to the effects of the BRAF inhibitor vemurafenib, whose resistance was driven in part by the functional PI3K/pAKT signaling axis, compared to ABCB5 WT status. In contrast to ABCB5 WT mice showing 100% vemurafenib resistant tumor formation, no tumor formation was observed after genetic induction in vemurafenib treated ABCB5 KO mice (see fig. 9, left panel), and survival was significantly prolonged in ABCB5 KO mice compared to ABCB5 WT mice (see fig. 9, right panel). These results reveal a crucial role for ABCB5 in tumor vemurafenib resistance through its function in the intact PIP2/PIP 3-dependent PI3K/pAKT signal transduction axis required to maintain vemurafenib resistance. These results also indicate that functional inhibition of ABCB5 can be therapeutically used to reverse melanoma BRAF inhibitor resistance.

Example 8: identification of a novel PIP2 structure accumulated in ABCB5 knock-out tissues identified a preferred physiological substrate for ABCB 5-dependent phosphorylation to its PIP3 form.

Quantitative lipid mass spectrometric analysis of skin tissues derived from ABCB5 Wild Type (WT) or ABCB5 knock-out (KO mouse) detected that a novel PIP2 form (PIP2 (6: 0/18: 0) @29.568051, i.e. PIP2 (6: 0/18: 0) -H, 24: 0 total fatty acid chain, unsaturated 0, formula C33H65O19P3) was specifically present in ABCB5 knock-out tissues, but not detected at the detection threshold of ABCB5 wild type skin (ABCB5 KO average: 7.79E +04+/-1.67E + 04; WT average: not detected), suggesting that this PIP2 molecular variant, previously unknown in the compound database, represents a preferred physiological substrate for ABCB 5-dependent phosphorylation to its PIP3 form, i.e. a biologically phosphorylated form of PIP3 involved in ABCB 5-dependent receptor tyrosine kinase or G-coupled receptor signaling. The bioinformatically generated structural model of this novel PIP2 (6: 0/18: 0) -H molecule (24: 0 total fatty acid chains, unsaturated 0, molecular formula C33H65O19P3) is illustrated.

This novel PIP2 (6: 0/18: 0) -H molecule, or its phosphorylated form, PIP3 (6: 0/18: 0) -H (formula C33H65O19P4), represents a composition useful as a therapeutic agent to enhance signaling through a variety of receptor tyrosine kinases or G protein-coupled receptors listed above in those disease conditions in which its signal transduction is impaired or in which ABCB5 function or ABCB5 expression levels are reduced, particularly in diseases associated with ABCB5+ stem cell deficiency, such as defects in skin wound healing, limbal stem cell deficiency, tissue regeneration deficiency in aging, and additional ABCB5 deficiency disorders, such as psoriasis, found in an ABCB5 knockout imiquimod-induced psoriasis mouse model compared to ABCB5 wild-type mice (see figure 10).

Example 9: functional profiling of the single nucleotide polymorphism of ABCB5 revealed a molecular ABCB5 ligand/substrate binding site involved in downstream molecular effector functions.

The structure of ABCB5 isoform 1(1257aa, NCBI reference sequence: NP-001157413.1) consists of two transmembrane domains (TMD) each with 6 transmembrane helices, i.e., it contains a total of 12 transmembrane helices (TM 1-12). ABCB5 isoform 2(812aa, NCBI reference sequence: NP-848654.3) consists of one TMD with 6 Transmembrane (TM) helices (TM 1-6). TM 1-6 of ABCB5 isoform 2 corresponds to TM 7-12 of ABCB5 isoform 1. TM12 of ABCB5 isoform 1 corresponds to TM6 of ABCB5 isoform 2. A Single Nucleotide Polymorphism (SNP) in the ABCB5 coding region (rs6461515) providing AA 970E > K in TM12 of ABCB5 isoform 1 and corresponding to AA525E > K in TM6 of ABCB5 isoform 2 is disclosed herein as being critical to ABCB5 function. The annotated reference E SNP (glutamate/E/GAA) is therefore conserved in various species including mice (mus musculus). However, the reference E SNP (glutamate/E/GAA) actually represents the minor codon in wisdom. Population diversity data showed that the E525 encoding allele (glutamate, G genotype, codon GAA) appeared at the highest frequency in african populations, where G/G homozygosity was rare, as opposed to the K525 encoding allele (lysine, a genotype, codon AAA) (data not shown).

The K SNP (lysine, codon AAA) is most frequently expressed from analysis of human cancers. Importantly, molecular switching from K (lysine, codon AAA) to E (glutamate, codon GAA) was experimentally induced in one allele at baseline by criprpr/Cas 9 mediated gene editing in wild type K525/K525 human melanoma cells expressing only ABCB5 isoform 2, resulting in a clonal hybrid ABCB5K 525/E525 melanoma cell variant with impaired ABCB5 signaling function and resulting in significant inhibition of ABCB5 driven tumor growth (P < 0.05) (see fig. 11).

These results suggest that amino acid residue 525 of ABCB5 isoform 2TM6 serves as an important molecular switch in the physiological ligand/substrate binding quality of ABCB5, particularly PIP2 and its phosphorylation product PIP3, where PIP2 and its phosphorylation product PIP3 are known to transmit extracellular RTK-mediated signals to activate the downstream PI3K/pAKT signaling pathway essential for tumor formation and progression, where K525(rs6461515) represents a more functional variant. Accordingly, residue 970 in TMl2 of ABCB5 isoform 1 is also associated with physiological ligand/substrate binding of ABCB 5. Two variant peptide sequences of ABCB5 isoform 2 and their coding RNA sequences (variant residues highlighted in red) are listed below, wherein the molecule comprising K525 is a more functional variant in PIP substrate/ligand binding and downstream signaling function compared to the reference comprising the E525 molecule (SNP rs 6461515):

ABCB5 isoform 2-E525 protein sequence (SEQ ID NO: 1):

ABCB5 isoforms 2-E525 coding RNA sequences (SEQ ID NO: 2):

ABCB5 isoform 2-K525 protein sequence (SEQ ID NO: 3):

ABCB5 isoforms 2-K525 coding RNA sequences (SEQ ID NO: 4):

based on bioinformatic analytical considerations, additional residues involved in ABCB5 substrate binding were N702 and H706 in TM7 of ABCB5 isoform 1, N257 and H261 in TM1 corresponding to ABCB5 isoform 2, and 857A > T in TM10 of ABCB5 isoform 1 (rs80123476) corresponding to 412A > T in TM4 of ABCB5 isoform 2 (rs 80123476).

In addition, the results to date have determined that a subset (but not all) of ABCB 5-specific monoclonal antibodies (antibodies that bind to the extracellular loop in 3-dimensional (i.e. circular form)) are capable of inhibiting PIP1, PIP2 or PIP3 binding to ABCB5 and thus inhibit ABCB 5-mediated PIP-dependent signal transduction and pAKT phosphorylation. Thus, the ABCB5 monoclonal antibody shown to inhibit ABCB 5-dependent PIP1, PIP2 or PIP3 binding and PIP-dependent signal transduction and pAKT phosphorylation demonstrated herein constitutes a uniquely useful novel composition, for example, by inhibiting therapeutically ABCB5 inhibition of ABCB 5-driven human cancer growth and progression and resulting in inhibition of ABCB 5-dependent receptor tyrosine kinase and G protein-coupled receptor signal transduction.

Example 10: and (5) molecular docking modeling.

Using bioinformatic methods and structural data available for ABCB5 homologous ABCB1 protein, model 3D structures were created for both the ABCB5 isoform 2-K525 and ABCB5 isoform 2-E525 polypeptide sequences listed above. In addition, 3D structures were created for the following ABCB5 ligands or competitive inhibitors to facilitate molecular docking/binding modeling using PyMOL software package:

(a) PIP2 (6: 0/18: 0) -H, 24: 0 total fatty acid chains, 0 unsaturation, formula C33H65O19P 3:

this is a novel PIP2 variant accumulated in the skin of ABCB5 KO mice first identified by mass spectrometry, suggesting that this novel molecule may be a physiological substrate for ABCB 5-dependent phosphorylation of PIP3, as described above. This molecule was previously unknown in the compound database (model structure as shown above).

(b) PI (4, 5) P2, diC8, molecular formula C25H49O19P 3: this PIP2 variant was shown to bind to ABCB5 by SPR. Obtained from Echelon Biosciences. Additional information on this molecule and its structure is found in CAS registry number (204858-53-7).

(c) Phosphatidylinositol C-8: PtdIns- (1, 2-dioctanoyl) is a synthetic analogue of natural phosphatidylinositol (PtdIns) comprising C8 at the sn-1 and sn-2 positions: 0 fatty acid. As described above, this molecule was shown to competitively inhibit PIP2 binding to ABCB 5. For more information on this molecule see CAS registry number 899827-36-2.

The results revealed that all the test molecules (i.e., the natural ligand PIP2 (6: 0/18: 0) -H, PI (4, 5) P2, diC8) and the competitive inhibitor phosphatidylinositol C-8 bind to either ABCB5 isoform 2-K525 or ABCB5 isoform 2-E525 structures in close proximity to the defined AA525 substrate binding site and TM6 of the ABCB5 molecule. In addition, the modeling results revealed that ABCB5 isoform 2-K525, in contrast to ABCB5 isoform 2-E525, had a higher binding affinity for PIP 2. These data further support experimental evidence of non-synonymous Single Nucleotide Polymorphisms (SNPs) in the ABCB5 coding region (rs6461515) that determine the critical role of AA525E relative to K residues in TM6 of ABCB5 isoform 2, which is disclosed herein as important for ABCB5 function, where ABCB5 isoform 2-K525 is a more functional ABCB5 variant with enhanced PIP2/PIP3 binding capacity and thus improved signal transduction capacity.

All references cited herein are incorporated by reference in their entirety. Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

Sequence listing

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gtgaagtgcc tcacatgagc catcccaaag attcattatt ccaaaccttg ggtttggcag 3660

tataagtcac aggcctacct gtttatgaaa acttacttac ttaaaataag agctactttt 3720

gggccgggtg cggtggctca cgcctgtaat cccagaactt tgggaggccg aggagggcgg 3780

atcacttgag gtcaggagtt cgagaccagc ctggccaaca tggtgaaacc ccgtctctac 3840

taaaaacaca aaaattagcc aatcttggtg gcgggcacct ggaatcccag ctacttggga 3900

ggctgaggca ggagaatcat ttgaacctag gaggcagagg ttgcagtgag ccgagatctc 3960

accactgcac tccagcctgc gcaacagagc gagactccat ctcaaaaaat aataaataag 4020

agctaatttt attgtgggtg aaaattttta aacgtctttc tctataataa aataatttcc 4080

ttaaatttta tatatacttt atcatatata atgtgtgaat gattttaaag ttctgtgtaa 4140

ataacaatat tggtaaaatg agttacattt tcaacttact taaatatgta atgtcacctg 4200

gtgattttat ctttattctt cagtgtattt tcttccattt acacatttag ctagcctccc 4260

taaagtgtac tctaccaata attgaaatct tgttaaacaa aattaaaacc atttatatat 4320

tatgctgctt tctttaaaat gcaaaataaa aataagattg gggacttgag aatca 4375

<210> 3

<211> 812

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 3

Met Val Asp Glu Asn Asp Ile Arg Ala Leu Asn Val Arg His Tyr Arg

1 5 10 15

Asp His Ile Gly Val Val Ser Gln Glu Pro Val Leu Phe Gly Thr Thr

20 25 30

Ile Ser Asn Asn Ile Lys Tyr Gly Arg Asp Asp Val Thr Asp Glu Glu

35 40 45

Met Glu Arg Ala Ala Arg Glu Ala Asn Ala Tyr Asp Phe Ile Met Glu

50 55 60

Phe Pro Asn Lys Phe Asn Thr Leu Val Gly Glu Lys Gly Ala Gln Met

65 70 75 80

Ser Gly Gly Gln Lys Gln Arg Ile Ala Ile Ala Arg Ala Leu Val Arg

85 90 95

Asn Pro Lys Ile Leu Ile Leu Asp Glu Ala Thr Ser Ala Leu Asp Ser

100 105 110

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

115 120 125

Arg Thr Thr Ile Val Val Ala His Arg Leu Ser Thr Ile Arg Ser Ala

130 135 140

Asp Leu Ile Val Thr Leu Lys Asp Gly Met Leu Ala Glu Lys Gly Ala

145 150 155 160

His Ala Glu Leu Met Ala Lys Arg Gly Leu Tyr Tyr Ser Leu Val Met

165 170 175

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

180 185 190

Ser Thr Glu Arg Lys Thr Asn Ser Leu Pro Leu His Ser Val Lys Ser

195 200 205

Ile Lys Ser Asp Phe Ile Asp Lys Ala Glu Glu Ser Thr Gln Ser Lys

210 215 220

Glu Ile Ser Leu Pro Glu Val Ser Leu Leu Lys Ile Leu Lys Leu Asn

225 230 235 240

Lys Pro Glu Trp Pro Phe Val Val Leu Gly Thr Leu Ala Ser Val Leu

245 250 255

Asn Gly Thr Val His Pro Val Phe Ser Ile Ile Phe Ala Lys Ile Ile

260 265 270

Thr Met Phe Gly Asn Asn Asp Lys Thr Thr Leu Lys His Asp Ala Glu

275 280 285

Ile Tyr Ser Met Ile Phe Val Ile Leu Gly Val Ile Cys Phe Val Ser

290 295 300

Tyr Phe Met Gln Gly Leu Phe Tyr Gly Arg Ala Gly Glu Ile Leu Thr

305 310 315 320

Met Arg Leu Arg His Leu Ala Phe Lys Ala Met Leu Tyr Gln Asp Ile

325 330 335

Ala Trp Phe Asp Glu Lys Glu Asn Ser Thr Gly Gly Leu Thr Thr Ile

340 345 350

Leu Ala Ile Asp Ile Ala Gln Ile Gln Gly Ala Thr Gly Ser Arg Ile

355 360 365

Gly Val Leu Thr Gln Asn Ala Thr Asn Met Gly Leu Ser Val Ile Ile

370 375 380

Ser Phe Ile Tyr Gly Trp Glu Met Thr Phe Leu Ile Leu Ser Ile Ala

385 390 395 400

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

405 410 415

Phe Ala Asn Lys Asp Lys Gln Glu Leu Lys His Ala Gly Lys Ile Ala

420 425 430

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

435 440 445

Lys Ala Phe Glu Gln Met Tyr Glu Glu Met Leu Gln Thr Gln His Arg

450 455 460

Asn Thr Ser Lys Lys Ala Gln Ile Ile Gly Ser Cys Tyr Ala Phe Ser

465 470 475 480

His Ala Phe Ile Tyr Phe Ala Tyr Ala Ala Gly Phe Arg Phe Gly Ala

485 490 495

Tyr Leu Ile Gln Ala Gly Arg Met Thr Pro Glu Gly Met Phe Ile Val

500 505 510

Phe Thr Ala Ile Ala Tyr Gly Ala Met Ala Ile Gly Lys Thr Leu Val

515 520 525

Leu Ala Pro Glu Tyr Ser Lys Ala Lys Ser Gly Ala Ala His Leu Phe

530 535 540

Ala Leu Leu Glu Lys Lys Pro Asn Ile Asp Ser Arg Ser Gln Glu Gly

545 550 555 560

Lys Lys Pro Asp Thr Cys Glu Gly Asn Leu Glu Phe Arg Glu Val Ser

565 570 575

Phe Phe Tyr Pro Cys Arg Pro Asp Val Phe Ile Leu Arg Gly Leu Ser

580 585 590

Leu Ser Ile Glu Arg Gly Lys Thr Val Ala Phe Val Gly Ser Ser Gly

595 600 605

Cys Gly Lys Ser Thr Ser Val Gln Leu Leu Gln Arg Leu Tyr Asp Pro

610 615 620

Val Gln Gly Gln Val Leu Phe Asp Gly Val Asp Ala Lys Glu Leu Asn

625 630 635 640

Val Gln Trp Leu Arg Ser Gln Ile Ala Ile Val Pro Gln Glu Pro Val

645 650 655

Leu Phe Asn Cys Ser Ile Ala Glu Asn Ile Ala Tyr Gly Asp Asn Ser

660 665 670

Arg Val Val Pro Leu Asp Glu Ile Lys Glu Ala Ala Asn Ala Ala Asn

675 680 685

Ile His Ser Phe Ile Glu Gly Leu Pro Glu Lys Tyr Asn Thr Gln Val

690 695 700

Gly Leu Lys Gly Ala Gln Leu Ser Gly Gly Gln Lys Gln Arg Leu Ala

705 710 715 720

Ile Ala Arg Ala Leu Leu Gln Lys Pro Lys Ile Leu Leu Leu Asp Glu

725 730 735

Ala Thr Ser Ala Leu Asp Asn Asp Ser Glu Lys Val Val Gln His Ala

740 745 750

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

755 760 765

Leu Ser Ala Ile Gln Asn Ala Asp Leu Ile Val Val Leu His Asn Gly

770 775 780

Lys Ile Lys Glu Gln Gly Thr His Gln Glu Leu Leu Arg Asn Arg Asp

785 790 795 800

Ile Tyr Phe Lys Leu Val Asn Ala Gln Ser Val Gln

805 810

<210> 4

<211> 4375

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic polynucleotide

<400> 4