Ligands and substrates of ABCB5

Abstract

The present invention is directed to methods and compositions for modulating the activity of ABCB5+ stem cells. The present invention also relates to assays and reagents for engineering and characterizing compounds that modulate ABCB5+ cell signaling.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 662,670, filed April 25, 2018, the entire contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THEINVENTION The present invention is directed to methods and compositions for regulating stem cell activity to treat disease, as well as related assays and reagents.The present invention is also directed to methods and compositions for wound healing and tissue engineering associated with ABCB5 positive cells. [Background technology]

[0003] 2. Background of the Invention Tumor initiation and progression have been linked to cumulative changes in oncogenes, tumor suppressors, and repair / stability genes at the DNA level. At the cellular level, human cancers are recognized to consist of phenotypically heterogeneous cell populations with diverse capabilities for self-renewal and tumor growth. This observation has led to the development of the cancer stem cell (CSC) model of tumor initiation and growth, which has been widely validated in multiple malignant lesions, including melanoma and colorectal cancer. CSCs have been shown to contribute to the failure of existing therapies to consistently eradicate malignancies through diverse molecular mechanisms that result in tumor progression and therapeutic resistance, including epithelial-mesenchymal transition (EMT), which is associated with the ability of human cancers to invade the vasculature and metastasize to new anatomical locations.

[0004] ABCB5 is a multidrug resistance (MDR) regulator expressed in a variety of human malignancies, where it is specifically overexpressed in treatment-resistant CD133(+) tumor subpopulations previously found to represent CSCs. ABCB5 confers drug resistance to chemotherapeutic agents such as 5-fluorouracil (5-FU) on cancer cells.

[0005] ABCB5+ stem cells are also found in normal tissues and have a role in tissue regeneration and aging. Regenerative medicine involves the repair, regeneration, maintenance and replacement of tissues and organs using foreign materials such as scaffolds. The scaffolds can be seeded with cells, such as primary or stem cells, and various factors that promote the growth of tissue. However, many problems remain in the design of suitable materials for regenerative medicine and tissue engineering. Summary of the Invention

[0006] Summary of the Invention The present invention, in some aspects, is directed to methods and compositions for modulating the activity of ABCB5+ stem cells. The present invention also relates to assays and reagents for engineering and characterizing compounds that modulate ABCB5+ cell signaling.

[0007] Aspects of the invention relate to methods of enhancing the function of ABCB5-positive cells, the methods 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 following administration of the composition. In some embodiments, the composition is a PIP2 or a PIP2 agonist. In some embodiments, the subject is a human or a non-human animal, including a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, cat, dog, llama, or a primate, such as a monkey.

[0008] In some embodiments, the composition comprises a phospholipid. In some embodiments, the composition comprises [PIP2(6:0 / 18:0)-H] - and a pharma- ceutically acceptable carrier. In some embodiments, the composition comprises a phospholipid comprising a compound having a structure described herein. In some embodiments, the structure comprises an R1 and an R2 group. In some embodiments, R1 and R2 are independent fatty acid chains. In some embodiments, the structure comprises that R1 and R2 are 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 a total fatty acid chain of 24:0.

[0009] 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, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular, or intrathecal routes.

[0010] Aspects of the invention relate to a method of inhibiting the function of ABCB5-positive cancer cells, the method 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 following administration of the composition.

[0011] Another aspect of the present invention relates to a method for inhibiting the function of ABCB5 positive cancer cells, 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 a small molecule, a lipid analogue, an anti-ABCB5 antibody or fragment having specificity for a cyclic or linear form of an extracellular polypeptide of a protein, an enzyme, and an anti-ABCB5 antibody or fragment thereof that modifies the conformation of the ABCB5 PIP2 binding site.

[0012] In some embodiments, the anti-ABCB5 antibody or fragment thereof that modifies the conformation of the ABCB5 PIP2 binding site inhibits the production of PIP3. In some embodiments, the anti-ABCB5 antibody or fragment thereof that modifies the conformation of the ABCB5 PIP2 binding site inhibits the PI3K pathway. In some embodiments, ABCB5-PIP2 binding is assessed following administration of the composition.

[0013] In some embodiments, the composition is a PIP2 antagonist. In some embodiments, the composition is selected from the group including a small molecule, a lipid analog, an anti-ABCB5 antibody or fragment with specificity for the cyclic or linear form of the extracellular polypeptide of the protein, and an enzyme. In some embodiments, the composition is a small molecule. In some embodiments, the composition is an ABCB5 antibody or fragment with specificity for the cyclic or linear form of the extracellular polypeptide of the protein. In some embodiments, the composition is an ABCB5 antibody or fragment that modifies 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.

[0014] In some embodiments, the subject is a human or a non-human animal, including a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, cat, dog, llama, or a primate, e.g., monkey. In some embodiments, the composition is administered by oral, intravenous, subcutaneous, topical, parenteral, intratumor, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular, or intrathecal routes.

[0015] Aspects of the invention relate to methods for identifying activators or inhibitors of the ABCB5-PIP2 pathway. In some embodiments, the invention includes contacting an ABCB5+ cell with a putative composition that modulates ABCB5-PIP2 binding; determining a level of a compound that is a product of the PIP2 pathway and comparing the level to a baseline level of the compound that is a product of the PIP2 pathway. In some embodiments, if the level is higher than the baseline level, the putative composition is identified as an ABCB5-PIP2 pathway activator. In some embodiments, if the level of the compound that is a product of the PIP2 pathway is lower than the baseline level, the putative composition is identified as an ABCB5-PIP2 pathway inhibitor, and the putative composition is ABCB5-PIP2.

[0016] In some embodiments, the putative composition that modulates the ABCB5-PIP2 pathway is PIP2 or a 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-ABCB5 antibody or a fragment thereof. In some embodiments, the PIP2 pathway compound is PIP3. In some embodiments, the PIP2 pathway compound is a member of the PI3K pathway. In some embodiments, the ABCB5+ cells comprise ABCB5 isoform 1, where the amino acid at position 970 is lysine. In some embodiments, the ABCB5+ cells comprise ABCB5 isoform 2, where the amino acid at position 525 is lysine.

[0017] In some embodiments, the assay involves determining the number of ABCB5 alleles and then testing how many are positive for K and how many are positive for E, i.e., an allele-specific quantification procedure that extracts both copy number and allele information.

[0018] Aspects of the invention relate to compositions comprising synthetic phospholipids, including compounds having a structure as described herein. In some embodiments, the structure comprises an R1 and an R2 group. In some embodiments, R1 and R2 are independent fatty acid chains. In some embodiments, R1 and R2 are at least twice as long as the other of R1 and R2. In some embodiments, the phospholipid has a total fatty acid chain of 22:0 to 26:0. In some embodiments, the phospholipid has a total fatty acid chain of 24:0. In some embodiments, the phospholipid has the formula: C33H65O19P3. In some embodiments, the phospholipid is [PIP2(6:0 / 18:0)-H] - and a pharma- ceutically acceptable carrier.

[0019] 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 is phosphorylated PIP3(6:0 / 18:0)-H - (C33H65O19P4) and a pharma- ceutically acceptable carrier.

[0020] Aspects of the invention relate to human anti-ABCB5 antibodies or ABCB5-binding fragments thereof that inhibit the ABCB5-PIP2 pathway, where the anti-ABCB5 antibodies or ABCB5-binding fragments thereof bind to an extracellular loop in the three-dimensional conformation of ABCB5.

[0021] In some embodiments, the human anti-ABCB5 antibody or ABCB5 binding fragment is preparable by a method including affinity maturation for specifically binding to an extracellular loop of a non-linear form of ABCB5. In some embodiments, the human anti-ABCB5 antibody or ABCB5 binding fragment has a sequence corresponding to an antibody preparable by a method including affinity maturation for specifically binding to an extracellular loop of a non-linear form of ABCB5.

[0022] Aspects of the invention relate to methods of preparing human anti-ABCB5 antibodies or ABCB5 binding fragments that inhibit the ABCB5-PIP2 pathway as described herein. In some embodiments, the anti-ABCB5 antibodies or ABCB5 binding fragments are subjected to affinity maturation to specifically bind to an extracellular loop of a non-linear form of the protein.

[0023] Aspects of the invention relate to methods for identifying antibodies or fragments that inhibit the ABCB5-PIP2 pathway. In some embodiments, antibodies or fragments that inhibit the ABCB5-PIP2 pathway are identified by contacting ABCB5+ cells with a putative antibody or fragment that binds ABCB5; assessing ABCB5-PIP2 binding after treatment with the antibody or fragment; determining the level of a compound that is a product of the PIP2 pathway and comparing the level to a baseline level of a compound that is a product of the PIP2 pathway.

[0024] In some embodiments, if the level of the compound that is a product of the PIP2 pathway is lower than the baseline level, the putative antibody or fragment is an inhibitor of the ABCB5-PIP2 pathway. In some embodiments, the PIP2 pathway compound is PIP3. In some embodiments, the PIP2 pathway compound is a member of the PI3K pathway.

[0025] Aspects of the invention relate to ABCB5 isoform 1, which comprises two transmembrane domains (TMDs) and 12 transmembrane helices (TM1-12). In some embodiments, the glutamic acid at position 970TM12 is mutated to a lysine or position 970TM12 is a glutamic acid.

[0026] Aspects of the invention relate to ABCB5 isoform 2, which comprises one transmembrane domain (TMD) and six transmembrane helices (TM1-6). In some embodiments, the glutamic acid at position 525TM6 is mutated to a lysine or position 525TM12 is a glutamic acid.

[0027] Aspects of the present invention relate to human anti-ABCB5 isoform antibodies or fragments thereof that inhibit the ABCB5-PIP2 pathway. In some embodiments, the anti-ABCB5 antibody or ABCB5-binding fragment thereof specifically binds to ABCB5 isoform 1 as described herein or ABCB5 isoform 2 as described herein.

[0028] Aspects of the present invention relate to human anti-ABCB5 isoform antibodies or fragments thereof that inhibit the ABCB5-PIP2 pathway. In some embodiments, the anti-ABCB5 antibody or ABCB5-binding fragment thereof specifically binds to ABCB5 isoform 1 or ABCB5 isoform 2 as described herein.

[0029] Aspects of the present invention relate to methods for identifying activators or inhibitors of the ABCB5-PIP2 pathway. In some embodiments, the methods include contacting ABCB5 isoform 1 or ABCB5 isoform 2 as described herein with a putative composition that modulates ABCB5-PIP2 binding; determining the level of a compound that is a product of the PIP2 pathway and comparing the level to a baseline level of a compound that is a product of the PIP2 pathway. In some embodiments, if the level is higher than the baseline level, the putative composition is an ABCB5-PIP2 pathway activator. In some embodiments, if the level of the PIP2 pathway compound is lower than the baseline level, the putative composition is an ABCB5-PIP2 inhibitor.

[0030] In some embodiments, the putative composition that modulates the ABCB5-PIP2 pathway is PIP2 or a 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-ABCB5 antibody or a fragment thereof. In some embodiments, the PIP2 pathway compound is PIP3. In some embodiments, the PIP2 pathway compound is a member of the PI3K pathway. In some embodiments, the ABCB5 isoform is recombinantly expressed.

[0031] Aspects of the present invention relate to a method for treating cancer in a subject. In some embodiments, the method comprises disrupting endogenous ABCB5 gene in a cell using gene editing. In some embodiments, the editing comprises contacting the cell with Cas protein, CRISPR RNA that hybridizes to the endogenous ABCB5 gene, and tracrRNA. In some embodiments, the endogenous ABCB5 gene is modified such that after contacting with Cas protein, CRISPR RNA, and tracrRNA, the AAA sequence in the region of the gene that codes for the terminal transmembrane helix of the ABCB5 gene is replaced with GAA. In some embodiments, the gene editing treats cancer in a subject.

[0032] In some embodiments, the subject has ABCB+ stem cells associated with the cancer prior to gene editing that is ABCB5 homozygous isoform 2 K525 / K525. In some embodiments, the cancer is melanoma or glioblastoma.

[0033] 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 effective amount to inhibit function of the ABCB5-PIP2 pathway to treat the cancer in the subject. In some embodiments, the cancer is comprised of cancer cells, the cancer cells express negligible or no ABCB1. 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 treated simultaneously with a chemotherapeutic agent. In some embodiments, the method further comprises assessing ABCB5-PIP2 binding following administration of the composition.

[0034] In some embodiments, the subject has ABCB+ stem cells associated with the cancer prior to gene editing that is ABCB5 homozygous isoform 2 K525 / K525. In some embodiments, the cancer is melanoma or glioblastoma.

[0035] Aspects of the present invention relate to methods for characterizing cancer. In some embodiments, the methods include isolating cancer cells from a subject and determining whether the cancer cells are ABCB5 homozygous isoform 2 K525 / K525, ABCB5 homozygous isoform 2 E525 / E525, or ABCB5 heterozygous isoform 2 K525 / E525 to characterize the cancer.

[0036] Each of the limiting factors of the present invention may include various aspects of the present invention. Therefore, it is understood that each of the limiting factors of the present invention, including any one element or combination of elements, may be included in each aspect of the present invention. The present invention is not limited in its application to the details of the configuration and arrangement of components described in the following description or illustrated in the drawings. The present invention is capable of other embodiments and can be practiced or carried out in various ways. In addition, the phraseology and terminology used in this specification are for the purpose of explanation and should not be considered as limiting. The words "including," "comprising," "having," "involving," and variations thereof in this specification are intended to include the items listed thereafter and their equivalents, as well as additional items.

[0037] 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 drawings is represented by the same numeral. For purposes of clarity, every component may not be labeled in every drawing. In the drawings: [Brief description of the drawings]

[0038] [Figure 1]Figures 1A-1C. PIP2 and PIP3 are biological ligands for ABCB5, which acts as a receptor for PIP2 and PIP3. Immunoblot with ABCB5 monoclonal antibody (Figure 1A, upper panel) shows that immunoprecipitation of PIP2 from human ABCB5-expressing melanoma cells with anti-PIP2 antibody pulldown revealed co-precipitation of ABCB5 protein. PIP2 pulldown was confirmed using immunoblot with PIP2 antibody (Figure 1A, lower panel). Immunoblot with ABCB5 monoclonal antibody (Figure 1B) shows that immunoprecipitation of PIP3 from human ABCB5-expressing melanoma cells with anti-PIP3 antibody pulldown revealed co-precipitation of ABCB5 protein. Figure 1C shows that PIP1, PIP2 and PIP3 all bound to recombinant human ABCB5 as well as mouse Abcb5, and there was no detection of binding in Abcb5 knockout mouse tissues. The binding of PIP2 and PIP3 to ABCB5 was thus more efficient than the binding of PIP1 to ABCB5.

[0039] [Diagram 2] Figure 2. Binding of PIP1, PIP2, and PIP3 to ABCB5 can be inhibited by competitive pharmacological ligands. The data show that PtdIns-(1,2-dioctanoyl) can competitively inhibit the binding of PIP1, PIP2, or PIP3 to ABCB5, with apparent saturation of the effect at concentrations as low as 0.1 mM.

[0040] [Figure 3-1] Figure 3. ABCB5 monoclonal antibodies can block the binding of PIP1, PIP2 or PIP3 to ABCB5. Surface plasmon resonance (SPR) analysis showed the binding of PIP1, PIP2 and PIP3 to ABCB5. Competition with anti-ABCB5 monoclonal antibodies resulted in a signal reduction in all three surfaces in a concentration-dependent manner (PIP3>PIP2>PIP1), up to 50% on the PIP3 surface. [Figure 3-2]ABCB5 monoclonal antibody can block the binding of PIP1, PIP2 or PIP3 to ABCB5. Surface plasmon resonance (SPR) analysis showed the binding of PIP1, PIP2 and PIP3 to ABCB5. Competition with anti-ABCB5 monoclonal antibody resulted in a signal reduction in all three surfaces in a concentration-dependent manner (PIP3>PIP2>PIP1), up to 50% on the PIP3 surface. [Figure 3-3] ABCB5 monoclonal antibody can block the binding of PIP1, PIP2 or PIP3 to ABCB5. Surface plasmon resonance (SPR) analysis showed the binding of PIP1, PIP2 and PIP3 to ABCB5. Competition with anti-ABCB5 monoclonal antibody resulted in a signal reduction in all three surfaces in a concentration-dependent manner (PIP3>PIP2>PIP1), up to 50% on the PIP3 surface.

[0041] [Figure 4] Figure 4. ABCB5 is functionally required for more efficient PIP2 to PIP3 conversion. ABCB5 monoclonal antibody significantly reduced the PIP3 / PIP2 ratio in human melanoma cells (left panel), whereas an isotype control antibody did not. Furthermore, examination of mouse ABCB5 knockout skin tissue also revealed that the presence of ABCB5 significantly reduced the PIP3 / PIP2 ratio compared to ABCB5 wild-type skin (right panel).

[0042] [Diagram 5]Figure 5. ABCB5 is required to maintain the PIP2 / PIP3-dependent PI3K / AKT signaling axis in malignant tumors, and inhibition of ABCB5 leads to inhibition of the PI3K / AKT signaling axis and dependent tumor growth and therapeutic resistance. Analysis of a 4-HT treatment-inducible Tyr::CreER;BrafCA;Ptenlox / lox genetic mouse melanoma model in an ABCB5 WT or ABCB5 KO background revealed a significant inhibition of the PI3K / AKT signaling axis and attenuated expression of p-AKT, p-mTOR and p-S6, among other dysregulated molecules, in ABCB5 KO versus ABCB5 WT tumors.

[0043] [Figure 6] Figure 6. Relative to the ABCB5 WT condition, the ABCB5 KO condition results in reduced tumor cell proliferation, as determined by determining the percentage of tumor cells staining positive for the proliferation marker Ki-67.

[0044] [Figure 7] 7A-7B. Relative to ABCB5 WT, ABCB5 KO resulted in downregulation of pro-angiogenic molecules (FIG. 7A, left panel) and, consequently, reduced CD31-positive microvessel density (FIG. 7A, right panel, and FIG. 7B).

[0045] [Figure 8]Figure 8. ABCB5(+) CRC cells express the EMT-sustaining receptor tyrosine kinase AXL, which acts as a regulator of ABCB5-dependent cancer invasion. Shown is a set of scatter plots representing representative flow cytometry analysis of AXL protein expression in ABCB5 KD vs. control transfected cell lines. Also shown is a bar graph illustrating AXL mRNA expression in ABCB5 KD vs. control transfected human CRC cells. Also shown is a Western blot analysis of AXL, AKT and phospho-AKT protein expression in either anti-ABCB5 mAb-treated or isotype control-treated CRC cells. Data were analyzed using unpaired t-test. Error bars indicate sem. *P<0.05, **P<0.01, ***P<0.001.

[0046] [Figure 9] Figure 9. ABCB5 plays a key role in tumor vemurafenib resistance through its function in maintaining an intact PIP2 / PIP3-dependent PI3K / pAKT signaling axis required for vemurafenib resistance. In a 4-HT treatment-inducible Tyr::CreER;BrafCA;Ptenlox / lox genetic mouse melanoma model in ABCB5 WT or ABCB5 KO background, the ABCB5 KO state, versus the ABCB5 WT state, resulted in complete sensitization to the effects of the BRAF inhibitor vemurafenib, the resistance to which is in part driven by a functional PI3K / pAKT signaling axis. Whereas ABCB5 WT mice showed 100% formation of vemurafenib-resistant tumors, upon gene induction in vemurafenib-treated ABCB5 KO mice, no tumor formation was observed (left panel) and survival was significantly extended in ABCB5 KO mice versus ABCB5 WT mice (right panel).

[0047] [Figure 10]Figure 10. Psoriasis was found to be exacerbated in a mouse model of imiquimod-induced psoriasis in ABCB5 knockout compared to ABCB5 wild-type mice.

[0048] [Figure 11] Figure 11. Amino acid residue 525 of TM6 of ABCB5 isoform 2 as a key molecular switch in the physiological ligand / substrate binding quality of ABCB5. A molecular switch from K (lysine, codon AAA) to E (glutamic acid, codon GAA) was experimentally induced in wild-type K525 / K525 human melanoma cells exclusively expressing ABCB5 isoform 2 at baseline through Crispr / Cas9-mediated gene editing in one allele, which resulted in clonal heterozygous melanoma cell variants with impaired ABCB5 signaling function and consequently significantly inhibited ABCB5-driven tumor growth (P<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Detailed Description of the Invention The present invention relates in some aspects to the discovery of ATP-binding cassette, subfamily B (MDR / TAP), member 5 (ABCB5) [Frank, NY et al. Regulation of progenitor cell fusion by ABCB5 P-glycoprotein, a novel human ATP-binding cassette transporter. J Biol Chem 278, 47156-65 (2003) and Schatton, T. et al. Identification of cells initiating human melanomas. Nature 451, 345-9 (2008)], which is preferentially expressed at high levels in the cell membrane of cancer stem cells and normal tissue-specific stem cells and serves as a receptor for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, also known simply as PIP2) and, to a lesser extent, for PIP1 and PIP3. "ABCB5(+) stem cells", as used herein, refer to cells that have the ability to self-renew and differentiate into mature cells of multiple adult cell lineages, and are characterized by the expression of ABCB5 on the cell surface. PIP2 is a minor phosphoinositol phospholipid component of cell membranes that is concentrated in cell membranes and is a substrate for many important signaling proteins, such as those that control signaling through receptor tyrosine kinases (RTKs) via the PI3K pathway or through the IP3 / DAG pathway of G protein-coupled receptors. Inhibition of the ABCB5-PIP2 pathway through inhibition of ABCB5 blocks the binding of PIP2 to ABCB5, and as a result, inhibits the phosphorylation of PIP2 to generate PIP3. Disruption of this pathway results in the inhibition of PI3K signaling downstream of tyrosine kinase receptors (e.g., VEGFR1, EGFR and AXL), abrogating 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 Figures 1-3).

[0050] An aspect of the present invention relates to a method for enhancing the function of ABCB5 positive cells. As used herein, "function of ABCB5 positive cells" refers to the activity of ABCB5 in a healthy subject and has a positive effect on the subject. For example, the function of ABCB5 positive cells includes wound healing, tissue regeneration, angiogenesis, promoting cell survival, and inhibiting cell death. It should be understood that wound healing, tissue regeneration, angiogenesis, cell survival, and cell death are determined by comparing the level or rate of each relative to the level or rate in a control sample. "Control sample" as used herein refers to a sample that lacks the function of ABCB5. "Healthy subject" as used herein is a subject that is otherwise disease-free.

[0051] The phrase "stem cell specific function" as used herein refers to the activity of ABCB5 associated with stem cells. For example, healthy ABCB5+ stem cells associated with skin use ABCB5-enhanced PI3K signaling and downstream AKT phosphorylation and mTOR signaling for angiogenesis and anti-apoptotic signaling, resulting in stem cell survival and vascular differentiation required for normal wound healing, among other downstream functions. ABCB5+ limbal stem cells utilize this pathway for anti-apoptotic signaling required for stem cell maintenance. For example, ABCB5+ cancer stem cells in melanoma or colorectal cancer utilize this pathway for cell survival, vascular mimicry, drug resistance, and EMT and metastatic invasion (as shown in Figure 1, i.e., ABCB5 blockage inhibits pAKT phosphorylation and EMT and invasiveness).

[0052] ABCB5 binding of PIP2 represents a stem cell-specific interaction that can serve, among other functions, to increase the rate of its PIP3 phosphorylation, thus enhancing the signaling role of PIP2 in cells that do not express ABCB5. ABCB5-PIP2 binding can also be inhibited by small molecule ABCB5 competitive ligands or substrates, or compositions containing same, which also inhibit signaling of important downstream ABCB5-dependent biological stem cell functions. Thus, the present invention has several important utilities. Thus, the invention described herein may be useful for promoting regeneration in healthy subjects.

[0053] The present invention may also be useful in treating subjects having or at risk of having a disease, such as subjects having or at risk of having cancer. The subject should mean a human or a vertebrate mammal, including but not limited to goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, cat, dog, llama and primate, such as monkey.Thus, the present invention can also be used to treat disease or condition in non-human subjects.For example, cancer is one of the main causes of death in pet animals (i.e., cats and dogs).Preferably, the subject is a human.

[0054] A subject at risk of developing cancer is a subject with a high probability of developing cancer.These subjects include, for example, subjects with genetic abnormalities whose existence has been shown to correlate with a higher probability of developing cancer, and subjects exposed to cancer-causing agents such as tobacco, asbestos or other chemical toxins, or subjects previously treated for cancer and in apparent remission.Subjects at risk of having cancer also include subjects with precancerous lesions.Precancerous lesions are areas of tissue that have changed characteristics and carry the risk of developing into skin cancer.Precancerous lesions can be caused by, for example, exposure to carcinogens such as UV radiation, genetics, arsenic, tar or X-ray radiation.

[0055] A subject with cancer is a subject with detectable cancerous cells.Cancer can be malignant or non-malignant cancer.Cancer or tumor includes, but is not limited to, biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasia; 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; sarcoma; skin cancer; testicular cancer; thyroid cancer; and kidney cancer, as well as other carcinomas and sarcomas.Preferably, cancer comprises cancer stem cells that express ABCB5.

[0056] Optionally, before treatment, the presence of ABCB5 positive stem cells can be detected using the binding molecules described herein.The detection or diagnosis method provided by the present invention generally comprises contacting one or more molecules of the present invention with a sample in or from a subject.Preferably, the sample is first taken from the subject, but in vivo detection methods are also envisioned.The sample can comprise any body tissue or fluid suspected to contain cancer stem cells.For example, stem cells are generally found in or around tumor masses.

[0057] ABCB5 or ATP-binding cassette subfamily B member 5 As its name suggests, ABCB5 is a member of the ATP-binding cassette transporter subfamily B. It is a transmembrane protein encoded by the ABCB5 gene. ATP-binding cassette (ABC) transporters play a pivotal role in physiology and pathology. They are involved in the transport of structurally diverse molecules ranging from small ions, sugars, and peptides to more complex organic molecules (Chen et al. 2005).

[0058] "ABCB5+ stem cells" or "ABCB5+ cells", as used herein, refer to cells that have the ability to self-renew and differentiate into mature cells of multiple adult cell lineages. In some embodiments, these cells are characterized by expression of ABCB5. 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.

[0059] Aspects of the present 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, an "ABCB5 isoform" is an ABCB5 protein having one variant of the structure of ABCB5. In some embodiments, the ABCB5 isoform is ABCB5 isoform 1 (1257 amino acids). ABCB5 isoform 1 contains two transmembrane domains (TMDs) each with six transmembrane (TM) helices, i.e., it contains a total of 12 transmembrane helices (TM1-12). In some embodiments, the ABCB5 isoform includes isoform 2 (812 amino acids). ABCB5 isoform 2 contains one TMD with six transmembrane (TM) helices (TM1-6). TM1-6 of ABCB5 isoform 2 corresponds to TM7-12 of ABCB5 isoform 1. In further embodiments, the presence of lysine residues at specific positions in the two isoforms is associated with the prevalence of cancer. In further embodiments, the residues are 970 in TM12 of ABCB5 isoform 1 and 525 in TM6 of ABCB5 isoform 2. Nonsynonymous single nucleotide polymorphisms (SNPs) in the coding region of ABCB5, which provide AA970E>K in TM12 of ABCB5 isoform 1 and correspond to AA525E>K in TM6 of ABCB5 isoform 2, have been found to be important for the function of ABCB5 in cancer cells. For example, 970 in TM12 of ABCB5 isoform 1 and 525 in TM6 of ABCB5 isoform 2 are important for the function of ABCB5-positive stem cells. In further embodiments, these residues are required for the function of ABCB5-positive cancer stem cells.

[0060] Additional residues involved in ABCB5 substrate binding are N702 and H706 in TM7 of ABCB5 isoform 1 (corresponding to N257 and H261 in TM1 of ABCB5 isoform 2) and 857A>T (rs80123476) in TM10 of ABCB5 isoform 1 (corresponding to 412A>T (rs80123476) in TM4 of ABCB5 isoform 2).

[0061] Genes encoding either the more functional ABCB5 isoform2-K525 protein sequence or the ABCB5 isoform2-K525 protein itself, as well as genes encoding the less functional ABCB5 isoform2-E525 protein sequence or the ABCB5 isoform2-E525 protein itself, are useful compositions. These compositions can be used, for example, for: 1. recombinantly expressing ABCB5 isoform2-K525 or ABCB5 isoform2-E525; 2. using ABCB5 isoform2-K525 and ABCB5 isoform2-E525 in docking and binding experiments to identify novel sequence-specific ABCB5 ligands and substrates; 3. using ABCB5 isoform2-K525 or ABCB5 isoform2-E525 to competitively inhibit the binding of PIP1, PIP2 or PIP3 to ABCB5 and thus also to inhibit ABCB5-dependent receptors. For use in molecular screens to identify synthetic compounds and naturally occurring substances that inhibit tyrosine kinase and G protein-coupled receptor signaling; ABCB5 isoform 2-K525 or ABCB5 isoform 2-E525 can be used for use in molecular screens to identify novel ABCB5 monoclonal antibodies that inhibit the binding of PIP1, PIP2 or PIP3 to ABCB5 and thus inhibit ABCB5-dependent receptor tyrosine kinase and G protein-coupled receptor signaling.

[0062] Compounds that competitively inhibit the binding of PIP1, PIP2 or PIP3 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 analogue that contains C8:0 fatty acid at the sn-1 and sn-2 positions of natural phosphatidylinositol (PtdIns) (CAS Registry Number 899827-36-2). These compounds are useful for treating cancers associated with ABCB5+ stem cells.

[0063] In some aspects, the present invention is a method for treating cancer by administering ABCB1 inhibitor to a subject having cancer.Herein, it is found that ABCB1 inhibitor is also useful for treating ABCB5+ cancer.These compounds competitively inhibit the binding of PIP1, PIP2 or PIP3 to ABCB5, and thus inhibit ABCB5-dependent signal transduction.

[0064] ABCB1 inhibitors, as used herein, are compounds that reduce or eliminate the function of ABCB1 in cells. ABCB1 inhibitors are known in the art and include anti-ABCB1 antibodies and their functional fragments and small molecules. Some ABCB1 inhibitors are ABCB1 agents for treating heart disease or vascular disease, ABCB1 agents for treating ABCB1+ cancer, ABCB1 agents for treating infectious diseases, ABCB1 agents for treating gastric diseases, and ABCB1 agents for treating various diseases. In some embodiments, ABCB1 inhibitors include, for example, PSC 833 (Valspodar), zosquidar, tariquidar and laniquidar, i.e., substrates and / or inhibitors of the relevant substrate binding sites of highly homologous ABCB1 molecules.

[0065] ABCB1 substrate or inhibitor is known for the treatment of various diseases.Based on the discovery of the novel ABCB5 isoform 2-AA525 substrate binding site for PIP1, PIP2 or PIP3, these compounds can be used to therapeutically inhibit the growth and progression of human cancers driven by ABCB5 through functional ABCB5 blockade, for example, in ABCB5 expressing cancers, and thus as small molecule inhibitors of ABCB5-dependent PIP1, PIP2 or PIP3 binding, and PIP-dependent signal transduction and pAKT phosphorylation.

[0066] In some embodiments, the ABCB1 inhibitors useful in the methods of treating cancer are ABCB1 agents for the treatment of heart / vascular disease. Non-limiting examples of these compounds are shown in the list below. ABCB1 agents for the treatment of cardiac / vascular diseases: [Table 1]

[0067] In some embodiments, the ABCB1 inhibitors useful in the methods of treating cancer are ABCB1 agents for the treatment of infectious diseases. Non-limiting examples of these compounds are shown in the list below. ABCB1 Agents for the Treatment of Infectious Diseases [Table 2]

[0068] In some embodiments, the ABCB1 inhibitor useful in the method of treating cancer is an ABCB1 agent for treating cancer. In some embodiments, the cancer is an ABCB5+ cancer, and the cancer does not have ABCB1 or has negligible ABCB1. Non-limiting examples of these compounds are shown in the following list. ABCB1 inhibitors for the treatment of ABCB5+ cancers [Table 3-1] [Table 3-2]

[0069] In some embodiments, the ABCB1 inhibitors useful in the methods of treating cancer are ABCB1 agents for the treatment of gastric diseases. Non-limiting examples of these compounds are shown in the list below. ABCB1 agents for gastric disorders: [Table 4]

[0070] Other miscellaneous ABCB1 inhibitors useful in the methods of the present invention include, but are not limited to, cyclosporine, cimetidine, aldosterone, tacrolimus, phenobarbital, dexamethasone, carbamazepine, colchicine, loperamide, imipramine, hydrocortisone, citalopram, taurocholic acid, fexofenadine, prednisone, estrone, diazepam, digitoxin, methylprednisolone, quetiapine, olanzapine, clozapine, prednisolone, betamethasone, alitretinoin, vecuronium, stanolone, epinastine, estriol, sphingosine, cerivastatin, levothyroxine ... Tiracetam, Phenytoin, Lamotrigine, Sitagliptin, Ketazolam, Silodosin, Rivaroxaban, Dabigatran Etexilate, Fesoterodine, Indacaterol, Clobazam, Linagliptin, Mirabegron, Bosutinib, Fluticasone Furoate, Mycophenolate Mofetil, Dapagliflozin, Umeclidinium, Edoxaban, Nintedanib, Ombitasvir, Elbasvir, Grazoprevir, Odanacatib, Baricitinib, Ubidecarenone, Ertugliflozin, Stanolone Acetate, Estradiol Acetate, Estradiol Benzoate, Estradiol Cypionate, Estradiol Dienanthate (Estradioldienanthate), estradiol valerate, testosterone propionate, asunaprevir, somatostatin, avatrombopag, venlafaxine, trimipramine, tacrine, eletriptan, sumatriptan, sirolimus, paritaprevir, dasabuvir, erythromycin, gramicidin D, itraconazole, tetracycline, valinomycin, topiramate, terfenadine, amprenavir, celiprolol, talinolol, flupentixol, trifluoperazine, rhodamine 6G, simvastatin, valspodar, seriponase alfa, curcumin, ascorbic acid, chlorpromazine, phenothiazine, atorvastatin, bromperidol, morphine ne, pentazocine, propranolol, neostigmine, moxidectin, mefloquine, fluticasone, fluticasone propionate, elagolix, chloroquine, paliperidone, lusutrombopag, posaconazole, dipyridamole, quinine, indomethacin, acetaminophen, haloperidol, naloxone, mannitol, betrixaban, clomiphene, omadacycline, grapiprant, latrolectinib, revefenacin, tenofovir disoproxil, tenofovir alafenamide, tenofovir, ledipasvir, sildenafil, vardenafil, cabergoline, prucalopride, risperidone, tramadol, azithromycin, fluconazole, ranolazine, cetirizine, tegaserod and doxepin.

[0071] PIP2 or phosphatidylinositol 4,5-bisphosphate PIP2 is a phospholipid that is present at low levels in cells but is involved in important cellular processes. Some of the cellular functions of PIP2 include endocytosis, exocytosis, phagocytosis, and the regulation of cell signaling (Czech et al., 2000). As used herein, "PIP2" refers to a phospholipid that binds to ABCB5.

[0072] An aspect of the present invention is a method for enhancing ABCB5 / PIP2-dependent signaling in normal stem cells through an ABCB5-PIP2 binding activator to enhance normal ABCB5 stem cell function. Such a method includes administering to a subject in need thereof an effective amount of a composition that enhances the ABCB5-PIP2 pathway, and evaluating 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 the structure: [ka] The present invention includes phospholipids, including compounds having the formula:

[0073] In some embodiments, R1 and R2 are independent fatty acid chains. In some embodiments, R1 and R2 are 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 a total fatty acid chain of 22:0, 23:0, 24:0, 25:0 or 26:0. In some embodiments of the invention, the composition is administered to a healthy subject. The subject may be a human or a 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. In some embodiments, the composition is used to promote wound healing, tissue regeneration, angiogenesis and cell survival, to reduce aging, and to inhibit cell death in a subject. 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 pharma- ceutically acceptable carrier.

[0074] In some embodiments, the composition comprises a PIP2 agonist. [ka] Includes.

[0075] One advantage of the method of the present invention is that it allows selective targeting of tissues with stem cells expressing ABCB5. Existing treatments that target tyrosine kinase pathways may have extensive side effects, while methods that target ABCB5 / PIP2 binding (activators or inhibitors) also regulate PI3K signaling and will have limited effects on a subset of cells that also express ABCB5. Therefore, the method should provide a lower rate of side effects. Targeting ABCB5 can be used as a stand-alone therapeutic approach for generalized disease or as an adjunctive treatment to sensitize cancer cells to chemotherapeutic agents, especially in patients with currently refractory metastatic disease.

[0076] Aspects of the present invention relate to a method for inhibiting ABCB5-PIP2 binding through an inhibitory molecule contained in a composition to inhibit the function of ABCB5-dependent cancer stem cells. Such a method further comprises evaluating ABCB5-PIP2 binding after administration of the composition, which represents functional blockage of ABCB5. In some embodiments, the composition inhibits PI3K pathway and suppresses tumorigenesis, metastasis and / or resistance to drugs that regulate PI3K signaling, such as melanoma resistant to vemurafenib mediated by PI3K signaling, or cancer resistant to EGFR inhibitors mediated through upregulation of PI3K signaling enhanced by ABCB5.

[0077] 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-ABCB5 antibodies or ABCB5 binding fragments with specificity for the cyclic or linear form of the extracellular polypeptide of a protein, and enzymes. In some embodiments, the composition comprises an ABCB5 antibody or ABCB5 binding fragment with specificity for the cyclic or linear form of the extracellular polypeptide of ABCB5. In some embodiments, the composition comprises an ABCB5 antibody or ABCB5 binding fragment that modifies the conformation of the ABCB5 PIP2 binding site. In some embodiments, the ABCB5 antibody is selected from the list comprising, for example, monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, F(ab')2, Fab, Fd, Fv, or single chain Fv fragments. In some embodiments, the ABCB5 antibody is a human anti-ABCB5 antibody or ABCB5 binding fragment that binds to an extracellular loop of the three-dimensional conformation of ABCB5. In some embodiments, the human anti-ABCB5 antibody is subjected to affinity maturation to specifically recognize and bind to the extracellular loops of the non-linear form of ABCB5. The human anti-ABCB5 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 loops of the non-linear form of ABCB5.

[0078] Aspects of the present invention relate to the generation (e.g., preparation) of human anti-ABCB5 antibodies or ABCB5 binding fragments that inhibit the ABCB5-PIP2 pathway. In some embodiments, the anti-ABCB5 antibodies or ABCB5 binding fragments are subjected to affinity maturation for specifically binding to the extracellular loops of a non-linear form of the protein. The affinity maturation process can occur by: a. phage display, yeast display or ribosome display; or b. panning techniques, e.g., antibodies are generated against linear extracellular loop peptides, and then the peptide protein is displayed and processed by antigen presenting cells, and the resulting antibodies can be matured using a display approach.

[0079] In some embodiments of the invention, the compositions are administered to healthy subjects. In some embodiments, the subject may be a human or a non-human animal, including a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, cat, dog, llama, or a primate, such as a 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.

[0080] Aspects of the present invention relate to a method for inhibiting ABCB5-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 a method further comprises evaluating ABCB5-PIP2 binding after administration of the composition, which represents functional blockade of ABCB5. In some embodiments, the composition inhibits PI3K pathway and suppresses tumorigenesis, metastasis and / or resistance to drugs that modulate PI3K signaling, such as melanoma resistant to vemurafenib mediated by PI3K signaling, or cancer resistant to EGFR inhibitors mediated through upregulation of PI3K signaling enhanced by ABCB5.

[0081] In some embodiments, the composition comprises a PIP2 antagonist. In some embodiments, the composition is selected from the group comprising a small molecule, a lipid analog, an anti-ABCB5 antibody or an ABCB5 binding fragment having specificity for the cyclic or linear form of the extracellular polypeptide of a protein, and an enzyme. In some embodiments, the composition comprises an ABCB5 antibody or an ABCB5 binding fragment having specificity for the cyclic or linear form of the extracellular polypeptide of ABCB5. In some embodiments, the composition comprises an ABCB5 antibody or an ABCB5 binding fragment that modifies the conformation of the ABCB5 PIP2 binding site. In some embodiments of the present invention, the composition is administered to a healthy subject.

[0082] In some embodiments, the subject may be a human or a non-human animal, including a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, cat, dog, llama, or a primate, such as a 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.

[0083] In one aspect, the present invention is useful as a screening tool in a method for the discovery of molecular compounds that inhibit or enhance ABCB5-PIP2 binding and thus represent functional ABCB5 blockers or activators. Such compounds include lipid analogs, PIP2 or PIP2 agonists, small molecule drugs such as PSC833, and a new subset of ABCB5 inhibitory monoclonal antibodies that bind to ABCB5 and block PIP2 binding through induction of molecular conformational changes, inhibiting pAKT phosphorylation and downstream signaling / effector pathways. The method of the present invention further includes contacting ABCB5+ cells with a putative composition comprising a compound that modulates ABCB5-PIP2 binding, and determining the level of a compound that is a product of the PIP2 pathway. If the level is greater than the baseline level, the putative composition is an ABCB5-PIP2 pathway activator, and if the level of the PIP2 pathway compound is less than the baseline level, the putative composition is an ABCB5-PIP2 inhibitor. In some embodiments, the compound that is a product of the PIP2 pathway is a compound that is a member of the PIP3 or PI3K pathway. "Baseline level," as used herein, refers to the level of a compound that is a product of the PIP2 pathway in a sample that has not been exposed to a putative composition that contains a compound that modulates ABCB5-PIP2 binding.

[0084] Aspects of the present invention disclose novel phospholipid analogs of PIP2, which have been characterized by mass spectrometry. The PIP2 analogs are novel endogenous PIP2 variant compounds (formula C 33 H 65 O 19 P3, which has a fatty acid chain composition representative of PIP2 with a total fatty acid chain of 24:0, identified as [PIP2(6:0 / 18:0)-H]. This analog was specifically enriched in ABCB5 knockout cells, indicating that ABCB5 is functionally required for efficient PIP2 conversion.

[0085] PIP2 has the chemical formula: C 47 H 80 O 19 P3 and the following structure: [ka] has.

[0086] In another aspect, the present invention provides a functional analog of PIP2, 33 H 65 O 19 Structure with P3 [PIP2(6:0 / 18:0)-H] - and 24:0 total fatty acid chain. In some embodiments, the compound inhibits the ABCB5-PIP2 pathway. In some embodiments of the present invention, the compound inhibits drug resistance, cell survival, epithelial-mesenchymal transition (EMT), and metastasis, and promotes cell death.

[0087] In some aspects, the present invention provides a compound having the structure: [ka] A compound having the formula: wherein R1 and R2 are independently fatty acid chains such that the structure has a total fatty acid chain of 22:0 to 26:0, and wherein one of R1 and R2 has at least twice the length of the other of R1 and R2. In some embodiments, the structure has a total fatty acid chain of 22:0, 23:0, 24:0, 25:0 or 26:0. In some embodiments, the compound inhibits the ABCB5-PIP2 pathway. In some embodiments of the invention, the compound inhibits drug resistance, cell survival, epithelial-mesenchymal transition (EMT), and metastasis, and promotes cell death.

[0088] In some aspects, the present invention is a method for screening for ABCB5 antagonists and activators using one of the novel compositions of the present invention or PIP2 or other PIP2 analogs.The method of the present invention further comprises contacting ABCB5+ cells with a putative composition that modulates ABCB5-PIP2 binding, and determining the level of a compound that is a product of the PIP2 pathway.If the level is greater than the baseline level, the putative composition is an ABCB5-PIP2 pathway activator, and if the level of the compound that is a product of the PIP2 pathway is lower than the baseline level, the putative composition is an ABCB5-PIP2 inhibitor.In some embodiments, the PIP2 pathway compound is a compound that is a member of the PIP3 or PI3K pathway.

[0089] In some embodiments, the ABCB5+ cells comprise ABCB5 isoform 1, which has a lysine at amino acid position 970. In some embodiments, the ABCB5+ cells comprise ABCB5 isoform 2, which has a lysine at amino acid position 525. ABCB5 expressing this SNP is most frequently found in human cancers.

[0090] In another aspect, the composition is a tool for such screening assays. In another aspect, the invention is a novel composition or method for the use of PIP2 or other PIP2 analogs as therapeutic compounds to enhance ABCB5-dependent stem cell function when exogenously administered.

[0091] Effective dose In the methods described herein, an "effective amount" refers to the amount of a composition that can achieve a desired therapeutic effect, such as enhancing or suppressing the ABCB5-PIP2 pathway.

[0092] In some embodiments, the composition comprises a PIP2, a PIP2 agonist, a PIP2 antagonist, a phospholipid, or [PIP2(6:0 / 18:0)-H] -In some embodiments, the composition comprises PIP2. In some embodiments, the amount of PIP2 in the composition is 1-100%. 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 PIP2 agonist in the composition is 1-100%. 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.

[0093] In some embodiments, the composition comprises a PIP2 antagonist. In some embodiments, the amount of the PIP2 antagonist in the composition is 1-100%. In some embodiments, the amount of the PIP2 antagonist 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.

[0094] In some embodiments, the composition comprises a phospholipid. In some embodiments, the amount of phospholipid in the composition is 1-100%. 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 composition comprises [PIP2(6:0 / 18:0)-H] - In some embodiments, the [PIP2(6:0 / 18:0)-H] in the composition -In some embodiments, the amount of [PIP2(6:0 / 18:0)-H] in the composition is 1 to 100%. - 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.

[0095] Pharmaceutical Compositions The compound, antibody, and encoding nucleic acid or nucleic acid set, the vector containing it, or the host cell containing the vector as described herein can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably be able to stabilize the active ingredient) and not harmful to the subject to be treated. Pharmaceutically acceptable excipients (carriers) include buffers well known in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (2000), Lippincott Williams and Wilkins, KE Hoover.

[0096] The pharmaceutical compositions to be used in the present methods may contain pharma- ceutically 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, KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic 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 methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polysaccharides, e.g., glycerol, sorbitol ... peptides; 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 non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0097] In some examples, the pharmaceutical compositions described herein include liposomes containing the compound or antibody (or encoding nucleic acid), which can be prepared by methods known in the art, such as those 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. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be made by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to produce liposomes with the desired diameter.

[0098] The compound or antibody, or encoding nucleic acid, may also be encapsulated in microcapsules, which may be, for example, those prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are known in the art; see, for example, Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing (2000).

[0099] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release form.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing compound or antibody (the matrices are in the form of shaped articles, for example, films), or microcapsules.Examples of sustained release matrices include polyester, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres made of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0100] In other examples, the pharmaceutical compositions described herein can be formulated in a sustained release format to affect selective binding to tissues or tumors by implementing specific protease biology techniques, for example, by peptide masking of the antigen binding site of an antibody (such as Probody™ or Conditionally Active Biologics™) to allow selective protease cleavage by one or more proteases in the tumor microenvironment. Activation may be formulated to be reversible in the normal microenvironment.

[0101] The pharmaceutical composition to be used for in vivo administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane. The therapeutic compound or antibody composition is generally placed into a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0102] The pharmaceutical compositions described herein may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral or rectal administration, or by inhalation or insufflation.

[0103] To prepare solid compositions such as tablets, the main active ingredient can be mixed with a pharmaceutical carrier, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogeneous mixture of the compound of the present invention, or a non-toxic pharma-ceutically acceptable salt thereof. When these preformulation compositions are referred to as homogeneous, it is meant that the active ingredient is evenly distributed throughout the composition, so that the composition can be easily subdivided into equally effective unit dosage forms, such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the above types, containing 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form that allows for a longer duration of action. For example, a tablet or pill may comprise an inner dosage component and an outer dosage component, the latter in the form of an envelope over the former. 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 can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0104] Suitable surfactants include, in particular, non-ionic agents such as polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions having a surfactant conveniently contain 0.05 to 5% surfactant, which may be 0.1 to 2.5%. It will be appreciated that other materials may be added, if required, such as mannitol or other pharma- ceutically acceptable vehicles.

[0105] Suitable emulsions can be prepared using commercially available lipid emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be dissolved in a premixed emulsion composition or in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and the emulsion may be formed by mixing phospholipids (e.g., egg phospholipids, soybean phospholipids or soy lecithin) with water. It will be understood that other materials, such as glycerol or glucose, may be added to adjust the osmotic pressure of the emulsion. Suitable emulsions will typically contain up to 20%, for example 5-20%, of an oil. The lipid emulsion may contain lipid droplets of 0.1 to 1.0.im, especially 0.1 to 0.5.im, and may have a pH in the range of 5.5 to 8.0.

[0106] An emulsion composition may be prepared by mixing the compound or antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water).

[0107] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0108] Compositions, preferably in sterile pharma- ceutically acceptable solvents, may be nebulized by use of a gas. Nebulized solutions may be breathed directly through the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

[0109] therapeutic application The compounds or antibodies described herein, as well as any of the encoding nucleic acids or sets of nucleic acids, vectors containing same, or host cells containing said vectors, are useful for treating cancer, inflammation, infectious diseases, or other malignancies requiring stimulation of an immune response.

[0110] To carry out the methods disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration (e.g., as a bolus or by continuous infusion over a period of time), intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation or topical. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, compounds or antibodies as described herein can be aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized products and fine powders.

[0111] The subject to be treated by the method described herein may be a human patient who has, is at risk of, or is suspected of having cancer or other malignant lesions that require the stimulation of immune response.Subjects with target disease or disorder can be identified by routine medical tests, such as clinical examinations, organ function tests, CT scans, or ultrasound.Subjects suspected of having any of these target diseases / disorders may show one or more symptoms of the disease / disorder.Subjects at risk of disease / disorder may be subjects who have one or more of the risk factors for the disease / disorder.

[0112] The methods and compositions described herein can be used to treat cancer.The examples of cancer that can be treated by 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, blood cancer, lymphoma, leukemia, skin cancer, ovarian cancer, bladder cancer, urothelial cancer, head and neck cancer, metastatic lesions of cancer, and all types of cancer that are diagnosed with high gene mutation number.In certain embodiments, cancer has high gene mutation number.Subjects with or at risk of various cancers can be identified by conventional medical methods.

[0113] In some instances, the human patient has frequent microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) found in soft tissue cancers, glioblastoma, esophageal and EGJ cancer, breast cancer, non-small cell lung cancer, ovarian surface epithelial carcinoma, cancer of unknown primary site, small cell lung cancer, non-epithelial ovarian cancer, pancreatic adenocarcinoma, other female genital tract malignancies, uveal melanoma, retroperitoneal or peritoneal sarcoma, thyroid cancer, uterine sarcoma, cholangiocarcinoma, prostate adenocarcinoma, hepatocellular carcinoma, neuroendocrine tumors, cervical cancer, colorectal adenocarcinoma, small intestinal malignancies, gastric adenocarcinoma, and endometrial cancer.

[0114] Effective amounts, as understood by those skilled in the art, vary depending on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex and weight, duration of treatment, nature of concomitant treatment (if any), specific route of administration, and similar factors within the knowledge and expertise of the health care professional. Empirical considerations such as half-life will generally contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized or fully humanized antibodies, can be used to extend the half-life of the antibody and to prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of treatment, generally, but not necessarily, based on the treatment and / or suppression and / or remission and / or delay of the target disease / disorder. Alternatively, sustained continuous release formulations of antibodies may be appropriate. A variety of formulations and devices for achieving sustained release are known in the art.

[0115] In one example, the dosage for a compound or antibody as described herein can be empirically determined in an individual given one or more doses of the compound or antibody. The individual is given incremental doses of the compound. Indicators of disease / disorder can be tracked to assess the efficacy of the compound.

[0116] In general, for administration of any of the compounds or antibodies described herein, the initial candidate dosage may be about 2 mg / kg. For purposes of this disclosure, a typical daily, weekly, biweekly, or triweekly dosage may range anywhere from about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 100 μg / kg to 300 μg / kg to 0.6 mg / kg, 1 mg / kg, 3 mg / kg, up to 10 mg / kg, 30 mg / kg to 100 mg / kg, or more, depending on the factors mentioned above. For repeated administration over several days, weeks, months, or longer, depending on the condition, treatment is sustained until a desired suppression of symptoms occurs or sufficient therapeutic levels are achieved to alleviate the target disease or disorder or its symptoms. An exemplary dosing regimen includes administering an initial dose of about 3 mg / kg every 3 weeks, followed by a maintenance dose of about 1 mg / kg of compound or antibody once every 6 weeks, or a maintenance dose of about 1 mg / kg every 3 weeks. However, other dosing regimens may be useful, depending on the pattern of pharmacokinetic decay the physician wishes to achieve. For example, dosing of 1 mg / kg once every 3 weeks in combination with treatment with at least one additional immunotherapeutic agent is contemplated. In some embodiments, dosing in the range of about 3 μg / mg to about 3 mg / kg may be used, such as about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 3 mg / kg. In some embodiments, the frequency of administration is once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks; or once a month, once every 2 months, once every 3 months, or once longer.The progress of this treatment is easily monitored by conventional techniques and assays.The administration regimen (including the compound or antibody used) can be changed over time.

[0117] In some embodiments, for an adult patient of normal body weight, a dose ranging from about 0.1 to 5.0 mg / kg may be administered. In some examples, the dosage described herein may be 10 mg / kg. The particular dosing regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as the characteristics of the individual agent (such as the half-life of the agent and other considerations well known in the art).

[0118] For purposes of this disclosure, the appropriate dosage of a compound or antibody as 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 treatments, the patient's clinical history and response to the antagonist, and the discretion of the attending physician. Typically, a physician 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 increase in progression-free survival, and / or overall survival. Methods for determining whether a dosage has produced the desired result will be apparent to one of skill in the art. Administration of one or more compounds or antibodies may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to a skilled physician. Administration of the compound or antibody may be essentially continuous or in a series of spaced doses over a preselected period of time, e.g., before, during, or after the onset of the target disease or disorder.

[0119] As used herein, the term "treating" refers to the application or administration to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition toward a disease / disorder, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disorder, the symptom of the disease, or the predisposition toward a disease or disorder. Alleviating a target disease / disorder includes delaying the onset or progression of the disease or reducing the severity of the disease. Treatment includes not only treatment to reduce the likelihood that a subject will develop a disease, but also treatment after a subject has developed a disease to combat the disease, prevent the disease from worsening, or delay the progression of the disease compared to that in the absence of treatment.

[0120] Alleviating a disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a target disease or disorder means to postpone, prevent, slow, retard, stabilize, and / or postpone the progression of the disease. This delay may be of various lengths of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating a disease or delaying the onset of a disease is a method that reduces the likelihood of developing one or more symptoms of the disease within a given time frame, or reduces the severity of symptoms within a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to obtain statistically significant results.

[0121] "Onset" or "progression" of a disease refers to the first signs and / or subsequent progression of the disease. Onset of a disease can be detected and evaluated using standard clinical techniques, as is well known in the art. However, onset also refers to progression, which may be undetectable. For the purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes occurrence, recurrence, and onset. As used herein, "onset" or "onset" of a target disease or disorder includes initial onset and / or recurrence.

[0122] In some embodiments, the compounds or antibodies described herein are 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 compounds or antibodies are administered in an amount effective in reducing the activity level 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).

[0123] Depending on the type of disease or site of disease to be treated, the pharmaceutical composition can be administered to the subject using conventional methods known to those skilled in the art of medicine.The composition can also be administered via other conventional routes, for example, parenterally, topically, orally, by inhalation spray, rectally, nasally, bucally, vaginally, or via implanted reservoir.The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intraperitoneal, intratumoral, intramuscular, intraarticular, intraarterial, intrasynovial, intrathecal, intralesional, and intracranial injection or infusion techniques.In addition, it can be administered to the subject via administration of injectable depot route, for example, using 1-month, 3-month, or 6-month depot injectable or biodegradable materials and methods.In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

[0124] Injectable compositions may contain a variety of carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol). For intravenous injection, water-soluble compounds or antibodies can be administered by drip infusion, in which a pharmaceutical formulation containing the compound or antibody and a physiologically acceptable excipient is injected. Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the compound or antibody can be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0125] In one embodiment, compound or antibody is administered via site-specific or targeted local delivery technology.Examples of site-specific or targeted local delivery technology include various implantable compound or antibody depot sources or local delivery catheters, such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application.See, for example, PCT Publication No. WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0126] Targeted delivery of therapeutic compositions containing antisense polynucleotides, expression vectors, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described, for example, in:Findeis et al., Trends Biotechnol. (1993) 11:202;Chiou et al., Gene Therapeutics: Methods and Applications of Direct Gene Transfer (JA Wolff, ed.) (1994);Wu et al., J. Biol. Chem. (1988) 263:621;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.

[0127] Therapeutic compositions containing polynucleotides (e.g., those encoding antibodies or other proteins described herein) are administered in the range of about 100 ng to about 200 mg of DNA for local administration in gene therapy protocols. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA or more can be used during gene therapy protocols.

[0128] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. 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). The expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. The expression of coding sequences can be constitutive or regulated.

[0129] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90 / 07936; WO94 / 03622; WO93 / 25698; WO93 / 25234; WO93 / 11230; WO93 / 10218; WO91 / 02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1247). ATCC VR-1250; ATCC VR 1249; ATCC VR-532) and adeno-associated virus (AAV) vectors (see, for example, PCT Publication Nos. WO 94 / 12649, WO 93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984 and WO 95 / 00655). Administration of DNA linked to inactivated adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be used.

[0130] Non-viral delivery vehicles and methods can also be used, including polycationic condensed DNA, either linked or not linked to inactivated 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 vehicles (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95 / 07994; WO96 / 17072; WO95 / 30763; and WO 97 / 42338) and neutralization of nuclear charge or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA transfer methods are described in PCT Publication No. WO 90 / 11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95 / 13796; WO94 / 23697; WO91 / 14445; and EP Patent No. 0524968, and further approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0131] The particular dosing regimen employed in the methods described herein, i.e., dosage, timing and repetition, will depend on 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 that act to enhance and / or complement the effectiveness of the agent. The efficacy of a treatment for a target disease / disorder can be assessed by methods well known in the art.

[0132] Treatment methods, including those described in this disclosure, may be used in combination with other types of treatment for the target disease or disorder disclosed herein. Examples include chemotherapy, immunotherapy (e.g., treatments that include therapeutic antibodies, antibodies, CAR T cells, or cancer vaccines), surgery, radiation, gene therapy, etc., or anti-infection treatments. Such treatments can be administered simultaneously with or sequentially (in any order) with the treatment according to the present disclosure. In some examples, the target disease is cancer (e.g., those disclosed herein), and the combination treatment includes an immune checkpoint (e.g., an inhibitory checkpoint) antagonist. Examples include PD-1 / PD-L1 antagonists (e.g., nivolumab, pembrolizumab, avelumab, durvalumab 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. Further examples include activators that enhance the activity of stimulatory checkpoints, such as CD122 (IL2) antagonists, 4-1BB, ICOS ligand, GITR and OX40.

[0133] For additional useful agents, see also: Physician's Desk Reference, 59.sup.th edition, (2005), Thomson PDR, Montvale NJ; 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 NJ. When co-administered with additional therapeutic agents, the preferred therapeutically effective dosage for each agent may be lower due to additive or synergistic effects.

[0134] The efficacy of the methods described herein can be evaluated by any method known in the art, and will be clear to skilled medical professionals.For example, the efficacy of antibody-based immunotherapy can be evaluated by subject survival, or the cancer burden in subject or tissue or its sample.In some embodiments, the method is evaluated based on the safety or toxicity in subject, for example, by subject's overall health and / or the presence of adverse events or serious adverse events.

[0135] The invention is not limited in its application to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and can be practiced or carried out in various ways. Also, the phraseology and terminology used herein are 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 are intended to encompass the items listed thereafter and equivalents thereof, as well as additional items.

[0136] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. The methods and techniques of this disclosure are generally carried out according to conventional methods well known in the art. In general, the nomenclature used in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are well known and commonly used in the art. The methods and techniques of this disclosure are generally carried out according to conventional methods as known in the art and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated.

[0137] The present invention is further illustrated by the following examples, which should not be construed as further limiting in any way. References cited throughout this application, including academic literature, published patents, published patent applications, and co-pending patent applications, are hereby expressly incorporated by reference.

[0138] example Example 1: ABCB5 promotes tumor invasion through regulation of AXL. Recent studies have shown that the receptor tyrosine kinase AXL, which correlates with adverse colorectal cancer (CRC) prognosis, is responsible for EMT induction in other malignancies. Herein, we show that AXL mRNA expression was reduced by more than 90% in COLO741 ABCB5 KD CRC cell cultures, and AXL protein expression was reduced by more than 50% in these cells compared to control-transfected cells (Figure 8). Furthermore, as determined by Western blot analysis, 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 ranging from 9% to 27%) (Figure 8), and also inhibited its downstream target phospho-AKT to a lesser extent, indicating a critical signaling pathway used by ABCB5+ cells. The functional relationship between ABCB5 and AXL was supported by the significantly upregulated AXL expression at both the mRNA and protein levels in untreated ABCB5(+) cells sorted by flow cytometry from all four cell lines (Fig. 8 ).

[0139] Furthermore, AXL expression (determined at both the mRNA and protein levels) as well as downstream signaling (p-AKT / AKT ratio) were enhanced in metastasis-induced COLO741MET compared to parental COLO741 cells (Figure 8).

[0140] Example 2: PIP2 and PIP3 were determined to be the natural in vivo binding ligands of ABCB5 by immunoprecipitation from human tissues. Immunoprecipitation of PIP2 from human ABCB5-expressing melanoma cells using anti-PIP2 antibody pull-down revealed co-precipitation of ABCB5 protein, as shown by immunoblot with ABCB5 monoclonal antibody in FIG. 1A (upper panel). PIP2 pull-down was confirmed by immunoblot with PIP2 antibody in FIG. 1A (lower panel). Similarly, immunoprecipitation of PIP3 from human ABCB5-expressing melanoma cells using anti-PIP3 antibody pull-down revealed co-precipitation of ABCB5 protein, as shown by immunoblot with ABCB5 monoclonal antibody in FIG. 1B. These data indicate that PIP2 and PIP3 are biological ligands for ABCB5, and ABCB5 serves as a receptor for PIP2 and PIP3.

[0141] Example 3: PIP1, PIP2 and PIP3 were determined to bind to ABCB5 by ELISA. ELISA plates coated with PIP1, PIP2 or PIP3 were incubated with either purified recombinant human ABCB5 isoform 2 (812aa, NCBI reference sequence: NP_848654.3), wild-type mouse Abcb5-expressing skin tissue, or skin tissue from ABCB5 knockout mice that does not express Abcb5 (as a specificity control), and then bound ABCB5 was detected using a mouse ABCB5-specific monoclonal antibody (Ksander et al Nature 2014). As shown in Figure 1C, PIP1, PIP2 and PIP3 all bound to recombinant human ABCB5 as well as mouse Abcb5, and no detection of binding was observed in Abcb5 knockout mouse tissue. The binding of PIP2 and PIP3 to ABCB5 was more efficient than that of PIP1 to ABCB5, as described herein. These data confirmed the binding of ABCB5 to PIP2 and PIP3, and also revealed its ability to bind PIP1, albeit with an apparently lower affinity. Similar results were obtained using the mouse ABCB5 mAb clone 3C2-1D12 (Frank NY et al. J Biol Chem. 2003) as the detection antibody.

[0142] 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) (CAS Registry No. 899827-36-2), a synthetic analogue of natural phosphatidylinositol (PtdIns) containing C8:0 fatty acids at the sn-1 and sn-2 positions. The data shown in Figure 2 indicate that this molecule can competitively inhibit the binding of PIP1, PIP2 or PIP3 to ABCB5, with an apparent saturation of effect at a concentration of only 0.1 mM.

[0143] These results provide proof of principle that analogs of PIP (including PIP2 analogs and chemical PIP2 variants that cannot be phosphorylated to biologically active PIP3), or additional synthetic chemical and biological agents, that compete with ABCB5 binding of PIP1, PIP2 or PIP3 but are not functional in signaling of various receptor tyrosine kinases (listed in the table below) or various G protein-coupled receptors, unlike PIP1, PIP2 or PIP3, can be used as therapeutic agents to modulate ABCB5 / PIP1, ABCB5 / PIP2 or ABCB5 / PIP3 receptor / ligand interactions associated with the PIP-dependent signaling mechanisms of such receptors in various disease states in which ABCB5 is functional, particularly, but not limited to, in the initiation and progression of human cancers.

[0144] [Table 5-1] [Table 5-2]

[0145] Example 5: Binding of PIP1, PIP2 and PIP3 to ABCB5 can be inhibited by ABCB5 monoclonal antibody. Surface plasmon resonance (SPR) analysis also showed the binding of PIP1, PIP2 and PIP3 to ABCB5. Competition with anti-ABCB5 monoclonal antibody resulted in a signal reduction of up to 50% on the PIP3 surface in a concentration-dependent manner (PIP3>PIP2>PIP1) on all three surfaces (see FIG. 3). An isotype control monoclonal antibody did not show any significant effect (not shown). These results indicate that ABCB5 monoclonal antibody can block the binding of PIP1, PIP2 or PIP3 to ABCB5.

[0146] Example 6: ABCB5 is required for more efficient conversion of PIP2 to PIP3. Functional experiments involving ABCB5 blockade with ABCB5 monoclonal antibody in human melanoma cells or loss of ABCB5 function in tissues from ABCB5 knockout mice revealed that ABCB5 is functionally required for more efficient PIP2 to PIP3 conversion, likely through its function as a PIP2 docking receptor. As illustrated in Figure 4, ABCB5 monoclonal antibody significantly reduced the PIP3 / PIP2 ratio in human melanoma cells, whereas an isotype control antibody did not (left). Furthermore, examination of mouse ABCB5 knockout skin tissues also revealed the presence of a significantly reduced PIP3 / PIP2 ratio compared to ABCB5 wild-type skin (right).

[0147] Together with findings from SPR analysis showing that ABCB5 monoclonal antibody can block ABCB5 / PIP2 receptor / ligand interaction, these results indicate that ABCB5 / PIP2 binding interaction is functionally required for more efficient phosphorylation of PIP2 to PIP3, implicating a critical role for ABCB5 in PIP2-dependent signaling for receptor tyrosine kinase and G protein-coupled receptor signaling in ABCB5-expressing cells, including cancer stem cells involved in tumorigenesis, cancer progression, and therapeutic resistance in diverse malignancies including melanoma, colorectal cancer, glioblastoma multiforme, Merkel cell carcinoma, SCC, and hepatocellular carcinoma, among others. Furthermore, physiological tissue-specific stem cells, including skin, eye, and intestinal stem cells, express high levels of ABCB5 and depend on receptor tyrosine kinase and G protein-coupled receptor signaling to execute their tissue regenerative functions. Thus, the current findings provide a means to block ABCB5-dependent PIP2 binding and phosphorylation of PIP3 in receptor tyrosine kinase signaling, or processing to IP3 and DAG in G protein-coupled receptor signaling, either with ABCB5 monoclonal antibodies or small molecule / chemical inhibitors of ABCB5 / PIP2 binding, leading to inhibition of receptor tyrosine kinase signaling (e.g., AXL (see Guo et al. J Biol Chem. 2018), EGFR) associated with cancer initiation / progression / therapy resistance, a G protein-coupled receptor signaling-dependent mechanism of cancer initiation / progression / therapy resistance. Furthermore, enhancing ABCB5 / PIP2 binding interaction, for example through enhanced ABCB5 expression / binding or by exogenous PIP2 addition, would enhance biological stem cell function to treat stem cell deficiency-related disorders.

[0148] Example 7: ABCB5 is required to maintain the PIP2 / PIP3-dependent PI3K / AKT signaling axis in malignant tumors, and inhibition of ABCB5 leads to inhibition of the PI3K / AKT signaling axis and dependent tumor growth and therapeutic resistance. Analysis of a 4-HT treatment-inducible Tyr::CreER;BrafCA;Ptenlox / lox genetic mouse melanoma model in an ABCB5 WT or ABCB5 KO background revealed a significant inhibition of the PI3K / AKT signaling axis in ABCB5 KO versus ABCB5 WT tumors, with attenuated expression of p-AKT, p-mTOR and p-S6, among other dysregulated molecules (see Figure 5).

[0149] Furthermore, in this model, the ABCB5 KO state, relative to the ABCB5 WT state, resulted in reduced tumor cell proliferation, as determined by determining the percentage of tumor cells staining positive for the proliferation marker Ki-67 (see FIG. 6): In addition, in this model, the ABCB5 KO state, relative to the ABCB5 WT state, not only led to downregulation of pro-angiogenic molecules (see Figure 7, left panel), but also resulted in a reduced density of CD31-positive microvessels (see Figure 7, right panel).

[0150] Certain ABCB5 monoclonal antibodies also disrupted the PIP2 / PIP3-dependent PI3K / pAKT signaling axis in colorectal cancer (see Guo et al. J Biol. Chem 2018), showing that the pAKT / AKT ratio was inhibited as a result of treatment, and similar results were obtained when investigating the effect of ABCB5 monoclonal antibodies on human melanoma cells, where the pAKT / AKT ratio was significantly inhibited as a result of treatment with these ABCB5 antibodies, which also inhibit the binding of PIP2 / PIP3 to ABCB5 and / or the conversion of PIP2 to PIP3. These data indicate that functional blockade of ABCB5 disrupts the PIP2 / PIP3-dependent PI3K / pAKT signaling axis, which is important for tumor growth and tumor angiogenesis, providing clear evidence for the anticancer therapeutic utility of functional blockade of ABCB5 / PIP2 / PIP3 receptor / ligand interaction.

[0151] Furthermore, in a 4-HT treatment-inducible Tyr::CreER;BrafCA;Ptenlox / lox genetic mouse melanoma model in an ABCB5 WT or ABCB5 KO background, the ABCB5 KO state, versus the ABCB5 WT state, resulted in complete sensitization to the effects of the BRAF inhibitor vemurafenib, the resistance to which is driven in part by a functional PI3K / pAKT signaling axis. No tumor formation was observed upon gene induction in vemurafenib-treated ABCB5 KO mice, which was the opposite to ABCB5 WT mice, which showed 100% formation of vemurafenib-resistant tumors (see Figure 9, left panel), and survival was significantly extended in ABCB5 KO mice versus ABCB5 WT mice (see Figure 9, right panel). These results reveal a critical role for ABCB5 in tumor vemurafenib resistance through its function in maintaining an intact PIP2 / PIP3-dependent PI3K / pAKT signaling axis required for vemurafenib resistance. These results also indicate that inhibition of ABCB5 function can be used therapeutically to reverse BRAF inhibitor resistance in melanoma.

[0152] Example 8: Identification of novel PIP2 structures accumulated in ABCB5 knockout tissues identifies preferred physiological substrates for ABCB5-dependent phosphorylation to its PIP3 form. Quantitative lipid mass analysis of skin tissues from ABCB5 wild-type (WT) or ABCB5 knockout (KO) mice detected 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, 0 unsaturation, formula C33H65O19P3), specifically present in ABCB5 knockout tissues but not detectable at the detection threshold in ABCB5 wild-type skin (ABCB5 KO mean: 7.79E+04+ / -1.67E+04; WT mean: not detected), indicating that this newly discovered PIP2 molecular variant, previously unknown in compound databases, represents a preferred physiological substrate for ABCB5-dependent phosphorylation to its PIP3 form, i.e., the biologically active phosphorylated PIP3 form involved in ABCB5-dependent receptor tyrosine kinase or G protein-coupled receptor signaling. A bioinformatically generated structural model of this novel PIP2(6:0 / 18:0)-H molecule (total fatty acid chains of 24:0, degree of unsaturation 0, formula C33H65O19P3) is described.

[0153] This PIP2(6:0 / 18:0)-H molecule, or its phosphorylated form PIP3(6:0 / 18:0)-H (formula C33H65O19P4), represents a composition that can be used as a therapeutic to enhance disease states in which signaling by the various receptor tyrosine kinases or G protein-coupled receptors listed above is impaired or the level of ABCB5 function or ABCB5 expression is reduced, particularly in diseases associated with ABCB5+ stem cell deficiency, such as impaired wound healing in the skin, limbal stem cell deficiency, impaired tissue regeneration in aging, and further ABCB5 deficiency disorders such as psoriasis, which has been found to be exacerbated in a mouse model of imiquimod-induced psoriasis in ABCB5 knockouts compared to ABCB5 wild-type mice (see FIG. 10).

[0154] Example 9: Functional dissection of single nucleotide polymorphisms in ABCB5 reveals molecular ABCB5 ligand / substrate binding sites linked to downstream molecular effector functions. The structure of ABCB5 isoform 1 (1257 aa, NCBI Reference Sequence: NP_001157413.1) consists of two transmembrane domains (TMDs) with six TM helices each, i.e., it contains a total of 12 transmembrane helices (TM1-12). ABCB5 isoform 2 (812 aa, NCBI Reference Sequence: NP_848654.3) consists of one TMD with six transmembrane helices (TM1-6). TM1-6 of ABCB5 isoform 2 correspond to TM7-12 of ABCB5 isoform 1. TM12 of ABCB5 isoform 1 corresponds to TM6 of ABCB5 isoform 2. A nonsynonymous single nucleotide polymorphism (SNP) in the coding region of ABCB5 (rs6461515), providing AA970E>K in TM12 of ABCB5 isoform 1 and corresponding to AA525E>K in TM6 of ABCB5 isoform 2, was herein found to be critically required for ABCB5 function. The annotated reference E SNP (glutamic acid / E / GAA) is hereby conserved across diverse species, including mice (mus musculus). However, the reference E SNP (glutamic acid / E / GAA) actually represents a minor codon in humans (homo sapiens). Population diversity data indicate that the allele encoding E525 (glutamic acid, G genotype, codon GAA) was found at the highest frequency in African populations, with G / G homozygosity being rare, which was the opposite to the allele encoding K525 (lysine, A genotype, codon AAA) (data not shown).

[0155] Analysis showed that human cancers most frequently express the K SNP (lysine, codon AAA). Importantly, in wild-type K525 / K525 human melanoma cells, which exclusively express ABCB5 isoform 2 at baseline, a molecular switch from K (lysine, codon AAA) to E (glutamic acid, codon GAA) experimentally induced through Crispr / Cas9-mediated gene editing in one allele resulted in clonal heterozygous melanoma cell variants with impaired ABCB5 signaling function and consequently significantly inhibited ABCB5-driven tumor growth (P<0.05) (see FIG. 11).

[0156] These results implicate amino acid residue 525 in TM6 of ABCB5 isoform 2 as a key molecular switch in the quality of physiological ligand / substrate binding of ABCB5, especially to PIP2 and its phosphorylated product PIP3 (which is known to transmit extracellular RTK-mediated signals to activate downstream PI3K / pAKT signaling pathways essential for tumor formation and progression), with K525 (rs6461515) representing the more functional variant. Similarly, residue 970 in TM12 of ABCB5 isoform 1 is also implicated in physiological ligand / substrate binding of ABCB5. The two variant peptide sequences of ABCB5 isoform 2 are listed below along with their coding RNA sequences (variant residues highlighted in red), with the molecule containing K525 being the more functional variant in terms of PIP substrate / ligand binding and downstream signaling function, compared to the molecule containing reference E525 (SNP rs6461515).

[0157] ABCB5 isoform 2-E525 protein sequence (SEQ ID NO:1):

number

number

[0158] RNA sequence encoding ABCB5 isoform 2-E525 (SEQ ID NO:2):

number

number

number

[0159] ABCB5 isoform 2-K525 protein sequence (SEQ ID NO:3):

number

number

[0160] RNA sequence encoding ABCB5 isoform 2-K525 (SEQ ID NO:4):

number

number

number

[0161] Further residues involved in substrate binding of ABCB5 based on consideration of bioinformatics analysis are N702 and H706 in TM7 of ABCB5 isoform 1 (corresponding to N257 and H261 in TM1 of ABCB5 isoform 2) and 857A>T (rs80123476) in TM10 of ABCB5 isoform 1 (corresponding to 412A>T (rs80123476) in TM4 of ABCB5 isoform 2).

[0162] In addition, results to date have established that a subset (but not all) of ABCB5-specific monoclonal antibodies (antibodies that bind to the three-dimensional (i.e., cyclic) form of the extracellular loops) can inhibit PIP1, PIP2 or PIP3 binding to ABCB5, and thus inhibit ABCB5-mediated PIP-dependent signaling and pAKT phosphorylation. Thus, the ABCB5 monoclonal antibodies shown herein to inhibit ABCB5-dependent PIP1, PIP2 or PIP3 binding and PIP-dependent signaling and pAKT phosphorylation constitute novel compositions that are uniquely useful for therapeutically inhibiting the growth and progression of ABCB5-driven human cancers, for example, through functional ABCB5 blockade and the resulting inhibition of ABCB5-dependent receptor tyrosine kinase and G protein-coupled receptor signaling.

[0163] Example 10: Molecular docking modeling. Using bioinformatics approaches and available data on the ABCB1 protein, which is homologous to ABCB5, 3D structural models were generated for the polypeptide sequences of both ABCB5 isoform2-K525 and ABCB5 isoform2-E525 listed above. In addition, 3D structures were generated for the following ABCB5 ligands or competitive inhibitors using the PyMOL software program package to facilitate molecular docking / binding modeling.

[0164] (a) PIP2(6:0 / 18:0)-H, total fatty acid chain of 24:0, degree of unsaturation 0, formula C33H65O19P3: This is the first new PIP2 variant identified by mass spectrometry to accumulate in the skin of ABCB5 KO mice, and as described above, this indicates that this novel molecule may be a physiological substrate for ABCB5-dependent phosphorylation to PIP3. This molecule was not previously known in compound databases (modeled structure shown above).

[0165] (b) PI(4,5)P2, diC8, formula C25H49O19P3: This PIP2 variant was shown by SPR to bind to ABCB5. It was obtained from Echelon Biosciences. Further information about this molecule and its structure can be found in CAS Registry Number (204858-53-7).

[0166] (c) Phosphatidylinositol C-8:PtdIns-(1,2-dioctanoyl) is a synthetic analog of natural phosphatidylinositol (PtdIns) containing C8:0 fatty acids at the sn-1 and sn-2 positions. As described above, this molecule has been shown to be capable of competitively inhibiting the binding of PIP2 to ABCB5. Further information about this molecule is as follows: CAS Registry Number 899827-36-2.

[0167] The results revealed binding of all the tested molecules, i.e., the natural ligands PIP2(6:0 / 18:0)-H, PI(4,5)P2, diC8, and the competitive inhibitor phosphatidylinositol C-8, to the structure of either ABCB5 isoform2-K525 or ABCB5 isoform2-E525, immediately adjacent to the determined AA525 substrate binding site and TM6 of the ABCB5 molecule. In addition, modeling results revealed high affinity binding of PIP2 for ABCB5 isoform2-K525 as opposed to ABCB5 isoform2-E525. These data further support experimental evidence for a critical role of a nonsynonymous single nucleotide polymorphism (SNP) in the coding region of ABCB5 (rs6461515) determining AA525E for the K residue in TM6 of ABCB5 isoform 2, which is shown herein to be important for ABCB5 function, with ABCB5 isoform 2-K525 being a more functional ABCB5 variant with enhanced PIP2 / PIP3 binding capacity and therefore improved signaling ability.

[0168] All references cited in this specification are fully incorporated by reference. It should be understood that having some aspects of at least one embodiment of the present invention thus described, various changes, modifications and improvements will be easily thought up by those skilled in the art. Such changes, modifications and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the present invention. Accordingly, the above description and drawings are merely examples.

Claims

1. PIP2 or the following structure: 【Chemistry 1】 where R1 and R2 are independent fatty acid chains; and wherein R1 and R2 are at least twice as long as the other of R1 and R2; A composition for enhancing the ABCB5-PIP2 pathway and thereby enhancing the function of ABCB5-positive cells in diseases related to defective skin wound healing, limbal stem cell deficiency, defective tissue regeneration in aging, and psoriasis, comprising a synthetic phospholipid comprising a compound having the formula: The composition, wherein the PIP2 or synthetic phospholipid has a total fatty acid chain length of 22:0 to 26:0, and optionally the phospholipid has a total fatty acid chain length of 24:

0.

2. The phospholipid has the formula: ３３ H ６５ O １９ P ３ The composition of claim 1 having the formula:

3. The phospholipid is [PIP2(6:0 / 18:0)-H] － and a pharma- ceutically acceptable carrier.

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