ABCB5 ligands and substrates

Abstract

This invention relates to methods and compositions for regulating the activity of ABCB5+ stem cells. The invention also relates to assays and reagents for manipulating and characterizing compounds that regulate ABCB5+ cell signal transduction.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 662,670, filed April 25, 2018, pursuant to 35 USC § 119(e), the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to methods and compositions for modulating stem cell activity to treat diseases, as well as related assays and reagents. The invention also relates to methods and compositions for wound healing and tissue engineering, involving ABCB5-positive cells. Background Technology

[0004] Tumorigenesis and progression are associated with the cumulative alteration of oncogenes, tumor suppressor genes, and repair / stability genes at the DNA level. At the cellular level, human cancers are widely recognized as being composed of phenotypic populations of cells with varying self-renewal and tumor-spreading capabilities. This observation has led to the development of cancer stem cell (CSC) models of tumor initiation and growth, which have been extensively validated in a variety of malignancies, including melanoma and colorectal cancer. CSCs have been shown to contribute to the inability of current therapies to consistently eradicate malignancies through multiple molecular mechanisms, including epithelial-mesenchymal transition (EMT), which is associated with the ability of human cancers to invade the vasculature and spread to new anatomical sites, leading to tumor progression and treatment resistance.

[0005] ABCB5 is a multidrug resistance (MDR) mediator expressed in a variety of human malignancies, with particular overexpression in a previously identified treatment-resistant CD133(+) tumor subset representing cancer cells (CSCs). ABCB5 confers resistance to chemotherapeutic agents such as 5-fluorouracil (5-FU).

[0006] ABCB5+ stem cells also exist in normal tissues and play a role in tissue regeneration and aging. Regenerative medicine involves using exogenous materials, such as scaffolds, to repair, regenerate, maintain, and replace tissues and organs. Scaffolds can be seeded with cells, such as primary cells or stem cells, as well as various factors that promote tissue growth. However, many challenges remain in designing suitable materials for regenerative medicine and tissue engineering. Invention Overview

[0008] In some aspects, the present invention relates to methods and compositions for regulating the activity of ABCB5+ stem cells. The invention also relates to assays and reagents for manipulating and characterizing compounds that regulate ABCB5+ cell signal transduction.

[0009] Some aspects of the present invention relate to a method for enhancing the function of ABCB5-positive cells, comprising administering to a subject in need an effective amount of a composition that enhances the ABCB5-PIP2 pathway.

[0010] In some embodiments, the invention further includes evaluating ABCB5-PIP2 binding after application of the composition.

[0011] In some embodiments, the composition is PIP2 or a PIP2 agonist.

[0012] In some implementations, the object is a human or non-human animal, including goats, sheep, bison, camels, cattle, pigs, rabbits, buffalo, horses, rats, mice, cats, dogs, llama, or primates such as monkeys.

[0013] In some embodiments, the composition comprises phospholipids.

[0014] In some embodiments, the composition comprises [PIP2(6:0 / 18:0)-H] - And a pharmaceutical carrier.

[0015] In some embodiments, the composition comprises a phospholipid, the phospholipid comprising a compound having the structure described herein. In some embodiments, the structure comprises R1 and R2 groups. In some embodiments, R1 and R2 are independent fatty acid chains. In some embodiments, the structure comprises R1 and R2, the length of which is at least twice the length of the other of R1 and R2. In some embodiments, the structure has a total fatty acid chain ratio of 22:0 to 26:0. In some embodiments, the structure has a total fatty acid chain ratio of 24:0.

[0016] 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 via oral, intravenous, subcutaneous, topical, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular, or intrathecal routes.

[0017] Some aspects of the present invention are methods for inhibiting the function of ABCB5-positive cancer cells, comprising administering to a subject in need an effective amount of a composition that inhibits the ABCB5-PIP2 pathway, and further comprising evaluating ABCB5-PIP2 binding after administration of the composition.

[0018] Other aspects of the present invention relate to a method for inhibiting the function of ABCB5-positive cancer cells, comprising administering to a subject in need an effective amount of a composition that inhibits ABCB5-PIP2 binding, wherein the composition is selected from the group comprising: small molecules, lipid analogs, anti-ABCB5 antibodies or fragments having specificity for cyclic or linear forms of extracellular polypeptides of the protein, enzymes, and anti-ABCB5 antibodies or fragments thereof that alter the conformation of the ABCB5 PIP2 binding site.

[0019] In some embodiments, anti-ABCB5 antibodies or fragments thereof that modify the conformation of the ABCB5 PIP2 binding site inhibit PIP3 production. In some embodiments, anti-ABCB5 antibodies or fragments thereof that modify the conformation of the ABCB5 PIP2 binding site inhibit the PI3K pathway.

[0020] In some embodiments, the ABCB5-PIP2 binding is evaluated after the composition is applied.

[0021] In some embodiments, the composition is a PIP2 antagonist. In some embodiments, the composition is selected from the group consisting of: small molecules, lipid analogs, anti-ABCB5 antibodies or fragments specific to the cyclic or linear form of the extracellular polypeptide of this protein, and enzymes. In some embodiments, the composition is a small molecule. In some embodiments, the composition is an anti-ABCB5 antibody or fragment specific to the cyclic or linear form of the extracellular polypeptide of this 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.

[0022] In some implementations, the object is a human or a non-human animal, including goats, sheep, bison, camels, cattle, pigs, rabbits, buffalo, horses, rats, mice, cats, dogs, llamas, or primates such as monkeys.

[0023] In some embodiments, the composition is administered via oral, intravenous, subcutaneous, topical, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular, or intrathecal routes.

[0024] Some aspects of this invention relate to methods for identifying enhancers or inhibitors of the ABCB5-PIP2 pathway, comprising: In some embodiments, the invention includes contacting ABCB5+ cells with a hypothetical composition that regulates ABCB5-PIP2 binding; determining a level of a PIP2 pathway product compound and comparing said level to a baseline level of the PIP2 pathway product compound. In some embodiments, if said level is greater than the baseline level, the hypothetical composition is identified as an ABCB5-PIP2 pathway enhancer. In some embodiments, if the level of the PIP2 pathway product compound is less than the baseline level, the hypothetical composition is identified as an ABCB5-PIP2 pathway inhibitor.

[0025] In some embodiments, the putative composition for regulating the ABCB5-PIP2 pathway is PIP2 or a PIP2 agonist. In some embodiments, the putative composition for regulating the ABCB5-PIP2 pathway is a small molecule. In some embodiments, the putative composition for regulating 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, ABCB5+ cells comprise ABCB5 isoform 1, wherein amino acid 970 is lysine. In some embodiments, ABCB5+ cells comprise ABCB5 isoform 2, wherein amino acid 525 is lysine.

[0026] In some implementations, the assay involves determining the number of ABCB5 alleles and then testing how many pairs of K are positive and how many pairs of E are positive, i.e., extracting copy number and allele type information using an allele-specific quantification procedure.

[0027] Some aspects of the present invention relate to compositions comprising synthetic phospholipids, said synthetic phospholipids comprising compounds having the structure described herein. In some embodiments, said structure comprises R1 and R2 groups. In some embodiments, R1 and R2 are independent fatty acid chains. In some embodiments, the lengths of R1 and R2 are at least twice the length of the other of R1 and R2. In some embodiments, said phospholipid has a total fatty acid chain ratio of 22:0 to 26:0. In some embodiments, said phospholipid has a total fatty acid chain ratio of 24:0. In some embodiments, said phospholipid has the formula: C33H65O19P3. In some embodiments, said phospholipid comprises [PIP2(6:0 / 18:0)-H] - And a pharmaceutical carrier.

[0028] 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.

[0029] In some embodiments, the phospholipid comprises phosphorylated PIP3(6:0 / 18:0)-H - (C33H65O19P4) and pharmaceutically usable carriers.

[0030] Some aspects of the present invention relate to human anti-ABCB5 antibodies or ABCB5-binding fragments thereof that inhibit the ABCB5-PIP2 pathway, wherein the anti-ABCB5 antibody or ABCB5-binding fragments thereof bind to an extracellular loop of a three-dimensional configuration of ABCB5.

[0031] In some embodiments, the human anti-ABCB5 antibody or ABCB5 binding fragment can be prepared by a method including affinity maturation to specifically bind to the 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 prepared by a method including affinity maturation to specifically bind to the extracellular loop of a non-linear form of ABCB5.

[0032] Some aspects of this invention relate to methods for preparing human anti-ABCB5 antibodies or ABCB5-binding fragments that inhibit the ABCB5-PIP2 pathway as described herein. In some embodiments, the anti-ABCB5 antibody or ABCB5-binding fragment undergoes affinity maturation to specifically bind to an extracellular loop of the protein in a non-linear form.

[0033] Some aspects of the present 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 to ABCB5; evaluating ABCB5-PIP2 binding after treatment with said antibody or fragment; determining the level of a PIP2 pathway product compound and comparing said level to a baseline level of a PIP2 pathway product compound.

[0034] In some embodiments, if the level of a PIP2 pathway product compound is below baseline, the antibody or fragment is assumed to be 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.

[0035] Some aspects of the present invention relate to ABCB5 isoform 1 comprising two transmembrane domains (TMDs) and 12 transmembrane helices (TM1-12). In some embodiments, the glutamic acid at position 970 of TM12 has been mutated to lysine, or the glutamic acid at position 970 of TM12 is glutamic acid.

[0036] Some aspects of the present invention relate to ABCB5 isoform 2 comprising a transmembrane domain (TMD) and six transmembrane helices (TM 1-6). In some embodiments, the glutamic acid at position 525 of TM6 has been mutated to lysine, or the glutamic acid at position 525 of TM12 has been mutated to glutamic acid.

[0037] Some aspects of this invention relate to human anti-ABCB5 isotype antibodies or binding fragments thereof that inhibit the ABCB5-PIP2 pathway. In some embodiments, the anti-ABCB5 antibody or its ABCB5 binding fragment specifically binds to either ABCB5 isotype 1 or ABCB5 isotype 2 as described herein.

[0038] Some aspects of this invention relate to human anti-ABCB5 isotype antibodies or binding fragments thereof that inhibit the ABCB5-PIP2 pathway. In some embodiments, the anti-ABCB5 antibody or its ABCB5 binding fragment specifically binds to either ABCB5 isotype 1 or ABCB5 isotype 2 as described herein.

[0039] Some aspects of this invention relate to methods for identifying enhancers or inhibitors of the ABCB5-PIP2 pathway. In some embodiments, the method includes contacting either ABCB5 isotype 1 or ABCB5 isotype 2 described herein with a hypothetical composition that modulates ABCB5-PIP2 binding; determining a level of a PIP2 pathway product compound and comparing said level to a baseline level of a PIP2 pathway product compound. In some embodiments, if said level is greater than a baseline level, the composition is assumed to be an ABCB5-PIP2 pathway enhancer. In some embodiments, if the level of a PIP2 pathway compound is less than a baseline level, the composition is assumed to be an ABCB5-PIP2 inhibitor.

[0040] In some embodiments, the presumed composition for regulating the ABCB5-PIP2 pathway is PIP2 or a PIP2 agonist. In some embodiments, the presumed composition for regulating the ABCB5-PIP2 pathway is a small molecule. In some embodiments, the presumed composition for regulating 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, recombinant expression of the ABCB5 isotype is performed.

[0041] Some aspects of the present invention relate to methods for treating cancer in a subject. In some embodiments, the method includes disrupting an endogenous ABCB5 gene in a cell using gene editing. In some embodiments, the editing includes contacting the cell with a Cas protein, CRISPR RNA, and tracrRNA that hybridize with the endogenous ABCB5 gene. In some embodiments, after contact with the Cas protein, CRISPR RNA, and tracrRNA, the endogenous ABCB5 gene is modified such that the AAA sequence in the terminal transmembrane helix region encoding the ABCB5 gene is replaced with GAA. In some embodiments, gene editing treats cancer in a subject.

[0042] In some implementations, the subject possesses cancer-associated ABCB+ stem cells prior to gene editing, which are homozygous ABCB5 congeners 2 K525 / K525. In some implementations, the cancer is melanoma or glioblastoma.

[0043] Some aspects of the present invention relate to methods for treating cancer in a subject. In some embodiments, the method includes administering an ABCB1 inhibitor to the subject in an effective amount that inhibits the function of the ABCB5-PIP2 pathway to treat cancer in the subject. In some embodiments, the cancer comprises cancer cells, and the cancer cells express negligible amounts of ABCB1 or do not express ABCB1. In some embodiments, the method further includes detecting the presence of ABCB5+ stem cells prior to the administration step. In some embodiments, the ABCB1 inhibitor is a pump inhibitor, and the cancer is not simultaneously treated with a chemotherapy agent. In some embodiments, the method further includes evaluating ABCB5-PIP2 binding after administration of the composition.

[0044] In some embodiments, the object has ABCB+ stem cells associated with the cancer prior to gene editing, which are ABCB5 homozygous congener 2 K525 / K525. In some embodiments, the cancer is melanoma or glioblastoma.

[0045] Some aspects of the present invention relate to methods for characterizing cancer. In some embodiments, the method includes isolating cancer cells from a subject and determining whether the cancer cells are ABCB5 homozygous congener 2 K525 / K525, ABCB5 homozygous congener 2 E525 / E525, or ABCB5 heterozygous congener 2 K525 / E525, to characterize the cancer.

[0046] Each limitation of the invention can encompass various embodiments of the invention. Therefore, it is contemplated that each limitation of the invention relating to any element or combination of elements can be included in every aspect of the invention. The application of the invention is not limited to the details of the construction and the arrangement of components set forth in the following description or shown in the drawings. The invention is capable of having other embodiments and can be practiced or performed in a variety of ways. Furthermore, the wording and terminology used herein are for descriptive purposes and should not be considered limiting. The use of “comprising,” “including,” or “having,” “containing,” “involving,” and variations thereof herein is intended to cover the items listed thereafter and their equivalents, as well as other items. Brief description of the attached diagram

[0048] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in multiple figures is indicated by the same reference numerals. For clarity, not every component is labeled in every drawing. In the drawings:

[0049] Figures 1A to 1C PIP2 and PIP3 are biological ligands of ABCB5, and ABCB5 acts as the receptor for PIP2 and PIP3. Immunoblotting using ABCB5 monoclonal antibodies (…) Figure 1A The image above shows that immunoprecipitation of PIP2 from human ABCB5-expressing melanoma cells using anti-PIP2 antibody revealed co-precipitation of ABCB5 protein. Immunoblotting using PIP2 antibody confirmed the co-precipitation of PIP2 from PIP2-expressing melanoma cells. Figure 1A (See image below). Immunoblotting using ABCB5 monoclonal antibody ( Figure 1B The results showed that immunoprecipitation of PIP3 from human ABCB5-expressing melanoma cells using anti-PIP3 antibody revealed co-precipitation of ABCB5 protein. Figure 1C The results showed that PIP1, PIP2, and PIP3 all bound to recombinant human ABCB5 and mouse Abcb5, while no binding was detected in Abcb5 knockout mouse tissues. Therefore, PIP2 and PIP3 bound to ABCB5 more efficiently than PIP1 bound to ABCB5.

[0050] Figure 2The binding of PIP1, PIP2, and PIP3 to ABCB5 can be inhibited by competitive pharmacological ligands. Data show that PtdIns-(1,2-dioctanoyl) competitively inhibits the binding of PIP1, PIP2, or PIP3 to ABCB5, with significant saturation at concentrations as low as 0.1 mM.

[0051] Figure 3 ABCB5 monoclonal antibodies can block the binding of PIP1, PIP2, or PIP3 to ABCB5. Surface plasmon resonance (SPR) analysis confirmed the binding of PIP1, PIP2, and PIP3 to ABCB5. Competition with anti-ABCB5 monoclonal antibodies resulted in a concentration-dependent reduction in signal intensity on all three surfaces (PIP3>PIP2>PIP1), with a reduction of up to 50% on the PIP3 surface.

[0052] Figure 4 ABCB5 is functionally required for a more efficient conversion of PIP2 to PIP3. ABCB5 monoclonal antibodies, rather than allotype control antibodies, significantly reduced the PIP3 / PIP2 ratio in human melanoma cells (left figure). Furthermore, examination of mouse ABCB5 gene knockout skin tissue showed a significantly reduced PIP3 / PIP2 ratio compared to ABCB5 wild-type skin (right figure).

[0053] Figure 5 ABCB5 is required to maintain the PIP2 / PIP3-dependent PI3K / AKT signaling axis in malignant tumors, and ABCB5 inhibition leads to suppression of the PI3K / AKT signaling axis, as well as associated tumor growth and treatment resistance. Analysis of 4-HT-induced Tyr::CreER; BrafCA; Ptenlox / lox genetic mouse melanoma models under ABCB5 WT or ABCB5 KO backgrounds showed significant inhibition of the PI3K / AKT signaling axis in ABCB5 KO compared to ABCB5 WT tumors, with attenuated expression of p-AKT, p-mTOR, and p-S6 among other dysregulated molecules.

[0054] Figure 6 Compared to the ABCB5 WT state, the ABCB5 KO state resulted in reduced tumor cell proliferation, as determined by identifying the percentage of tumor cells positive for the proliferation marker Ki-67.

[0055] Figures 7A-7B Compared to the ABCB5 WT state, the ABCB5 KO state resulted in a downregulation of pro-angiogenic molecules. Figure 7A (left image), and the results showed a decrease in CD31-positive microvessel density ( Figure 7A The right image and Figure 7B ).

[0056] Figure 8 ABCB5(+) CRC cells express the receptor tyrosine kinase AXL, which maintains EMT and acts as a mediator of ABCB5-dependent cancer invasion. A set of scatter plots is shown, depicting a representative flow cytometry analysis of AXL protein expression in ABCB5 KD compared to control transfected cell lines. Bar plots are also shown, illustrating AXL mRNA expression in ABCB5 KD compared to control transfected human CRC cells. Western blot analysis of AXL, AKT, and phosphorylated AKT protein expression in CRC cells treated with anti-ABCB5 mAb or isotype control is also shown. Data were analyzed using unpaired t-tests. Error bars represent sem *P < 0.05, **P < 0.01, ***P < 0.001.

[0057] Figure 9 ABCB5 plays a crucial role in tumor vemurafenib resistance through its function in the intact PIP2 / PIP3-dependent PI3K / pAKT signaling axis required to maintain vemurafenib resistance. In Tyr::CreER; BrafCA; Ptenlox / lox genetically modified mouse melanoma models under ABCB5 WT or ABCB5 KO backgrounds, ABCB5 KO status resulted in complete sensitivity to the BRAF inhibitor vemurafenib compared to ABCB5 WT status, with vemurafenib resistance partially driven by the functional PI3K / pAKT signaling axis. No tumor formation was observed in vemurafenib-treated ABCB5 KO mice after genetic induction, compared to 100% vemurafenib-resistant tumor formation in ABCB5 WT mice (left panel), and survival was significantly prolonged in ABCB5 KO mice compared to ABCB5 WT mice (right panel).

[0058] Figure 10 Compared with ABCB5 wild-type mice, psoriasis was exacerbated in ABCB5 knockout imiquimod-induced psoriasis mouse models.

[0059] Figure 11The 525th amino acid residue of TM6 in ABCB5 isotype 2 is a crucial molecular switch in the physiological ligand / substrate binding quality of ABCB5. At baseline, in wild-type K525 / K525 human melanoma cells expressing only ABCB5 isotype 2, an experimental induction of a molecular switch from K (lysine, AAA codon) to E (glutamate, GAA codon) in one allele via Crispr / Cas9-mediated gene editing yielded a clonal heterozygous ABCB5K525 / E525 melanoma cell variant with impaired ABCB5 signal transduction function, and resulted in a significant inhibition of ABCB5-driven tumor growth (P<0.05). Invention Details

[0061] This invention relates in several aspects to the discovery that 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 plasma membrane of cancer stem cells and normal tissue-specific stem cells, acts as a receptor for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, also known as PIP2), and to a lesser extent as a receptor for PIP1 and PIP3. As used herein, “ABCB5(+) stem cells” refers to cells capable of self-renewal and differentiation into mature cells of various adult cell lineages and characterized by the expression of ABCB5 on their cell surface. PIP2 is a cell membrane-rich, low-phosphoinositol phospholipid component, a substrate for many important signal transduction proteins, regulating signal transduction via the PI3K pathway or the IP3 / DAG pathway of G protein-coupled receptors, for example, through receptor tyrosine kinases (RTKs). Inhibition of the ABCB5-PIP2 pathway by inhibiting ABCB5 blocks the binding of PIP2 to ABCB5 and subsequent phosphorylation of PIP2 to produce PIP3. Therefore, disruption of this pathway leads to inhibition of downstream PI3K signaling of tyrosine kinase receptors (e.g., VEGFR1, EGFR, and AXL) and eliminates their stem cell-specific function. ABCB5-PIP2 binding can be assessed using various methods known in the art. For example, ABCB5 PIP2 binding can be assessed using methods including immunoprecipitation, Western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, microscopy, and spectroscopy (see Figures 1-3).

[0062] Some aspects of this invention relate to methods for enhancing the function of ABCB5-positive cells. As used herein, "ABCB5-positive cell function" refers to the activity of ABCB5 in a healthy subject and has a positive effect on the subject. For example, ABCB5-positive cell function includes promoting wound healing, tissue regeneration, angiogenesis, 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 to the level or rate in a control sample. "Control sample" as used herein refers to a sample lacking ABCB5 function. As used herein, "healthy subject" is another subject without disease.

[0063] As used herein, the phrase “stem cell-specific function” refers to stem cell-related ABCB5 activity. For example, skin-associated healthy ABCB5+ stem cells utilize ABCB5-enhanced PI3K signaling and downstream AKT phosphorylation, as well as mTOR signaling, for angiogenesis and anti-apoptotic signaling, leading to stem cell survival and vascular differentiation, and other downstream functions required for normal wound healing. ABCB5+ limbal stem cells utilize this pathway for anti-apoptotic signaling required for stem cell maintenance. ABCB5+ cancer stem cells (e.g., in melanoma or colorectal cancer) utilize this pathway for cell survival, vasculogenic mimicry, drug resistance and EMT, and metastatic invasion (as shown in Figure 1, i.e., through ABCB5 blockade inhibiting pAKT phosphorylation and EMT, and invasiveness).

[0064] Among other functions, ABCB5 binding to PIP2 can be used to increase the rate of its phosphorylation to PIP3, and thus represents a stem cell-specific interaction to enhance the signal transduction 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 them, which also inhibit downstream signal transduction in key ABCB5-dependent stem cell functions. Therefore, this invention has several important applications.

[0065] Therefore, the invention described herein can be used to promote regeneration in healthy individuals.

[0066] This invention can also be used to treat objects that have a disease or are at risk of having a disease, such as objects that have cancer or are at risk of having cancer.

[0067] The object should refer to humans or vertebrate mammals, including but not limited to goats, sheep, bison, camels, cattle, pigs, rabbits, buffalo, horses, rats, mice, cats, dogs, llamas, and primates such as monkeys. Therefore, the invention can also be used to treat diseases or conditions in non-human objects. For example, cancer is one of the leading causes of death in companion animals (i.e., cats and dogs). Preferably, the object is a human.

[0068] Subjects at risk of cancer are those with a high probability of developing cancer. These subjects include, for example, those with genetic abnormalities that have been shown to be associated with a higher probability of developing cancer, those exposed to carcinogens (such as tobacco, asbestos, or other chemical toxins), or those who have previously undergone cancer treatment and achieved significant remission. Subjects at risk of cancer also include those with precancerous lesions. Precancerous lesions are areas of tissue with altered characteristics and a risk of developing into skin cancer. Precancerous lesions can be caused by, for example, UV radiation, genetics, or exposure to carcinogens (such as arsenic, tar, or X-rays).

[0069] A person suffering from cancer is a person with detectable cancer cells. Cancer can be malignant or non-malignant. Cancers or tumors include, but are not limited to, bile duct cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; stomach 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 cancers and sarcomas. Preferably, cancers include cancer stem cells expressing ABCB5.

[0070] Optionally, prior to treatment, the presence of ABCB5-positive stem cells can be detected using the binding molecules described herein. The detection or diagnostic methods provided by this invention generally involve contacting one or more molecules of this invention with a sample from or within the subject. Preferably, the sample is first harvested from the subject; however, in vivo detection methods are also contemplated. The sample may include any body tissue or fluid suspected of containing cancer stem cells. For example, stem cells are typically present in or around a tumor mass.

[0071] ABCB5 or ATP binding cassette B subfamily 5

[0072] 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 crucial role in physiology and pathology. They are involved in the transport of structurally diverse molecules, from small ions, sugars, and peptides to more complex organic molecules (Chen et al. 2005).

[0073] As used herein, "ABCB5+ stem cells" or "ABCB5+ cells" refers to cells capable of self-renewal and differentiation into mature cells of various adult cell lineages. In some embodiments, these cells are characterized by the expression of ABCB5. In some embodiments of the invention, ABCB5+ cells are cancer stem cells. In some embodiments of the invention, ABCB5+ cells are healthy stem cells.

[0074] Some aspects of this invention relate to the identification of ABCB5 isotypes involved in cancer. In some embodiments, the ABCB5 isotype relates to melanoma or glioblastoma. As used herein, “ABCB5 isotype” is a variant of the ABCB5 protein having the ABCB5 structure. In some embodiments, the ABCB5 isotype is ABCB5 isotype 1 (1257 amino acids). ABCB5 isotype 1 comprises two transmembrane domains (TMDs), each having six transmembrane (TM) helices, i.e., it comprises a total of 12 transmembrane helices (TM 1-12). In some embodiments, the ABCB5 isotype isotype 2 (812 amino acids). ABCB5 isotype 2 comprises a TMD having six transmembrane (TM) helices (TM 1-6). TM 1-6 of ABCB5 isotype 2 corresponds to TM 7-12 of ABCB5 isotype 1. In other embodiments, the presence of lysine residues at specific positions in both isotypes is associated with cancer incidence. In other embodiments, this residue is 970 in TM12 of ABCB5 isotype 1 and 525 in TM6 of ABCB5 isotype 2. It is shown herein that non-synonymous single nucleotide polymorphisms (SNPs) in the ABCB5 coding region providing AA 970 E>K in TM12 of ABCB5 isotype 1 and corresponding to AA 525 E>K in TM6 of ABCB5 isotype 2 are important for ABCB5 function in cancer cells. For example, 970 in TM12 of ABCB5 isotype 1 and 525 in TM6 of ABCB5 isotype 2 are important for ABCB5-positive stem cell function. In other embodiments, these residues are required for ABCB5-positive cancer stem cell function.

[0075] Other residues involved in ABCB5 substrate binding are N702 and H706 in TM7 of ABCB5 isotype 1, corresponding to N257 and H261 in TM1 of ABCB5 isotype 2, and 857 A>T (rs80123476) in TM10 of ABCB5 isotype 1, corresponding to 412 A>T (rs80123476) in TM4 of ABCB5 isotype 2.

[0076] Genes encoding the higher-functioning ABCB5 isotype 2-K525 protein sequence or the ABCB5 isotype 2-K525 protein itself, as well as genes encoding the lower-functioning ABCB5 isotype 2-E525 protein sequence or the ABCB5 isotype 2-E525 protein itself, are useful combinations. These compositions can be used, for example: 1. recombinantly expressing ABCB5 isotype 2-K525 or ABCB5 isotype 2-E525; 2. using ABCB5 isotype 2-K525 and ABCB5 isotype 2-E525 in docking and binding assays to identify novel sequence-specific ABCB5 ligands and substrates; using ABCB5 isotype 2-K525 or ABCB5 isotype 2-E525 in molecular screening to identify synthetic compounds and naturally occurring substances that competitively inhibit the binding of PIP1, PIP2, or PIP3 to ABCB5 and thus also inhibit ABCB5-dependent receptor tyrosine kinase and G protein-coupled receptor signaling; using ABCB5 isotype 2-K525 or ABCB5 isotype 2-E525 in molecular screening to identify novel ABCB5 monoclonal antibodies that inhibit the binding of PIP1, PIP2, or PIP3 to ABCB5 and thus also inhibit ABCB5-dependent receptor tyrosine kinase and G protein-coupled receptor signaling.

[0077] 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, synthetic analogs of natural phosphatidylinositol (PtdIns) containing C8:0 fatty acids at the n-1 and sn-2 positions (CAS Registry No. 899827-36-2). These compounds can be used to treat cancers associated with ABCB5+ stem cells.

[0078] In some aspects, the present invention relates to a method of treating cancer by administering an ABCB1 inhibitor to a subject suffering from cancer. It has been found herein that ABCB1 inhibitors can also be used to treat ABCB5+ cancers. These compounds competitively inhibit the binding of PIP1, PIP2, or PIP3 to ABCB5, and thus inhibit ABCB5-dependent signal transduction.

[0079] As used herein, ABCB1 inhibitors are compounds that reduce or eliminate ABCB1 function in cells. ABCB1 inhibitors are known in the art and include anti-ABCB1 antibodies and their functional fragments, as well as small molecules. Some ABCB1 inhibitors are ABCB1 agents for the treatment of heart or vascular diseases, ABCB1 agents for the treatment of ABCB1+ cancers, ABCB1 agents for the treatment of infectious diseases, ABCB1 agents for the treatment of gastric diseases, and ABCB1 agents for the treatment of other diseases. In some embodiments, ABCB1 inhibitors include, for example, PSC 833 (Valspodar), Zosuquidar, Tariquidar, and Laniquidar, i.e., inhibitors of substrate and / or related substrate binding sites of highly homologous ABCB1 molecules.

[0080] ABCB1 substrates or inhibitors are known to be used to treat a variety of diseases. Based on the discovery of novel ABCB5 isoform 2-AA525 substrate binding sites for PIP1, PIP2, or PIP3, these compounds can therefore be used as small molecule inhibitors of ABCB5-dependent PIP1, PIP2, or PIP3 binding, as well as PIP-dependent signal transduction and pAKT phosphorylation, to, for example, by blocking functional ABCB5 to therapeutically inhibit ABCB5-expressing cancers and thus ABCB5-driven human cancer growth and progression.

[0081] In some implementations, ABCB1 inhibitors that can be used to treat cancer are ABCB1 agents used to treat heart / vascular diseases. Non-limiting examples of these compounds are shown in the following list.

[0082] ABCB1, a medication used to treat heart / vascular diseases.

[0083] Verapamil Reserpine Nifedipine Digoxin Quinidine Nicardipine Prazosin Diltiazem Amitriptyline Losartan Pravastatin Acebutolol Acetylsalicylic acid Timolol Nadolol Debrisoquine Ezetimibe Tolvaptan Pitavastatin Canagliflozin Clopidogrel Ticagrelor Apixaban Cobimetinib Selexipag Ambrisentan Metoprolol Atenolol Bromocriptine Amlodipine

[0084] In some implementations, ABCB1 inhibitors that can be used to treat cancer are ABCB1 agents used to treat infectious diseases. Non-limiting examples of these compounds are shown in the following list.

[0085] ABCB1 agents used to treat infectious diseases

[0086] Ivermectin Clarithromycin Ketoconazole Ritonavir Saquinavir Nelfinavir Indinavir Rifampicin Ciprofloxacin, Rifamycin, Sparfloxacin, Levofloxacin, Grepafloxacin Levomilnacipran Simeprevir Zidovudine Atazanavir Telaprevir Fidaxomicin and Lamivudine Sofosbuvir Voxiprevir Pibrentasvir Glecaprevir Letemurovir Dolutegravir

[0087] In some embodiments, the ABCB1 inhibitors used in methods for treating cancer are ABCB1 agents for treating cancer. In some embodiments, the cancer is 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.

[0088] ABCB1 inhibitors used to treat ABCB5+ cancers

[0089] Vinblastine Tamoxifen Mitoxantrone Doxorubicin Daunorubicin Etoposide Paclitaxel Dactinomycin Dasatinib Gefitinib Nilotinib Cisplatin Camptothecin Diethylstilbestrol Clonidine Estradiol Docetaxel Methotrexate Gemcitabine Topotecan Erlotinib Conjugated estrogens Ethinylestradiol Cabazitaxel Temsirolimus Romidepsin Afatinib Dabrafenib Crizotinib Pazopanib Axitinib Trastuzumab emtansine Irinotecan Ceritinib Lenvatinib Osimertinib Rucaparib Abemaciclib Fluciclovine (18F) Octreotide Ondansetron Regorafenib Melphalan Changchun Vinorelbine Vemurafenib Duvelisib Progesterone Ibuprofen Gilteritinib Talazoparib Toremifene Dacomitinib Glasdegib Olaparib Palbociclib

[0090] In some implementations, ABCB1 inhibitors that can be used to treat cancer are ABCB1 agents used to treat gastric ailments. Non-limiting examples of these compounds are shown in the following list.

[0091] ABCB1 for stomach ailments

[0092] Omeprazole Nizatidine Domperidone Lansoprazole Ranitidine Pantoprazole

[0093] Other ABCB1 inhibitors that can be used in the methods of this invention include, but are not limited to: cyclosporine, cimetidine, aldosterone, tacrolimus, phenobarbital, dexamethasone, carbamazepine, colchicine, loperamide, imipramine, hydrocortisone, citalopram, and taurocholic acid. Acid, Fexofenadine, Prednisone, Estrone, Diazepam, Digitoxin, Methylprednisolone, Quetiapine, Olanzapine, Clozapine, Prednisolone, Betamethasone, Alitretinoin, Vecuronium bromide Onion, Stanolone, Epinastine, Estriol, Sphingosine, Cerivastatin, Levetiracetam, Phenytoin, Lamotrigine, Sitagliptin, Ketazolam, Silodosin, Rivaroxaban, Dabigatran etexilate, Fesoterodine, Indacaterol, Clobazam, Linagliptin, Mirabegron, Bosutinib, Fluticasone furoate, MycophenolateThe following are listed: mofetil, dapagliflozin, umeclinium, edoxaban, nintedanib, ometasvir, elbasvir, gzoprevir, odanacatib, baricitinib, ubidecarenone, ertugliflozin, stanolone acetate, estradiol acetate, estradiol benzoate, estradiol cypionate, estradiol diheptanoate, estradiol valerate, testosterone propionate, asunaprevir, and 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 6G), Simvastatin, Valspodar, Cerliponase alfa, Curcumin, Ascorbic acidacid), chlorpromazine, phenothiazine, atorvastatin, bromperidol, morphine, pentazocine, propranolol, neostigmine, moxidectin, mefloquine, fluticasone, fluticasone propionate. propionate, Elagolix, Chloroquine, Paliperidone, Lusutrombopag, Posaconazole, Dipyridamole, Quinine, Indometacin, Acetaminophen, Haloperidol, Naloxone, Mannitol, Betrixaban, Clomifene, Omadacycline, Grapiprant, Larotrectinib, Revefenacin, Tenofovir disoproxil, Tenofovir alafenamide The following are listed: alafenamide, tenofovir, ledipasvir, sildenafil, vardenafil, cabergoline, prucalopride, risperidone, tramadol, azithromycin, fluconazole, ranolazine, cetirizine, tegaserod, and doxepin.

[0094] PIP2 or phosphatidylinositol 4,5-bisphosphate

[0095] PIP2 is a phospholipid present in low levels in cells but involved in a variety of important cellular processes. Some of the cellular functions of PIP2 include regulating endocytosis, exocytosis, phagocytosis, and cell signal transduction (Czech et al., 2000).

[0096] As used in this article, “PIP2” refers to phospholipids bound to ABCB5.

[0097] Some aspects of the present invention are methods for enhancing ABCB5 / PIP2-dependent signaling transduction in normal stem cells via an ABCB5-PIP2 binding enhancer to enhance ABCB5-normal stem cell function. Such a method includes administering an effective amount of a composition enhancing the ABCB5-PIP2 pathway to a subject in need, and evaluating ABCB5-PIP2 binding after administration of said composition. In some embodiments, the composition comprises PIP2, a PIP2 agonist, a phospholipid, and [PIP2(6:0 / 18:0)-H]. In some embodiments, the composition comprises a phospholipid containing a compound having the following structure:

[0098]

[0099] In some embodiments, R1 and R2 are independent fatty acid chains. In some embodiments, the lengths of R1 and R2 are at least twice the length of the other of R1 and R2. In some embodiments, the structure has a total fatty acid chain ratio of 22:0 to 26:0. In some embodiments, the structure has a total fatty acid chain ratio of 22:0, 23:0, 24:0, 25:0, or 26:0. In some embodiments of the invention, the composition is applied to a healthy subject. The subject can be a human or a non-human animal, including goats, sheep, bison, camels, cattle, pigs, rabbits, buffalo, horses, rats, mice, cats, dogs, llamas, or primates such as monkeys. In some embodiments, the composition is used in the subject to promote wound healing, tissue regeneration, angiogenesis and cell survival, reduce aging, and inhibit cell death. In some embodiments of the invention, the composition is administered via oral, intravenous, subcutaneous, topical, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular, or intrathecal routes. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0100] In some embodiments, the composition comprises a PIP2 agonist. In some embodiments, the composition comprises:

[0101] .

[0102] One advantage of the method of this invention is that it allows selective targeting of tissues with stem cells expressing ABCB5. While existing therapies targeting the tyrosine kinase pathway have potentially broad side effects, methods that also modulate PI3K signaling by targeting ABCB5 / PIP2 binding (enhancer or inhibitor) limit their action to a subpopulation of cells that also express ABCB5. Therefore, this method should provide a lower potential rate of side effects. ABCB5 targeting can be used as a standalone treatment for disseminated disease or as adjuvant therapy to sensitize cancer cells to chemotherapy agents, particularly in patients currently suffering from refractory metastatic disease.

[0103] Some aspects of the present invention relate to methods for inhibiting ABCB5-PIP2 binding by means of an inhibitory molecule contained in a composition to suppress ABCB5-dependent cancer stem cell function. Such methods represent a functional blockade of ABCB5 and also include evaluating ABCB5-PIP2 binding after administration of the composition. In some embodiments, the composition inhibits the PI3K pathway and suppresses tumorigenesis, metastasis, and / or resistance to drugs that regulate PI3K signaling, such as melanoma resistance to vemurafenib mediated by PI3K signaling, or cancer resistance to EGFR inhibitors mediated by ABCB5-enhanced PI3K signaling upregulation.

[0104] In some embodiments, the composition comprises a PIP2 antagonist. In some embodiments, the composition is selected from the group consisting of: small molecules, lipid analogs, anti-ABCB5 antibodies or ABCB5 binding fragments specific to the cyclic or linear form of the extracellular polypeptide of the protein, and enzymes. In some embodiments, the composition comprises an anti-ABCB5 antibody or ABCB5 binding fragment specific to 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, for example, the list including: 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 the extracellular loop of the three-dimensional conformation of ABCB5. In some embodiments, human anti-ABCB5 antibodies undergo affinity maturation to recognize and specifically bind to the non-linear extracellular loop of ABCB5. The human anti-ABCB5 antibodies or ABCB5 binding fragments described herein have sequences corresponding to antibodies that can be prepared by methods including affinity maturation to specifically bind to the non-linear extracellular loop of ABCB5.

[0105] Some aspects of this 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 antibody or ABCB5-binding fragment undergoes affinity maturation to specifically bind to an extracellular loop of the protein in a non-linear form. The affinity maturation process can occur via: a. phage display, yeast display, or ribosome display; or b. panning techniques. For example, once an antibody has been generated against a linear extracellular cyclic peptide, the resulting antibody can be matured using a display method by presenting and allowing the peptide protein to be processed by an antigen-presenting cell.

[0106] In some embodiments of the invention, the composition is applied to a healthy subject.

[0107] In some embodiments, the object can be a human or a non-human animal, including goats, sheep, bison, camels, cattle, pigs, rabbits, buffalo, horses, rats, mice, cats, dogs, llamas, or primates such as monkeys. 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 via oral, intravenous, subcutaneous, topical, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular, or intrathecal routes.

[0108] Some aspects of the present invention relate to a method for inhibiting ABCB5-dependent cancer stem cell function by administering an effective amount of a composition that inhibits ABCB5-PIP2 binding to a subject in need. Such a method represents a functional blockade of ABCB5 and further includes evaluating ABCB5-PIP2 binding after administration of the composition. In some embodiments, the composition inhibits the PI3K pathway and suppresses tumorigenesis, metastasis, and / or resistance to drugs that regulate PI3K signaling, such as melanoma resistance to vemurafenib mediated by PI3K signaling, or cancer resistance to EGFR inhibitors mediated by ABCB5-enhanced PI3K signaling upregulation.

[0109] In some embodiments, the composition comprises a PIP2 antagonist. In some embodiments, the composition is selected from the group consisting of: small molecules, lipid analogs, anti-ABCB5 antibodies or ABCB5 binding fragments specific to the cyclic or linear form of the extracellular polypeptide of ABCB5, and enzymes. In some embodiments, the composition comprises an anti-ABCB5 antibody or ABCB5 binding fragment specific to 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 of the invention, the composition is administered to a healthy subject.

[0110] In some embodiments, the object can be a human or a non-human animal, including goats, sheep, bison, camels, cattle, pigs, rabbits, buffalo, horses, rats, mice, cats, dogs, llamas, or primates such as monkeys. 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 via oral, intravenous, subcutaneous, topical, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, ocular, or intrathecal routes.

[0111] In one aspect, the present invention can be used as a screening tool in a method for developing molecular compounds that inhibit or enhance ABCB5-PIP2 binding and thus represent functional ABCB5 blockers or enhancers. Such compounds include lipid analogs, PIP2 or PIP2 agonists, small molecule drugs (e.g., PSC833), and a new subset of ABCB5-inhibiting monoclonal antibodies that bind to ABCB5 and block PIP2 binding and inhibit pAKT phosphorylation and downstream signal transduction / effect pathways by inducing spatial changes in the molecule. The method of the present invention also includes contacting ABCB5+ cells with a hypothetical composition containing a compound that regulates ABCB5-PIP2 binding, determining the level of the PIP2 pathway product compound. If the level is greater than a baseline level, the composition is assumed to be an ABCB5-PIP2 pathway enhancer, and if the level of the PIP2 pathway compound is lower than a baseline level, the composition is assumed to be an ABCB5-PIP2 inhibitor. In some embodiments, the PIP2 pathway product compound is a member of the PIP3 or PI3K pathway. As used herein, “baseline level” refers to the level of PIP2 pathway product compounds in a sample that has not been exposed to a hypothetical composition containing compounds that regulate ABCB5-PIP2 binding.

[0112] Some aspects of this invention disclose novel phospholipid analogs of PIP2 that have been characterized by mass spectrometry. The fatty acid chain composition of the PIP2 analogs represents novel endogenously present PIP2 variant compounds (Formula C). 33 H 65 O 19 P3, a PIP2 with a total fatty acid chain of 24:0, is labeled [PIP2(6:0 / 18:0)-H]). This analogue is particularly enriched in ABCB5 knockout cells, suggesting that ABCB5 is functionally required for efficient PIP2 conversion.

[0113] PIP2 has the chemical formula C 47 H 80 O 19 P3 and the following structures:

[0114]

[0115] In other aspects, the present invention relates to novel compounds that are functional analogs of PIP2 and have the structure [PIP2(6:0 / 18:0)-H]- and the formula C 33 H 65 O 19 The total fatty acid chains are P3 and 24:0. 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.

[0116] In some respects, the present invention is a compound having the following structure:

[0117]

[0118] R1 and R2 are independently fatty acid chains, such that the structure has a total fatty acid chain ratio of 22:0 to 26:0, and wherein the length of one of R1 and R2 is at least twice the length of the other. In some embodiments, the structure has a total fatty acid chain ratio 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.

[0119] In some aspects, the present invention is a method for screening ABCB5 antagonists and enhancers using the novel compositions of the present invention or PIP2 or other PIP2 analogues. The method of the present invention further includes contacting ABCB5+ cells with a hypothetical composition that regulates ABCB5-PIP2 binding, determining the level of a PIP2 pathway product compound. If the level is greater than a baseline level, the composition is assumed to be an ABCB5-PIP2 pathway enhancer, and if the level of a PIP2 pathway product compound is less than a baseline level, the composition is assumed to be an ABCB5-PIP2 inhibitor. In some embodiments, the PIP2 pathway compound is PIP3, or a member of the PI3K pathway.

[0120] In some embodiments, ABCB5+ cells comprise ABCB5 isotype 1, which has a lysine residue at amino acid position 970. In some embodiments, ABCB5+ cells comprise ABCB5 isotype 2, which has a lysine residue at amino acid position 525. ABCB5 expressing this SNP is most commonly found in human cancers.

[0121] In others, the composition is a tool used for such screening determinations.

[0122] In other aspects, the present invention is a method of using novel compositions or PIP2 or other PIP2 analogues as therapeutic compounds to enhance ABCB5-dependent stem cell function upon exogenous application.

[0123] Effective dose

[0124] In the methods described herein, the term "effective amount" refers to the amount of composition that can achieve the desired therapeutic effect (e.g., enhance or inhibit the ABCB5-PIP2 pathway).

[0125] In some embodiments, the composition comprises PIP2, a PIP2 agonist, a PIP2 antagonist, a phospholipid, or [PIP2(6:0 / 18:0)-H]. In some embodiments, the composition comprises PIP2. In some embodiments, the amount of PIP2 in the composition is from 1% to 100%. In some embodiments, the amount of PIP2 in the composition is at least 1%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more. In some embodiments, the composition comprises a PIP2 agonist. In some embodiments, the amount of the PIP2 agonist in the composition is from 1% to 100%. In some embodiments, the amount of the 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.

[0126] In some embodiments, the composition comprises a PIP2 antagonist. In some embodiments, the amount of the PIP2 antagonist in the composition is from 1% to 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.

[0127] In some embodiments, the composition comprises phospholipids. In some embodiments, the amount of phospholipids in the composition is from 1% to 100%. In some embodiments, the amount of phospholipids 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 amount of [PIP2(6:0 / 18:0)-H] in the composition is from 1% to 100%. In some embodiments, the amount of [PIP2(6:0 / 18:0)-H] in the composition is at least 1%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more.

[0128] Pharmaceutical Composition

[0129] The compounds, antibodies, and those encoding nucleic acids or sets of nucleic acids, carriers containing these, or host cells containing said carriers described herein can be mixed with pharmaceutically acceptable excipients to form pharmaceutical compositions for treating a target disease. "Acceptable" means that the carrier must be compatible with (and preferably able to stabilize) the active ingredient of the composition and harmless to the subject of treatment. Pharmaceutically acceptable excipients (carriers) include buffers known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0130] Pharmaceutical compositions used in this method may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and may contain buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexachlorocyclohexane quaternary ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residuals). (Based on) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG).

[0131] In some embodiments, the pharmaceutical compositions described herein comprise liposomes containing a compound or antibody (or encoding a nucleic acid), said liposomes being 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. Patent Nos. 4,485,045 and 4,544,545. U.S. Patent No. 5,013,556 discloses liposomes with extended cycle times. Particularly usable liposomes can be produced by a reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter defining a pore size to produce liposomes with a desired diameter.

[0132] Compounds, antibodies, or encoding nucleic acids can also be encapsulated in microcapsules prepared, for example, by coagulation techniques or by interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively; encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or encapsulated in macroemulsions. Such techniques are known in the art, for example, see Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0133] In other embodiments, the pharmaceutical compositions described herein may be formulated in a sustained-release form. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer comprising a compound or antibody, said matrix being in the form of a molded article, such as a membrane or microcapsule. Some examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid ester, non-degradable ethylene-vinyl acetate, and degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT. TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose isobutyrate acetate and poly-D-(-)-3-hydroxybutyric acid.

[0134] In other embodiments, the pharmaceutical compositions described herein may be formulated in a sustained-release form that influences selective binding to tissues or tumors by implementing certain protease biology techniques, such as peptide masking of antibody-antigen binding sites, to allow delivery within the tumor microenvironment via one or more proteases, such as Probody. TM or Conditionally Active Biologics TM Selective protease cleavage. In the normal microenvironment, activation can be described as reversible.

[0135] Pharmaceutical compositions intended for internal administration must be sterile. This can be easily accomplished, for example, by filtration through a sterile filter membrane. Therapeutic compounds or antibody compositions are typically placed in containers with sterile inlets, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle.

[0136] The pharmaceutical compositions described herein may be in unit dose form for oral, parenteral, or rectal administration, or for administration by inhalation or blowing, such as tablets, pills, capsules, powders, granules, solutions, or suspensions, or suppositories.

[0137] For the preparation of solid compositions (e.g., tablets), the main active ingredient can be mixed with a pharmaceutical carrier (e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gum) and other pharmaceutical diluents (e.g., water) to form a solid preformed composition comprising a homogeneous mixture of the compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When these preformed compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition, such that the composition can be easily further divided into equivalent unit dose forms, such as tablets, pills, and capsules. The solid preformed composition is then further divided into unit dose forms of the type described above, containing 0.1 to about 500 mg of the active ingredient of the present invention. The new composition can be coated or otherwise compounded into tablets or pills to provide a dose form that offers the advantage of prolonged action. For example, tablets or pills may comprise an inner dose component and an outer dose component, the latter being a coating over the former. These two components can be separated by an enteric coating layer, which resists disintegration in the stomach and allows the inner component to enter the duodenum intact or be released with a delay. A variety of materials can be used for such enteric coatings or coatings, including a variety of polymeric acids, as well as mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0138] Suitable surfactants specifically contain nonionic agents, such as polyoxyethylene sorbitol (e.g., Tween). TM 20, 40, 60, 80 or 85) and other anhydrous sorbitols (e.g., Span) TM 20, 40, 60, 80, or 85). Compositions containing surfactants will conveniently contain 0.05% to 5% surfactant, and may be 0.1% to 2.5%. It should be understood that other ingredients, such as mannitol or other pharmaceutically acceptable carriers, may be added if desired.

[0139] Suitable emulsions can be prepared using commercially available fat emulsions, such as Intralipid. TM Liposyn TM Infonutrol TM Lipofundin TM and Lipiphysan TMThe active ingredient may be dissolved in a premixed emulsion composition, or alternatively, dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and mixed with phospholipids (e.g., lecithin, soybean lecithin, or soybean lecithin) and water to form an emulsion. It should be understood that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, 5% to 20%. Fat emulsions may contain 0.1 to 1.0 μm, particularly 0.1 to 0.5 μm, of fat droplets, and have a pH of 5.5 to 8.0.

[0140] Emulsion compositions can be made by combining compounds or antibodies with Intralipids TM Those prepared by mixing with its components (soybean oil, lecithin, glycerin and water).

[0141] Pharmaceutical compositions for inhalation or inhalation include solutions and suspensions, as well as powders, that are pharmaceutically acceptable in aqueous or organic solvents or mixtures thereof. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the composition is administered via oral or nasal inhalation for local or systemic action.

[0142] Compositions in a preferably sterile pharmaceutically acceptable solvent can be nebulized using a gas. The nebulized solution can be inhaled directly from the nebulizer, or the nebulizer can be connected to a mask, tent, or intermittent positive pressure ventilator. The solution, suspension, or powder composition can be administered from a device that delivers the formulation in a suitable manner, preferably orally or nasally.

[0143] Therapeutic applications

[0144] Any compound or antibody described herein, as well as those encoding nucleic acids or sets of nucleic acids, vectors containing these, or host cells containing said vectors, may be used to treat cancer, inflammation, infectious diseases, or other malignancies that require stimulation of an immune response.

[0145] To implement 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, as a bolus, or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or surface route. Commercially available nebulizers (including jet nebulizers and ultrasonic nebulizers) for liquid formulations can be used for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, fluorocarbon formulations and metered-dose inhalers can be used to atomize the compounds or antibodies described herein, or inhale them as lyophilized and ground powders.

[0146] Subjects to be treated using the methods described herein may be patients who have, are at risk of having, or are suspected of having cancer or other malignancies requiring immune response stimulation. Subjects with the target disease or disorder may be identified through routine medical examinations, such as laboratory tests, organ function tests, CT scans, or ultrasound. Subjects suspected of having any such target disease / disorder may exhibit one or more symptoms of that disease / disorder. Subjects at risk of a disease / disorder may be those who have one or more risk factors for that disease / disorder.

[0147] The methods and compositions described herein can be used to treat cancer. Examples of cancers that can be treated with the methods and compositions described herein include, but are not limited to: lung cancer, melanoma, kidney cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, stomach cancer, pancreatic cancer, thyroid cancer, hematologic malignancies, lymphoma, leukemia, skin cancer, ovarian cancer, bladder cancer, urothelial carcinoma, head and neck cancer, metastatic lesions of cancer, and all types of cancer diagnosed with a high mutation burden. In one specific embodiment, the cancer has a high mutation burden. Subjects who have or are at risk of having multiple cancers can be identified through routine medical procedures.

[0148] In some cases, patients exhibit high microsatellite instability (MSI-H) or mismatch repair deficient (dMMR), which has been found in soft tissue cancers, glioblastomas, esophageal and EGJ cancers, breast cancers, non-small cell lung cancers, ovarian surface epithelial cancers, cancers of unknown primary origin, small cell lung cancers, non-epithelial ovarian cancers, pancreatic cancers, other malignancies of the female reproductive tract, uveal melanomas, retroperitoneal or peritoneal sarcomas, thyroid cancers, uterine sarcomas, bile duct cancers, prostate adenocarcinomas, hepatocellular carcinomas, neuroendocrine tumors, cervical cancers, colorectal adenocarcinomas, small bowel malignancies, gastric adenocarcinomas, and endometrial cancers.

[0149] As those skilled in the art will recognize, the effective dose varies depending on: the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, height, sex, and weight, duration of treatment, the nature of concurrent treatments (if any), the specific route of administration, and similar factors within the knowledge and expertise of the health practitioner. Empirical considerations, such as half-life, often aid in dose determination. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to prolong the antibody's half-life and prevent the antibody from being attacked by the host immune system. The frequency of administration can be determined and adjusted during the treatment process and is typically (but not necessarily) based on the treatment and / or suppression and / or improvement and / or delay of the target disease / disorder. Alternatively, a sustained-release formulation of the antibody may be suitable. Various formulations and devices for achieving sustained release are known in the art.

[0150] In one instance, the dosage of the compound or antibody described herein may be determined empirically in an individual who has already received one or more administrations of the compound or antibody. The individual is then given escalating doses of the compound. To assess the efficacy of the compound, indicators of the disease / disorder may be tracked.

[0151] Generally, for the administration of any compound or antibody described herein, the initial candidate dose may be about 2 mg / kg. For the purposes of this disclosure, typical daily, weekly, bi-weekly, or tri-weekly doses may be any of the following: 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, up to 30 mg / kg to 100 mg / kg, or higher, depending on the foregoing factors. For repeated administration over days, weeks, months, or longer, treatment continues, depending on the condition, until desired symptom suppression is achieved, or until an adequate level of treatment is reached to alleviate the target disease or disorder or its symptoms. Exemplary dosing regimens include an initial dose of about 3 mg / kg every 3 weeks, followed by a maintenance dose of about 1 mg / kg of the compound or antibody once every 6 weeks, or a maintenance dose of about 1 mg / kg every 3 weeks thereafter. However, other dosing regimens may be available depending on the pharmacokinetic decay pattern desired by the practitioner. For example, combination therapy with 1 mg / kg every 3 weeks in combination with at least one additional immunotherapeutic agent may be considered. In some embodiments, dosing may be used at approximately 3 µg / mg to approximately 3 mg / kg (e.g., approximately 3 µg / mg, approximately 10 µg / mg, approximately 30 µg / mg, approximately 100 µg / mg, approximately 300 µg / mg, approximately 1 mg / kg, and approximately 3 mg / kg). In some embodiments, dosing may be performed weekly, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or monthly, every 2 months, or every 3 months, or for longer periods. The progress of this treatment can be easily monitored using routine techniques and assays. Dosing regimens (including the compounds or antibodies used) may vary over time.

[0152] In some implementations, a dose of about 0.1 to 5.0 mg / kg may be administered to adult patients of normal weight. In some instances, the dose described herein may be 10 mg / kg. The specific dosing regimen, i.e., dose, time, and repetition, will depend on the individual and their medical history, as well as the characteristics of the individual drug (e.g., the half-life of the drug, and other considerations known in the art).

[0153] For the purposes of this disclosure, the appropriate dose of the compound or antibody described herein will depend on the specific compound or antibody, antibody and / or non-antibody peptide (or combination thereof) used, the type and severity of the disease / disorder, whether the compound or antibody is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to antagonists, and the attending physician's judgment. Typically, clinicians will administer the compound or antibody up to a dose that achieves the desired outcome. In some embodiments, the desired outcome is a reduction in tumor size, prolongation of progression-free survival, and / or improvement in overall survival. Methods for determining whether a dose produces the desired outcome will be apparent to those skilled in the art. Administration of one or more compounds or antibodies can be continuous or intermittent, depending on factors such as the recipient's physiological condition, whether the administration is therapeutic or prophylactic, and other factors known to a skilled practitioner. Administration of the compound or antibody can be substantially continuous over a preselected time period or can be performed at a series of intervals, such as before, during, or after the occurrence of the target disease or disorder.

[0154] As used herein, the term "treatment" means the application or administration of a composition comprising one or more active agents to a subject suffering from, having symptoms of, or being predisposed to a target disease or disorder, with the aim of curing, treating, alleviating, relieving, altering, remedying, improving, or influencing the disease, its symptoms, or the predisposition to the disease or disorder. Alleviating a target disease or disorder includes delaying the onset or progression of the disease, or reducing its severity. Compared to no treatment, treatment reduces the likelihood that the subject will develop the disease and, if so, to receive treatment to combat it after it has occurred, prevents the disease from worsening or slows its progression.

[0155] Disease mitigation does not necessarily require a curative outcome. As used in this article, “delaying” the onset of a target disease or disorder means postponing, hindering, slowing, halting, stabilizing, and / or delaying the progression of the disease. This delay can vary in duration, depending on the history of the disease and / or the individual being treated. Methods for “delaying” or mitigating the onset of disease or delaying the onset of disease are those that, when compared to not using the method, reduce the likelihood of one or more symptoms of the disease occurring within a given timeframe and / or reduce the severity of symptoms within a given timeframe. Such comparisons are typically based on clinical studies using a number of subjects sufficient to provide statistically significant results.

[0156] The term "occurrence" or "progression" of a disease refers to the initial presentation and / or subsequent progression of the disease. The occurrence of a disease can be detected and assessed using standard clinical techniques known in the art. However, occurrence also refers to progression that may be undetectable. For the purposes of this disclosure, occurrence or progression refers to the biological process of symptoms. "Occurrence" includes appearance, relapse, and onset. As used herein, "onset" or "occurrence" of a target disease or disorder includes initial onset and / or relapse.

[0157] 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 higher) in vivo. In other embodiments, the compounds or antibodies are administered in an amount that effectively reduces 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 higher).

[0158] Depending on the type or location of the disease to be treated, the pharmaceutical composition may be administered to the subject using conventional methods known to a person skilled in the medical field. The composition may also be administered via other conventional routes, such as parenteral, topical, oral, via inhalation spray, rectal, nasal, sublingual, vaginal, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intraperitoneal, intratumoral, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Additionally, the composition may be administered via an injectable reservoir, for example, using a 1-month, 3-month, or 6-month injectable reservoir or biodegradable materials and methods. In some instances, the pharmaceutical composition is administered intraocularly or intravitreal.

[0159] Injectable compositions may contain a variety of carriers, such as vegetable oils, dimethyl lactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble compounds or antibodies may be administered via infusion, which involves infusing a pharmaceutical formulation containing the compound or antibody and a physiologically acceptable excipient. Physiologically acceptable excipients may include, for example, 5% dextran, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular formulations, such as sterile formulations in the form of suitable soluble salts of compounds or antibodies, may be dissolved in pharmaceutical excipients such as water for injection, 0.9% saline, or 5% glucose solution and administered.

[0160] In one embodiment, the compound or antibody is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable reservoir sources or local delivery catheters for the compound or antibody, such as infusion catheters, indwelling catheters, or needle catheters; synthetic grafts; adventitial wraps, shunts, 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.

[0161] Targeted delivery can also be achieved using therapeutic compositions containing antisense polynucleotides, expression vectors, or subgenomic polynucleotides. Receptor-mediated DNA delivery techniques are described in, for example, 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.

[0162] Therapeutic compositions comprising polynucleotides (e.g., those encoding antibodies or other proteins described herein) are administered at doses of about 100 ng to about 200 mg of DNA for topical application in a gene therapy regimen. In some embodiments, concentrations 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 higher may also be used during a gene therapy regimen.

[0163] The therapeutic polynucleotides and peptides described herein can be delivered using gene delivery carriers. These carriers can be viral or non-viral in origin (see Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of the coding sequences can be constitutive or regulated.

[0164] Virus-based vectors for delivering desired polynucleotides and expressing them in desired cells are well known in the art. Exemplary virus-based vectors include, but are not limited to, recombinant retroviruses (see, for example, PCT Publications No. WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO91 / 02805; U.S. Patents Nos. 5,219,740 and 4,777,127; UK Patent No. 2,200,651; and European Patent No. 0 345 242); vectors based on the alphavirus genus (e.g., Sindbis virus vector, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus). equineencephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532) and adeno-associated virus (AAV) vectors (see, for example, PCT Publications Nos. WO 94 / 12649, WO93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655). Application of DNA linked to a killed adenovirus, as described in Curiel, Hum. Gene Ther. (1992) 3:147, is also possible.

[0165] Non-viral delivery carriers and methods may also be used, including, but not limited to, polycationic condensed DNA linked to or not linked to a single, killable adenovirus (see, for example, Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, for example, Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery carriers (see, for example, U.S. Patent No. 5,814,482; PCT Publications Nos. WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338); and nuclear charge neutralization or fusion with the cell membrane. Naked DNA may also be used. Exemplary methods for introducing naked DNA are described in PCT Publication No. WO 90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can be used as gene delivery carriers are described in: U.S. Patent No. 5,422,120; PCT Publication Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and European Patent No. 0524968. Other methods are described in Philip, Mol. Cell. Biol. (1994) 14:2411 and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0166] The specific dosing regimen used in the methods described in this article, namely the dose, time, and repetition, will depend on the specific subject and the subject's medical history.

[0167] In some implementations, more than one compound or antibody, or a combination of a compound or antibody with 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 to enhance and / or complement the effectiveness of the therapeutic agent.

[0168] The therapeutic efficacy of a target disease / disorder can be assessed using methods known in the art.

[0169] Treatments described in this disclosure, for example, may be used in combination with other types of treatments targeting the target disease or disorder disclosed herein. Examples include chemotherapy, immunotherapy (e.g., treatments involving therapeutic antibodies, CAR T-cells, or cancer vaccines), surgery, radiation, gene therapy, or anti-infective treatments. Such treatments may be administered concurrently with or sequentially (in any order) with treatments according to this disclosure. In some cases, the target disease is cancer (e.g., those disclosed herein) and the combination treatment includes immune checkpoint (e.g., checkpoint inhibitor) antagonists. Some 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. Other examples include activators that enhance the activity of stimulated checkpoints, such as CD122 (IL2) agonists, 4-1BB, ICOS ligands, GITR, and OX40.

[0170] For additional available agents, see Physician's Desk Reference, 59.sup.th edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al., Eds. Remington's TheScience and Practice of Pharmacy 20th edition, (2000), Lippincott Williamsand 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.

[0171] When used in combination with other therapeutic agents, the appropriate therapeutic dose of each agent may be reduced due to additive or synergistic effects.

[0172] The efficacy of the methods described herein can be assessed by any method known in the art and will be readily apparent to a skilled medical professional. For example, the efficacy of antibody-based immunotherapy can be assessed by the survival of the subject or the cancer burden in the subject or its tissues or samples. In some embodiments, the method is assessed based on the safety or toxicity of the treatment in the subject, for example by the subject's overall health and / or the presence of adverse or serious adverse events.

[0173] The application of this invention is not limited to the details of the construction and arrangement of the components set forth in the following description or illustrated in the accompanying drawings. The invention may be implemented in other ways and can be practiced or carried out in a variety of manners. Furthermore, the phrases and terms used herein are for descriptive purposes and should not be construed as limiting. The use of “comprising,” “including,” or “having,” “containing,” “involving,” and variations thereof herein is intended to cover the items listed below and their equivalents, as well as additional items.

[0174] Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless required by context, nouns without quantifiers will include one / more / agents. The methods and techniques of this disclosure are generally practiced according to conventional methods known in the art. Generally, the nomenclature and techniques described herein in relation to biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are those known and commonly used in the art. Unless otherwise indicated, the methods and techniques of this disclosure are generally practiced according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification.

[0175] The invention is further illustrated by the following examples, which should not be construed as further limitations. The entire contents of all references cited throughout this application (including bibliographic references, authorized patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference. Example

[0176] Example 1: ABCB5 promotes tumor invasion by regulating AXL.

[0177] Recent studies have shown that receptor tyrosine kinase AXL, associated with poor prognosis in colorectal cancer (CRC), is a cause of EMT induction in other malignancies. This article demonstrates that, compared to control transfected cells, AXL mRNA expression was reduced by >90% in COLO741 ABCB5 KD CRC cell cultures, and AXL protein expression was reduced by >50% in these cells. Figure 8 Furthermore, mAb-mediated ABCB5 blockade consistently inhibited AXL expression in all four CRC cell lines examined (COLO741, SW620, HT29, and HCT116, with ABCB5(+) tumor cell frequencies ranging from 9% to 27%), as determined by Western blot analysis. Figure 8 Furthermore, it also inhibits its downstream target phosphate-AKT to a lesser extent, demonstrating the important signaling pathway used by ABCB5+ cells. The functional relationship between ABCB5 and AXL is supported by the significantly upregulated expression of AXL at both the mRNA and protein levels in untreated ABCB5(+) cells sorted from all four cell lines by flow cytometry. Figure 8 ).

[0178] Furthermore, compared to parental COLO741 cells, AXL expression (as determined at the mRNA and protein levels) and downstream signaling (p-AKT / AKT ratio) were enhanced in metastasis-derived COLO741 MET cells. Figure 8 ).

[0179] Example 2: PIP2 and PIP3, as determined by immunoprecipitation from human tissue, are natural in vivo binding ligands of ABCB5.

[0180] Immunoprecipitation from human ABCB5-expressing melanoma cells treated with anti-PIP2 antibody revealed co-precipitation of ABCB5 protein, such as... Figure 1A The immunoblotting using the ABCB5 monoclonal antibody is shown in the image above. Figure 1A Immunoblotting of the PIP2 antibody confirmed the presence of PIP2 pull-down (see figure below). Similarly, immunoprecipitation of human ABCB5-expressing melanoma cells treated with anti-PIP3 antibody revealed co-precipitation of ABCB5 protein, such as... Figure 1B The results are shown in the immunoblotting using the ABCB5 monoclonal antibody. These data indicate that PIP2 and PIP3 are biological ligands of ABCB5, and that ABCB5 acts as a receptor for PIP2 and PIP3.

[0181] Example 3: PIP1, PIP2 and PIP3, as determined by ELISA, are combined with ABCB5.

[0182] ELISA plates coated with PIP1, PIP2, or PIP3 were incubated with purified recombinant human ABCB5 isotype 2 (812 aa, NCBI reference sequence: NP_848654.3), wild-type mouse Abcb5-expressing skin tissue, or Abcb5-non-expressing skin tissue from ABCB5 knockout mice (as a specific control). ABCB5 binding was then detected using a mouse ABCB5-specific monoclonal antibody (Ksander et al Nature 2014). Figure 1C As shown, all PIP1, PIP2, and PIP3 bound to recombinant human ABCB5 and mouse Abcb5, with no binding detection observed in Abcb5 knockout mouse tissues. Therefore, PIP2 and PIP3 bound to ABCB5 more efficiently than PIP1. These data confirm the binding of ABCB5 to PIP2 and PIP3 and also reveal the ability of ABCB5 to bind to PIP1, albeit with a significantly lower affinity. Similar results were obtained using mouse ABCB5 mAb clone 3C2-1D12 (Frank NY et al. J Biol Chem. 2003) as the detection antibody.

[0183] Example 4: The binding of PIP1, PIP2 and PIP3 to ABCB5 can be inhibited by competitive pharmacological ligands.

[0184] 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 with an increased dose of PtdIns-(1,2-dioctanoyl), a synthetic analog of natural phosphatidylinositol (PTDIns), containing C8:0 fatty acids at sn-1 and sn-2 positions (CAS Registry No. 899827-36-2). Figure 2 The data illustrated in the example show that the molecule can competitively inhibit the binding of PIP1, PIP2, or PIP3 to ABCB5, exhibiting a significant saturation effect at concentrations as low as 0.1 mM.

[0185] These results provide evidence that PIP analogs (including PIP2 analogs and chemical PIP2 variants that cannot be phosphorylated to biologically active PIP3), or other synthetic chemical or biological agents that compete with PIP1, PIP2, or PIP3 binding to ABCB5 but are different from PIP1, PIP2, or PIP3 that do not play a role in signal transduction of different receptor tyrosine kinases (listed in the table below) or multiple G protein-coupled receptors, can be used as therapeutic agents to modulate ABCB5 / PIP1, ABCB5 / PIP2, or ABCB5 / PIP3 receptor / ligand interactions that are associated with PIP-dependent signal transduction mechanisms of such receptors in multiple disease states, where ABCB5 is functional, particularly but not limited to the initiation and progression of human cancer.

[0186]

[0187]

[0188]

[0189]

[0190] Example 5: The binding of PIP1, PIP2 and PIP3 to ABCB5 can be inhibited by the ABCB5 monoclonal antibody.

[0191] Surface plasmon resonance (SPR) analysis also confirmed the binding of PIP1, PIP2, and PIP3 to ABCB5. Competition with the anti-ABCB5 monoclonal antibody resulted in a concentration-dependent reduction in signal on all three surfaces (PIP3 > PIP2 > PIP1), with a reduction of up to 50% on the PIP3 surface (see [link to original text]). Figure 3 The isotype control monoclonal antibody did not show significant effect (no examples provided). These results indicate that the ABCB5 monoclonal antibody can block the binding of PIP1, PIP2, or PIP3 to ABCB5.

[0192] Example 6: ABCB5 is required for a more efficient conversion of PIP2 to PIP3.

[0193] Functional experiments involving ABCB5 blockade using ABCB5 monoclonal antibodies in human melanoma cells, or ABCB5 functional ablation in tissues derived from ABCB5 knockout mice, revealed that ABCB5 is functionally required for more efficient conversion of PIP2 to PIP3, possibly through its function as a PIP2 docking receptor. Figure 4As illustrated in the example, the ABCB5 monoclonal antibody, rather than the allotype control antibody, significantly reduced the PIP3 / PIP2 ratio in human melanoma cells (left). Furthermore, examination of mouse ABCB5 knockout skin tissue also revealed a significantly reduced PIP3 / PIP2 ratio compared to ABCB5 wild-type skin (right).

[0194] Similarly, SPR analysis results indicate that the ABCB5 monoclonal antibody blocks the ABCB5 / PIP2 receptor / ligand interaction. These results suggest that the ABCB5 / PIP2 binding interaction is functionally required for more efficient phosphorylation of PIP2 to PIP3, implying that ABCB5 plays a crucial role in PIP2-dependent signaling transduction involving receptor tyrosine kinase and G protein-coupled receptor signaling in ABCB5-expressing cells, including cancer stem cells involved in tumorigenesis, cancer progression, and treatment resistance in various malignancies, including melanoma, colorectal cancer, glioblastoma multiforme, Merkel cell carcinoma, SCC, and hepatocellular carcinoma. Furthermore, physiological tissue-specific stem cells, including skin, eye, and intestinal stem cells, express ABCB5 at high levels and rely on receptor tyrosine kinase and G protein-coupled receptor signaling to perform their tissue regeneration functions. Therefore, the current findings provide a means to block ABCB5-dependent PIP2 binding and phosphorylation to PIP3 in receptor tyrosine kinase signaling, or to block processing into IP3 and DAG in G protein-coupled receptor signaling, via ABCB5 monoclonal antibodies or small molecule / chemical inhibitors of ABCB5 / PIP2 binding. This results in inhibition of receptor tyrosine kinase signaling associated with cancer initiation / progression / treatment resistance (e.g., AXL (see Guo et al. J Biol Chem. 2018), EGFR), or inhibition of G protein-coupled receptor signaling-dependent mechanisms of cancer initiation / progression / treatment resistance. Furthermore, enhancing the ABCB5 / PIP2 binding interaction, for example through increased ABCB5 expression / binding or through exogenous PIP2 addition, will enhance the function of biological stem cells to treat stem cell deficiency-related disorders.

[0195] Example 7: ABCB5 is required to maintain the PIP2 / PIP3-dependent PI3K / AKT signaling axis in malignant tumors, and ABCB5 inhibition leads to inhibition of the PI3K / AKT signaling axis as well as tumor growth-dependent and treatment-resistant tumors.

[0196] Analysis of 4-HT treatment-induced Tyr::CreER;BrafCA;Ptenlox / lox genetically modified mouse melanoma models in the ABCB5 WT or ABCB5 KO background revealed significant inhibition of the PI3K / AKT signaling axis in ABCB5 KO compared to ABCB5 WT tumors, with attenuated expression of p-AKT, p-mTOR, and p-S6 among other dysregulated molecules (see [link to relevant documentation]). Figure 5 ).

[0197] Furthermore, in this model, the ABCB5 KO state resulted in reduced tumor cell proliferation compared to the ABCB5 WT state, as determined by identifying the percentage of tumor cells positive for the proliferation marker Ki-67 (see [link to model]). Figure 6 ):

[0198] In addition, in this model, the ABCB5 KO state led to a downregulation of pro-angiogenic molecules compared to the ABCB5 WT state (see Figure 7, left panel), and as a result, reduced CD31-positive microvessel density (see Figure 7, right panel).

[0199] It was also shown that specific ABCB5 monoclonal antibodies disrupt the PIP2 / PIP3-dependent PI3K / pAKT signaling axis in colorectal cancer (see Guo et al. J Biol. Chem 2018), where treatment resulted in inhibition of the pAKT / AKT ratio. Similar results were obtained when examining the effects of ABCB5 monoclonal antibodies on human melanoma cells, where treatment with ABCB5 antibodies that also inhibit PIP2 / PIP3 binding to ABCB5 and / or PIP2-to-PIP3 conversion resulted in significant inhibition of the pAKT / AKT ratio. These data demonstrate that functional blockade of ABCB5 disrupts the PIP2 / PIP3-dependent PI3K / pAKT signaling axis, which is crucial for tumor growth and angiogenesis, providing clear evidence for the anticancer therapeutic efficacy of functional blockade of ABCB5 / PIP2 / PIP3 receptor / ligand interactions.

[0200] Furthermore, in ABCB5 WT or ABCB5 KO mouse models of melanoma induced by 4-HT treatment in Tyr::Creer;BrafCA;Ptenlox / lox genetic mice, the ABCB5 KO state resulted in complete sensitivity to the BRAF inhibitor vemurafenib compared to the ABCB5 WT state, with vemurafenib resistance partially driven by the functional PI3K / pAKT signaling axis. In contrast to ABCB5 WT mice exhibiting 100% vemurafenib-resistant tumor formation, no tumor formation was observed after genetic induction in vemurafenib-treated ABCB5 KO mice (see [link to ABCB5 WT mice]). Figure 9(left figure), and survival was significantly prolonged in ABCB5 KO mice compared to ABCB5 WT mice (see left figure). Figure 9 (See right figure). These results reveal the crucial role of ABCB5 in tumor vemurafenib resistance through its function in the intact PIP2 / PIP3-dependent PI3K / pAKT signaling axis required to maintain vemurafenib resistance. These results also suggest that functional inhibition of ABCB5 could be therapeutically used to reverse BRAF inhibitor resistance in melanoma.

[0201] Example 8: Identification of novel PIP2 structures accumulated in ABCB5 knockout tissues. Preferred physiological substrates for ABCB5-dependent phosphorylation to its PIP3 form were identified.

[0202] Quantitative lipid mass spectrometry analysis of skin tissue 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, total fatty acid chain 24:0, unsaturated 0, molecular formula C33H65O19P3) specifically present in ABCB5 knockout tissues, but not detected at the detection threshold in ABCB5 wild-type skin (ABCB5 KO mean: 7.79E+04 + / - 1.67E+04; WT mean: not detected). This suggests 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, namely the bioactively phosphorylated PIP3 form involved in ABCB5-dependent receptor tyrosine kinase or G protein-coupled receptor signal transduction. An example illustrates the structural model generated by bioinformatics for this novel PIP2(6:0 / 18:0)-H molecule (total fatty acid chain is 24:0, unsaturated 0, molecular formula C33H65O19P3).

[0203] This novel PIP2(6:0 / 18:0)-H molecule, or its phosphorylated form PIP3(6:0 / 18:0)-H (molecular formula C33H65O19P4), represents a composition that can be used as a therapeutic agent to enhance signal transduction via the various receptor tyrosine kinases or G protein-coupled receptors listed above in those disease conditions in which ABCB5 signal transduction is impaired or in which ABCB5 function or ABCB5 expression levels are reduced, particularly in diseases associated with ABCB5+ stem cell defects, such as skin wound healing defects, limbal stem cell defects, tissue regeneration defects in aging, and other ABCB5 deficiency disorders, such as psoriasis, which has been found to exacerbate psoriasis in ABCB5 knockout imiquimod-induced psoriasis mouse models compared to ABCB5 wild-type mice (see [link to relevant documentation]). Figure 10 ).

[0204] Example 9: Functional profiling of ABCB5 single nucleotide polymorphisms reveals molecular ABCB5 ligand / substrate binding sites involved in downstream molecular effector functions.

[0205] ABCB5 isotype 1 (1257 aa, NCBI reference sequence: NP_001157413.1) consists of two transmembrane domains (TMDs), each with six transmembrane (TM) helices, for a total of 12 transmembrane helices (TM 1-12). ABCB5 isotype 2 (812 aa, NCBI reference sequence: NP_848654.3) consists of a single TMD with six transmembrane (TM) helices (TM 1-6). TM 1-6 of ABCB5 isotype 2 corresponds to TM 7-12 of ABCB5 isotype 1. TM 12 of ABCB5 isotype 1 corresponds to TM 6 of ABCB5 isotype 2. A non-synonymous single nucleotide polymorphism (SNP) in the ABCB5 coding region (rs6461515) providing AA 970 E>K in TM12 of ABCB5 isotype 1 and corresponding to AA 525 E>K in TM6 of ABCB5 isotype 2, is revealed to be crucial for ABCB5 function in this study. The annotated reference E SNP (glutamate / E / GAA) is therefore conserved across multiple species, including the house mouse (mus musculus). However, the reference E SNP (glutamate / E / GAA) actually represents a minor codon in Homo sapiens. Population diversity data show that the E525-encoded allele (glutamate, G genotype, codon GAA) occurs at the highest frequency in African populations, where G / G homozygosity is rare, in contrast to the K525-encoded allele (lysine, A genotype, codon AAA) (data not shown).

[0206] Based on analysis of the most frequently expressed K SNP (lysine, codon AAA) in human cancers, and importantly, in wild-type K525 / K525 human melanoma cells expressing only ABCB5 isotype 2 at baseline, experimental induction of a molecular transition from K (lysine, codon AAA) to E (glutamate, codon GAA) in one allele via CRISPR / Cas9-mediated gene editing yielded a clonal heterozygous ABCB5 K525 / E525 melanoma cell variant with impaired ABCB5 signaling function, and resulted in significant inhibition of ABCB5-driven tumor growth (P < 0.05) (see [link to article]). Figure 11 ).

[0207] These results suggest that amino acid residue 525 of ABCB5 isotype 2 TM6 acts as an important molecular switch in the physiological ligand / substrate binding quality of ABCB5, particularly PIP2 and its phosphorylated product PIP3, which is known to transmit extracellular RTK-mediated signals to activate the downstream PI3K / pAKT signaling pathway essential for tumorigenesis and progression. K525 (rs6461515) represents a more functional variant. Correspondingly, residue 970 of ABCB5 isotype 1 TM12 is also associated with physiological ligand / substrate binding of ABCB5. The peptide sequences and their encoding RNA sequences of the two variants of ABCB5 isotype 2 (variant residues are highlighted in red) are listed below, where the molecule containing K525 is a more functional variant in terms of PIP substrate / ligand binding and downstream signaling compared to the reference molecule containing E525 (SNPrs6461515).

[0208] ABCB5 isotype 2-E525 protein sequence (SEQ ID NO: 1):

[0209]

[0210]

[0211] The RNA sequence encoding ABCB5 isotype 2-E525 (SEQ ID NO: 2):

[0212]

[0213]

[0214]

[0215] ABCB5 isotype 2-K525 protein sequence (SEQ ID NO: 3):

[0216]

[0217]

[0218] The ABCB5 isotype 2-K525 encoding RNA sequence (SEQ ID NO: 4):

[0219]

[0220]

[0221]

[0222] Based on bioinformatics analysis, the other residues involved in ABCB5 substrate binding are N702 and H706 in TM7 of ABCB5 isotype 1, corresponding to N257 and H261 in TM1 of ABCB5 isotype 2, and 857 A>T (rs80123476) in TM10 of ABCB5 isotype 1, corresponding to 412 A>T (rs80123476) in TM4 of ABCB5 isotype 2.

[0223] Furthermore, results to date have identified a subset (but not all) of ABCB5-specific monoclonal antibodies (antibodies that bind to the extracellular loop in a 3-dimensional (i.e., circular) form) manner that can inhibit the binding of PIP1, PIP2, or PIP3 to ABCB5, and thus inhibit ABCB5-mediated PIP-dependent signaling and pAKT phosphorylation. Therefore, ABCB5 monoclonal antibodies demonstrating inhibition of ABCB5-dependent PIP1, PIP2, or PIP3 binding and PIP-dependent signaling and pAKT phosphorylation as demonstrated herein constitute uniquely usable novel compositions, for example, that therapeutically inhibit ABCB5-driven human cancer growth and progression through functional ABCB5 blockade and result in inhibition of ABCB5-dependent receptor tyrosine kinase and G protein-coupled receptor signaling.

[0224] Example 10: Molecular docking modeling.

[0225] Using bioinformatics methods and structural data applicable to ABCB5 homologous ABCB1 proteins, model 3D structures were created for both the ABCB5 isotype 2-K525 and ABCB5 isotype 2-E525 polypeptide sequences listed above. Additionally, 3D structures were created for the following ABCB5 ligands or competitive inhibitors to facilitate molecular docking / binding modeling using the PyMOL software package:

[0226] (a) PIP2(6:0 / 18:0)-H, total fatty acid chain ratio 24:0, unsaturated 0, molecular formula C33H65O19P3:

[0227] This is the first time a novel PIP2 variant accumulated in the skin of ABCB5 KO mice has been identified by mass spectrometry, suggesting that this new molecule may be a physiological substrate for ABCB5-dependent phosphorylation to PIP3, as described above. This molecule was previously unknown in compound databases (model structure shown above).

[0228] (b) PI(4,5)P2, diC8, molecular formula C25H49O19P3: This PIP2 variant is shown to bind to ABCB5 via SPR. It was obtained from Echelon Biosciences. Further information about this molecule and its structure can be found in CAS registry number (204858-53-7).

[0229] (c) Phosphatidylinositol C-8:PtdIns-(1,2-dioctanoyl) is a synthetic analog of natural phosphatidylinositol (PtdIns) containing C8:0 fatty acids at sn-1 and sn-2 positions. As described above, this molecule competitively inhibits the binding of PIP2 to ABCB5. More information about this molecule can be found in CAS Registry No. 899827-36-2.

[0230] The results revealed that all tested molecules (i.e., the natural ligands PIP2(6:0 / 18:0)-H, PI(4,5)P2, and diC8) and the competitive inhibitor phosphatidylinositol C-8 bind tightly to either the ABCB5 isoform 2-K525 or the ABCB5 isoform 2-E525 structure, closely approaching the identified AA525 substrate binding site and the TM6 of the ABCB5 molecule. Furthermore, modeling results revealed that, unlike ABCB5 isoform 2-E525, ABCB5 isoform 2-K525 exhibits a higher affinity for PIP2. These data further support experimental evidence that non-synonymous single nucleotide polymorphisms (SNPs) in the ABCB5 coding region (rs6461515) determine the critical role of AA 525 E relative to the K residue in TM6 of ABCB5 isotype 2, which in this paper reveals that it is important for ABCB5 function, where ABCB5 isotype 2-K525 is a more functional ABCB5 variant with enhanced PIP2 / PIP3 binding ability, and thus improved signal transduction ability.

[0231] All references cited herein are incorporated herein by reference in their entirety. Therefore, several aspects of at least one embodiment of the invention have been described, and it should be understood that various changes, modifications, and improvements will readily occur to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to fall within the spirit and scope of the invention. Therefore, the foregoing description and figures are by way of example only. sequence list <110> Children's Medical Center Company <120> ABCB5 ligands and substrates <130> C0875.70074WO00 <140> Not yet allocated <141> at the same time <150> US 62 / 662,670 <151> April 25, 2018 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 812 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 1 Met Val Asp Glu Asn Asp Ile Arg Ala Leu Asn Val Arg His Tyr Arg 1 5 10 15 Asp His Ile Gly Val Val Ser Gln Glu Pro Val Leu Phe Gly Thr Thr 20 25 30<00006?8>Ile Ser Asn Asn Ile Lys Tyr Gly Arg Asp Asp Val Thr Asp Glu Glu 35 40 45 Met Glu Arg Ala Ala Arg Glu Ala Asn Ala Tyr Asp Phe Ile Met Glu 50 55 60 Phe Pro Asn Lys Phe Asn Thr Leu Val Gly Glu Lys Gly Ala Gln Met 65 70 75 80 Ser Gly Gly Gln Lys Gln Arg Ile Ala Ile Ala Arg Ala Leu Val Arg 85 90 95 Asn Pro Lys Ile Leu Ile Leu Asp Glu Ala Thr Ser Ala Leu Asp Ser 100 105 ;110 It should be noted that there is a possible error in the original text where is likely incomplete. This has been reflected in the translation as <00006?8. Also, there seems to be a semicolon in the original at "100 105 ;110" which has been left as-is in the translation for accuracy.Glu Ser Lys Ser Ala Val Gln Ala Ala Leu Glu Lys Ala Ser Lys Gly 115 120 125 Arg Thr Thr Ile Val Val Ala His Arg Leu Ser Thr Ile Arg Ser Ala 130 135 140 Asp Leu Ile Val Thr Leu Lys Asp Gly Met Leu Ala Glu Lys Gly Ala 145 150 155 160 His Ala Glu Leu Met Ala Lys Arg Gly Leu Tyr Tyr Ser Leu Val Met 165 170 175 Ser Gln Asp Ile Lys Lys Ala Asp Glu Gln Met Glu Ser Met Thr Tyr 180 185 190 Ser Thr Glu Arg Lys Thr Asn Ser Leu Pro Leu His Ser Val Lys Ser 195 200 205 Ile Lys Ser Asp Phe Ile Asp Lys Ala Glu Glu Ser Thr Gln Ser Lys 210 215 220 Glu Ile Ser Leu Pro Glu Val Ser Leu Leu Lys Ile Leu Lys Leu Asn 225 230 235 240 Lys Pro Glu Trp Pro Phe Val Val Leu Gly Thr Leu Ala Ser Val Leu 245 250 255 Asn Gly Thr Val His Pro Val Phe Ser Ile Ile Phe Ala Lys Ile Ile 260 265 270 Thr Met Phe Gly Asn Asn Asp Lys Thr Thr Leu Lys His Asp Ala Glu 275 280 285 Ile Tyr Ser Met Ile Phe Val Ile Leu Gly Val Ile Cys Phe Val Ser 290 295 300 Tyr Phe Met Gln Gly Leu Phe Tyr Gly Arg Ala Gly Glu Ile Leu Thr 305 310 315 320 Met Arg Leu Arg His Leu Ala Phe Lys Ala Met Leu Tyr Gln Asp Ile 325 330 335 Ala Trp Phe Asp Glu Lys Glu Asn Ser Thr Gly Gly Leu Thr Thr Ile 340 345 350 Leu Ala Ile Asp Ile Ala Gln Ile Gln Gly Ala Thr Gly Ser Arg Ile 355 360 365 Gly Val Leu Thr Gln Asn Ala Thr Asn Met Gly Leu Ser Val Ile Ile 370 375 380 Ser Phe Ile Tyr Gly Trp Glu Met Thr Phe Leu Ile Leu Ser Ile Ala 385 390 395 400 Pro Val Leu Ala Val Thr Gly Met Ile Glu Thr Ala Ala Met Thr Gly 405 410 415 Phe Ala Asn Lys Asp Lys Gln Glu Leu Lys His Ala Gly Lys Ile Ala 420 425 430 Thr Glu Ala Leu Glu Asn Ile Arg Thr Ile Val Ser Leu Thr Arg Glu 435 440 445 Lys Ala Phe Glu Gln Met Tyr Glu Glu Met Leu Gln Thr Gln His Arg 450 455 460 Asn Thr Ser Lys Lys Ala Gln Ile Ile Gly Ser Cys Tyr Ala Phe Ser 465 470 475 480 His Ala Phe Ile Tyr Phe Ala Tyr Ala Ala Gly Phe Arg Phe Gly Ala 485 490 495 Tyr Leu Ile Gln Ala Gly Arg Met Thr Pro Glu Gly Met Phe Ile Val 500 505 510 Phe Thr Ala Ile Ala Tyr Gly Ala Met Ala Ile Gly Glu Thr Leu Val 515 520 525 Leu Ala Pro Glu Tyr Ser Lys Ala Lys Ser Gly Ala Ala His Leu Phe 530 535 540 Ala Leu Leu Glu Lys Lys Pro Asn Ile Asp Ser Arg Ser Gln Glu Gly 545 550 555 560 Lys Lys Pro Asp Thr Cys Glu Gly Asn Leu Glu Phe Arg Glu Val Ser 565 570 575 Phe Phe Tyr Pro Cys Arg Pro Asp Val Phe Ile Leu Arg Gly Leu Ser 580 585 590 Leu Ser Ile Glu Arg Gly Lys Thr Val Ala Phe Val Gly Ser Ser Gly 595 600 605 Cys Gly Lys Ser Thr Ser Val Gln Leu Leu Gln Arg Leu Tyr Asp Pro 610 615 620 Val Gln Gly Gln Val Leu Phe Asp Gly Val Asp Ala Lys Glu Leu Asn 625 630 635 640 Val Gln Trp Leu Arg Ser Gln Ile Ala Ile Val Pro Gln Glu Pro Val 645 650 655 Leu Phe Asn Cys Ser Ile Ala Glu Asn Ile Ala Tyr Gly Asp Asn Ser 660 665 670 Arg Val Val Pro Leu Asp Glu Ile Lys Glu Ala Ala Asn Ala Ala Asn 675 680 685 Ile His Ser Phe Ile Glu Gly Leu Pro Glu Lys Tyr Asn Thr Gln Val 690 695 700 Gly Leu Lys Gly Ala Gln Leu Ser Gly Gly Gln Lys Gln Arg Leu Ala 705 710 715 720 Ile Ala Arg Ala Leu Leu Gln Lys Pro Lys Ile Leu Leu Leu Asp Glu 725 730 735 Ala Thr Ser Ala Leu Asp Asn Asp Ser Glu Lys Val Val Gln His Ala 740 745 750 Leu Asp Lys Ala Arg Thr Gly Arg Thr Cys Leu Val Val Thr His Arg 755 760 765 Leu Ser Ala Ile Gln Asn Ala Asp Leu Ile Val Val Leu His Asn Gly 770 775 780 Lys Ile Lys Glu Gln Gly Thr His Gln Glu Leu Leu Arg Asn Arg Asp 785 790 795 800 Ile Tyr Phe Lys Leu Val Asn Ala Gln Ser Val Gln 805 810 <210> 2 <211> 4375 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 2 attgcttctc ggccttttgg ctaagatcaa gtgtaatctg tgttcttttt tatttggtca 60 tatcttccat tctttcttac ctaattcctc taatatctct ctgtgagcct aaaccaataa 120 ttatatatta cattctattg tctttcttat ataactgcag aaagataaat atcactttgt 180 ttgttcctgt aggttttctt tagtgtaatc catagcagtt attgcattgg agcagcagtc 240 cctcactttg aaaccttcgc aatagcccga ggagctgcct ttcatatttt ccaggttatt 300 gataagaaac ccagtataga taactttcc acagctggat ataaacctga atccatagaa 360 ggaactgtgg aatttaaaaa tgtttctttc aattatccat caagaccatc tatcaagatt 420 ctgaaaggtc tgaatctcag aattaagtct ggagagacag tcgccttggt cggtctcaat 480 ggcagtggga agagtacggt agtccagctt ctgcagaggt tatatgatcc ggatgatggc 540 tttatcatgg tggatgagaa tgacatcaga gctttaaatg tgcggcatta tcgagaccat 600 attggagtgg ttagtcaaga gcctgttttg ttcgggacca ccatcagtaa caatatcaag 660 tatggacgag atgatgtgac tgatgaagag atggagagag cagcaaggga agcaaatgcg 720 tatgatttta tcatggagtt tcctaataaa tttaatacat tggtagggga aaaaggagct 780 caaatgagtg gagggcagaa acagaggatc gcaattgctc gtgccttagt tcgaaacccc 840 aagattctga ttttagatga ggctacgtct gccctggatt cagaaagcaa gtcagctgtt 900 caagctgcac tggagaaggc gagcaaaggt cggactacaa tcgtggtagc acaccgactt 960 tctactattc gaagtgcaga tttgattgtg accctaaagg atggaatgct ggcggagaaa 1020 ggagcacatg ctgaactaat ggcaaaacga ggtctatatt attcacttgt gatgtcacag 1080 gatattaaaa aagctgatga acagatggag tcaatgacat attctactga aagaaagacc 1140 aactcacttc ctctgcactc tgtgaagagc atcaagtcag acttcattga caaggctgag 1200 gaatccaccc aatctaaaga gataagtctt cctgaagtct ctctattaaa aattttaaag 1260 ttaaacaagc ctgaatggcc ttttgtggtt ctggggacat tggcttctgt tctaaatgga 1320 actgttcatc cagtattttc catcatcttt gcaaaaatta taaccatgtt tggaaataat 1380 gataaaacca cattaaagca tgatgcagaa atttattcca tgatattcgt cattttgggt 1440 gttattgct ttgtcagtta tttcatgcag ggattatttt acggcagagc aggggaaatt 1500 ttaacgatga gattaagaca cttggccttc aaagccatgt tatatcagga tattgcctgg 1560 ttgatgaaa aggaaaacag cacaggaggc ttgacaacaa tattagccat agatatagca 1620 caaattcaag gagcaacagg ttccaggatt ggcgtcttaa cacaaaatgc aactaacatg 1680 ggactttcag ttatcatttc ctttatatat ggatgggaga tgacattcct gattctgagt 1740 attgctccag tacttgccgt gacaggaatg attgaaaccg cagcaatgac tggatttgcc 1800 aacaaagata agcaagaact taagcatgct ggaaagatag caactgaagc tttggagaat 1860 atacgtacta tagtgtcatt aacaagggaa aaagccttcg agcaaatgta tgaagagatg 1920 cttcagactc aacacagaaa tacctcgaag aaagcacaga ttattggaag ctgttatgca 1980 ttcagccatg cctttatata ttttgcctat gcggcagggt ttcgatttgg agcctattta 2040 attcaagctg gacgaatgac cccagagggc atgttcatag tttttactgc aattgcatat 2100 ggagctatgg ccatcggaga aacgctcgtt ttggctcctg aatattccaa agccaaatcg 2160 ggggctgcgc atctgtttgc cttgttggaa aagaaaccaa atatagacag ccgcagtcaa 2220 gaagggaaaa agccagacac atgtgaaggg aatttagagt ttcgagaagt ctctttcttc 2280 tatccatgtc gcccagatgt tttcatcctc cgtggcttat ccctcagtat tgagcgagga 2340 aagacagtag catttgtggg gagcagcggc tgtgggaaaa gcacttctgt tcaacttctg 2400 cagagacttt atgaccccgt gcaaggacaa gtgctgtttg atggtgtgga tgcaaaagaa 2460 ttgaatgtac agtggctccg ttcccaaata gcaatcgttc ctcaagagcc tgtgctcttc 2520 aactgcagca ttgctgagaa catcgcctat ggtgacaaca gccgtgtggt gccattagat 2580 gagatcaaag aagccgcaaa tgcagcaaat atccattctt ttattgaagg tctccctgag 2640 aaatacaaca cacaagttgg actgaaagga gcacagcttt ctggcggcca gaaacaaaga 2700 ctagctattg caagggctct tctccaaaaa cccaaaattt tattgttgga tgaggccact 2760 tcagccctcg ataatgacag tgagaaggtg gttcagcatg cccttgataa agccaggacg 2820 ggaaggacat gcctagtggt cactcacagg ctctctgcaa ttcagaacgc agatttgata 2880 gtggttctgc acaatggaaa gataaaggaa caaggaactc atcaagagct cctgagaaat 2940 cgagacatat atttaagtt agtgaatgca cagtcagtgc agtgatgctg ttgaggtagc 3000 acatattttg atgttcgtgt aatgcaaaga aggagtactt aataattact tggcaagctt 3060 tgatctcttt tattgcatat atcaatacct agaatcatgc tactcaagta catacatgtt 3120 ctattcacac accatctgac cttcagattt ttaaaaggaa gcaaaaattt gcttatttca 3180 tgtaagtgaa ataatgctta tatccttcac tttataaaac tattctagca catttgcttg 3240 taaagcagtt ttctacaagg tgaatttatt tcccatcaac ttctgctata aaatcggaaa 3300 tatgtttcca gggggaatat tatccaatta accatgttga aggttttagc aaaggcagtg 3360 taagatagag tggggcctgt agcattgcag ggagagtgtc tttcacttgg aattttgttt 3420 tgcagcacat attacagtag ttttgctagt cccttttctc cagaccgtag ggatttctct 3480 caataagtat tcactatttc tctaaatttt attctatttt tttgttgagc agggaataga 3540 aaggattacg atgtaaaatt tctgggagga ttaggtagct atctcctact tcaccagtaa 3600 gtgaagtgcc tcacatgagc catcccaaag attcattatt ccaaaccttg ggtttggcag 3660 tataagtcac aggcctacct gtttatgaaa acttacttac ttaaaataag agctactttt 3720 gggccgggtg cggtggctca cgcctgtaat cccagaactt tgggaggccg aggagggcgg 3780 atcacttgag gtcaggagtt cgagaccagc ctggccaaca tggtgaaacc ccgtctctac 3840 taaaaacaca aaaattagcc aatcttggtg gcgggcacct ggaatcccag ctacttggga 3900 ggctgaggca ggagaatcat ttgaacctag gaggcagagg ttgcagtgag ccgagatctc 3960 accactgcac tccagcctgc gcaacagagc gagactccat ctcaaaaaat aataaataag 4020 agctaatttt attgtgggtg aaaattttta aacgtctttc tctataataa aataatttcc 4080 ttaaatttta tatatacttt atcatatata atgtgtgaat gattttaaag ttctgtgtaa 4140 ataacaatat tggtaaaatg agttacattt tcaacttact taaatatgta atgtcacctg 4200 gtgattttat ctttattctt cagtgtattt tcttccattt acacatttag ctagcctccc 4260 taaagtgtac tctaccaata attgaaatct tgttaaacaa aattaaaacc atttatatat 4320 tatgctgctt tctttaaaat gcaaaataaa aataagattg gggacttgag aatca 4375 <210> 3[[ID=!19]]** **<211> 812 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 3 Met Val Asp Glu Asn Asp Ile Arg Ala Leu Asn Val Arg His Tyr Arg 1 5 10 15 <0000!865> Note: There seems to be an issue with the tag in the original text as it's not clear if it should be included as is or if there's a typo. I've preserved it as it is for translation but flagged it for your reference. Also, the tag in the original has been marked as it might be a typo (extra exclamation mark added for clarity).Asp His Ile Gly Val Val Ser Gln Glu Pro Val Leu Phe Gly Thr Thr 20 25 30 Ile Ser Asn Asn Ile Lys Tyr Gly Arg Asp Asp Val Thr Asp Glu Glu 35 40 45 Met Glu Arg Ala Ala Arg Glu Ala Asn Ala Tyr Asp Phe Ile Met Glu 50 55 60 Phe Pro Asn Lys Phe Asn Thr Leu Val Gly Glu Lys Gly Ala Gln Met 65 70 75 80 Ser Gly Gly Gln Lys Gln Arg Ile Ala Ile Ala Arg Ala Leu Val Arg 85 90 95 Asn Pro Lys Ile Leu Ile Leu Asp Glu Ala Thr Ser Ala Leu Asp Ser 100 105 110 Glu Ser Lys Ser Ala Val Gln Ala Ala Leu Glu Lys Ala Ser Lys Gly 115 120 125 Arg Thr Thr Ile Val Val Ala His Arg Leu Ser Thr Ile Arg Ser Ala 130 135 140 Asp Leu Ile Val Thr Leu Lys Asp Gly Met Leu Ala Glu Lys Gly Ala 145 150 155 160 His Ala Glu Leu Met Ala Lys Arg Gly Leu Tyr Tyr Ser Leu Val Met 165 170 175 Ser Gln Asp Ile Lys Lys Ala Asp Glu Gln Met Glu Ser Met Thr Tyr 180 185 190 Ser Thr Glu Arg Lys Thr Asn Ser Leu Pro Leu His Ser Val Lys Ser 195 200 205 Ile Lys Ser Asp Phe Ile Asp Lys Ala Glu Glu Ser Thr Gln Ser Lys 210 215 220 Glu Ile Ser Leu Pro Glu Val Ser Leu Leu Lys Ile Leu Lys Leu Asn 225 230 235 240 Lys Pro Glu Trp Pro Phe Val Val Leu Gly Thr Leu Ala Ser Val Leu 245 250 255 Asn Gly Thr Val His Pro Val Phe Ser Ile Ile Phe Ala Lys Ile Ile 260 265 270 Thr Met Phe Gly Asn Asn Asp Lys Thr Thr Leu Lys His Asp Ala Glu 275 280 285 Ile Tyr Ser Met Ile Phe Val Ile Leu Gly Val Ile Cys Phe Val Ser 290 295 300 Tyr Phe Met Gln Gly Leu Phe Tyr Gly Arg Ala Gly Glu Ile Leu Thr 305 310 315 320 Met Arg Leu Arg His Leu Ala Phe Lys Ala Met Leu Tyr Gln Asp Ile 325 330 335 Ala Trp Phe Asp Glu Lys Glu Asn Ser Thr Gly Gly Leu Thr Thr Ile 340 345 350 Leu Ala Ile Asp Ile Ala Gln Ile Gln Gly Ala Thr Gly Ser Arg Ile 355 360 365 Gly Val Leu Thr Gln Asn Ala Thr Asn Met Gly Leu Ser Val Ile Ile 370 375 380 Ser Phe Ile Tyr Gly Trp Glu Met Thr Phe Leu Ile Leu Ser Ile Ala 385 390 395 400 Pro Val Leu Ala Val Thr Gly Met Ile Glu Thr Ala Ala Met Thr Gly 405 410 415 Phe Ala Asn Lys Asp Lys Gln Glu Leu Lys His Ala Gly Lys Ile Ala 420 425 430 Thr Glu Ala Leu Glu Asn Ile Arg Thr Ile Val Ser Leu Thr Arg Glu 435 440 445 Lys Ala Phe Glu Gln Met Tyr Glu Glu Met Leu Gln Thr Gln His Arg 450 455 460 Asn Thr Ser Lys Lys Ala Gln Ile Ile Gly Ser Cys Tyr Ala Phe Ser 465 470 475 480 His Ala Phe Ile Tyr Phe Ala Tyr Ala Ala Gly Phe Arg Phe Gly Ala 485 490 495 Tyr Leu Ile Gln Ala Gly Arg Met Thr Pro Glu Gly Met Phe Ile Val 500 505 510 Phe Thr Ala Ile Ala Tyr Gly Ala Met Ala Ile Gly Lys Thr Leu Val 515 520 525 Leu Ala Pro Glu Tyr Ser Lys Ala Lys Ser Gly Ala Ala His Leu Phe 530 535 540 Ala Leu Leu Glu Lys Lys Pro Asn Ile Asp Ser Arg Ser Gln Glu Gly 545 550 555 560 Lys Lys Pro Asp Thr Cys Glu Gly Asn Leu Glu Phe Arg Glu Val Ser 565 570 575 Phe Phe Tyr Pro Cys Arg Pro Asp Val Phe Ile Leu Arg Gly Leu Ser 580 585 590 Leu Ser Ile Glu Arg Gly Lys Thr Val Ala Phe Val Gly Ser Ser Gly 595 600 605 Cys Gly Lys Ser Thr Ser Val Gln Leu Leu Gln Arg Leu Tyr Asp Pro 610 615 620 Val Gln Gly Gln Val Leu Phe Asp Gly Val Asp Ala Lys Glu Leu Asn 625 630 635 640 Val Gln Trp Leu Arg Ser Gln Ile Ala Ile Val Pro Gln Glu Pro Val 645 650 655 Leu Phe Asn Cys Ser Ile Ala Glu Asn Ile Ala Tyr Gly Asp Asn Ser 660 665 670 Arg Val Val Pro Leu Asp Glu Ile Lys Glu Ala Ala Asn Ala Ala Asn 675 680 685 Ile His Ser Phe Ile Glu Gly Leu Pro Glu Lys Tyr Asn Thr Gln Val 690 695 700 Gly Leu Lys Gly Ala Gln Leu Ser Gly Gly Gln Lys Gln Arg Leu Ala 705 710 715 720 Ile Ala Arg Ala Leu Leu Gln Lys Pro Lys Ile Leu Leu Leu Asp Glu 725 730 735 Ala Thr Ser Ala Leu Asp Asn Asp Ser Glu Lys Val Val Gln His Ala 740 745 750 Leu Asp Lys Ala Arg Thr Gly Arg Thr Cys Leu Val Val Thr His Arg 755 760 765 Leu Ser Ala Ile Gln Asn Ala Asp Leu Ile Val Val Leu His Asn Gly 770 775 780 Lys Ile Lys Glu Gln Gly Thr His Gln Glu Leu Leu Arg Asn Arg Asp 785 790 795 800 Ile Tyr Phe Lys Leu Val Asn Ala Gln Ser Val Gln 805 810 <210> 4 <211> 4375 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 4 attgcttctc ggccttttgg ctaagatcaa gtgtaatctg tgttcttttt tatttggtca 60 tatcttccat tctttcttac ctaattcctc taatatctct ctgtgagcct aaaccaataa 120 ttatatatta cattctattg tctttcttat ataactgcag aaagataaat atcactttgt 180 ttgttcctgt aggttttctt tagtgtaatc catagcagtt attgcattgg agcagcagtc 240 cctcactttg aaaccttcgc aatagcccga ggagctgcct ttcatatttt ccaggttatt 300 gataagaaac ccagtataga taacttttcc acagctggat ataaacctga atccatagaa 360 ggaactgtgg aatttaaaaa tgtttctttc aattatccat caagaccatc tatcaagatt 420 ctgaaaggtc tgaatctcag aattaagtct ggagagacag tcgccttggt cggtctcaat 480 ggcagtggga agagtacggt agtccagctt ctgcagaggt tatatgatcc ggatgatggc 540 tttatcatgg tggatgagaa tgacatcaga gctttaaatg tgcggcatta tcgagaccat 600 attggagtgg ttagtcaaga gcctgttttg ttcgggacca ccatcagtaa caatatcaag 660 tatggacgag atgatgtgac tgatgaagag atggagagag cagcaaggga agcaaatgcg 720 tatgatttta tcatggagtt tcctaataaa tttaatacat tggtagggga aaaaggagct 780 caaatgagtg gagggcagaa acagaggatc gcaattgctc gtgccttagt tcgaaacccc 840 aagattctga ttttagatga ggctacgtct gccctggatt cagaaagcaa gtcagctgtt 900 caagctgcac tggagaaggc gagcaaaggt cggactacaa tcgtggtagc acaccgactt 960 tctactattc gaagtgcaga tttgattgtg accctaaagg atggaatgct ggcggagaaa 1020 ggagcacatg ctgaactaat ggcaaaacga ggtctatatt attcacttgt gatgtcacag 1080 gatattaaaa aagctgatga acagatggag tcaatgacat attctactga aagaaagacc 1140 aactcacttc ctctgcactc tgtgaagagc atcaagtcag acttcattga caaggctgag 1200 gaatccaccc aatctaaaga gataagtctt cctgaagtct ctctattaaa aattttaaag 1260 ttaaacaagc ctgaatggcc ttttgtggtt ctggggacat tggcttctgt tctaaatgga 1320 actgttcatc cagtattttc catcatcttt gcaaaaatta taaccatgtt tggaaataat 1380 gataaaacca cattaaagca tgatgcagaa atttattcca tgatattcgt cattttgggt 1440 gttattgct ttgtcagtta tttcatgcag ggattatttt acggcagagc aggggaaatt 1500 ttaacgatga gattaagaca cttggccttc aaagccatgt tatatcagga tattgcctgg 1560 ttgatgaaa aggaaaacag cacaggaggc ttgacaacaa tattagccat agatatagca 1620 caaattcaag gagcaacagg ttccaggatt ggcgtcttaa cacaaaatgc aactaacatg 1680 ggactttcag ttatcatttc ctttatatat ggatgggaga tgacattcct gattctgagt 1740 attgctccag tacttgccgt gacaggaatg attgaaaccg cagcaatgac tggatttgcc 1800 aacaaagata agcaagaact taagcatgct ggaaagatag caactgaagc tttggagaat 1860 atacgtacta tagtgtcatt aacaagggaa aaagccttcg agcaaatgta tgaagagatg 1920 cttcagactc aacacagaaa tacctcgaag aaagcacaga ttattggaag ctgttatgca 1980 ttcagccatg cctttatata ttttgcctat gcggcagggt ttcgatttgg agcctattta 2040 attcaagctg gacgaatgac cccagagggc atgttcatag tttttactgc aattgcatat 2100 ggagctatgg ccatcggaaa aacgctcgtt ttggctcctg aatattccaa agccaaatcg 2160 ggggctgcgc atctgtttgc cttgttggaa aagaaaccaa atatagacag ccgcagtcaa 2220 gaagggaaaa agccagacac atgtgaaggg aatttagagt ttcgagaagt ctctttcttc 2280 tatccatgtc gcccagatgt tttcatcctc cgtggcttat ccctcagtat tgagcgagga 2340 aagacagtag catttgtggg gagcagcggc tgtgggaaaa gcacttctgt tcaacttctg 2400 cagagacttt atgaccccgt gcaaggacaa gtgctgtttg atggtgtgga tgcaaaagaa 2460 ttgaatgtac agtggctccg ttcccaaata gcaatcgttc ctcaagagcc tgtgctcttc 2520 aactgcagca ttgctgagaa catcgcctat ggtgacaaca gccgtgtggt gccattagat 2580 gagatcaaag aagccgcaaa tgcagcaaat atccattctt ttattgaagg tctccctgag 2640 aaatacaaca cacaagttgg actgaaagga gcacagcttt ctggcggcca gaaacaaaga 2700 ctagctattg caagggctct tctccaaaaa cccaaaattt tattgttgga tgaggccact 2760 tcagccctcg ataatgacag tgagaaggtg gttcagcatg cccttgataa agccaggacg 2820 ggaaggacat gcctagtggt cactcacagg ctctctgcaa ttcagaacgc agatttgata 2880 gtggttctgc acaatggaaa gataaaggaa caaggaactc atcaagagct cctgagaaat 2940 cgagacatat atttaagtt agtgaatgca cagtcagtgc agtgatgctg ttgaggtagc 3000 acatattttg atgttcgtgt aatgcaaaga aggagtactt aataattact tggcaagctt 3060 tgatctcttt tattgcatat atcaatacct agaatcatgc tactcaagta catacatgtt 3120 ctattcacac accatctgac cttcagattt ttaaaaggaa gcaaaaattt gcttatttca 3180 tgtaagtgaa ataatgctta tatccttcac tttataaaac tattctagca catttgcttg 3240 taaagcagtt ttctacaagg tgaatttatt tcccatcaac ttctgctata aaatcggaaa 3300 tatgtttcca gggggaatat tatccaatta accatgttga aggttttagc aaaggcagtg 3360 taagatagag tggggcctgt agcattgcag ggagagtgtc tttcacttgg aatttgttt 3420 tgcagcacat attacagtag ttttgctagt cccttttctc cagaccgtag ggatttctct 3480 caatagtat tcactatttc tctaaattttt attctatttt ttgttgagc aggaataga 3540 aaggattacg atgtaaatt tctgggagga ttaggtagct atctcctact tcaccagtaa 3600 gtgaagtgcc tcacatgagc catcccaag attcattatt cacaaccttg ggtttggcag 3660 tataagtcac aggcctacct gtttatgaa acttacttac ttaaataag agctactttt 3720 gggccgggtg cggtggctca cgcctgtaat cccagaactt tgggaggccg aggaggggcgg 3780 atcacttgag gtcaggagtt cgagaccagc ctggccaca tggtgaaacc ccgtctctac 3840 taaaaacaca aaaattagcc aatctggtg gcggcacct ggaatcccag ctacttggga 3900 ggctgaggca ggagaatcat ttgaactag gaggcagagg tgcagtgag ccgagatctc 3960 accactgcac tccagcctgc gcaacagagc gagactccat ctcaaaaat aaaatag 4020 agctaatttt attgtgggtg aaattttta aacgtctttc tctataataa ataatttcc 4080 ttaaatttta tatatacttt atcatatata atgtgtgaat gattttaaag ttctgtgtaa 4140 atacaat tgtaaatg agttacattt tcacttact taatatgta atgtcacctg 4200 gtgattttat cttattctt cagtgtattt tcttccattt acacatttag ctagcctccc 4260 taaagtgtac tctaccaata attgaaatct tgttaaacaa attaaaacc attatatat 4320 tatgctgctt tctttaaaat gcaaaataaa aataagattg gggacttgag aatca 4375

Claims

1. A composition comprising a synthetic phospholipid and a pharmaceutically acceptable carrier, wherein the structure of the synthetic phospholipid is: R1 refers to a 6:0 fatty acid chain, and R2 refers to an 18:0 fatty acid chain.

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