INHIBITION OF FKBP1A FOR THE TREATMENT OF TRIPLE-NEGATIVE MAMMARY CARCINOMA

Abstract

The invention relates to a method for finding inhibitors of the peptidyl-prolyl cis-trans isomerase FKBP1A or antibodies, proteins or molecules having a specific affinity to FKBP1A. The invention also relates to FKBP1A-specific siRNA, inhibitors of the expression of FKBP1A, inhibitors of the enzymatic activity of FKBP1A, and inhibitors of the interaction(s) of FKBP1A with interaction partner(s), in each case for the treatment of diseases, in particular cancers or neurodegenerative diseases. The invention further relates to the use of FKBP1A as a prognostic or diagnostic marker for cancers. The cancers are preferably mammary carcinoma, in particular triple-negative mammary carcinoma, in this case very particularly the mesenchymal stem-like sub-type.

Claims

1. A method for finding inhibitors of the peptidyl-prolyl cis-trans isomerase FKBP1A, which are suitable for the treatment of triple-negative mammary carcinoma, comprising steps of: (a) providing an inhibitor of: (i) an expression of FKBP1A; and/or (ii) an enzymatic activity of FKBP1A; and/or (iii) interaction(s) of FKBP1A with interaction partner(s); (b) incubating cancer cells of a cancer cell line with the inhibitor of FKBP1A, and determining a characteristic of the cancer cells; (c) incubating cancer cells of the same cancer cell line as in step (b) in an absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); (d) comparing the determined characteristic of the cancer cells according to steps (b) and (c).

2. (canceled)

3. The method according to claim 1, wherein the characteristic determined in step (b) is selected from a group consisting of (A) cellular differentiation status, (B) epithelial characteristics, (C) cellular immunogenicity, (D) apoptosis induction or apoptosis ability, (E) cellular stem cell character, and/or (F) cellular metastatic ability.

4. The method according to claim 1, wherein the inhibitor provided in step (a) inhibits the interaction(s) of FKBP1A with interaction partner(s), with at least one interaction partner being selected from the group consisting of interaction partners which effect a reduction or arrest of (A) the cellular differentiation status, (B) the epithelial characteristics, (C) the cellular immunogenicity (D), the induction of apoptosis or apoptosis ability, (E) the cellular stem cell character, and/or (F) cellular metastatic ability.

5. The method according to claim 1, wherein the inhibitor provided in step (a) inhibits the interaction(s) of FKBP1A with interaction partner(s), at least one interaction partner being selected from the group consisting of interacting proteins, interacting peptides, and interacting nucleic acids.

6. The method according to claim 1, wherein in step (b) the characteristic is determined by quantifying differentiation markers, epithelial markers, (tumor) stem cell markers, immunomodulating proteins, immunomediating proteins or combinations thereof.

7. The method according to claim 1, wherein step (b) comprises incubating a mixture of cancer cells of a cancer cell line and immune cells of an immune cell line with the inhibitor of FKBP1A and wherein, as a characteristic of the cancer cells, their cellular immunogenicity is determined by quantifying the immune cell activation and/or by quantifying the immune cell-mediated cytotoxic effect on the cancer cells; and wherein step (c) comprises incubating a mixture of cancer cells of the same cancer cell line and immune cells of the same immune cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b).

8. The method according to claim 1, wherein the inhibitor provided in step (a) inhibits (i) the expression of FKBP1A and at the same time does not inhibit (ii) the enzymatic activity of FKB1A.

9. (canceled)

10. The method according to claim 1, wherein steps (a) is: providing the inhibitor of (i) the expression of FKBP1A.

11. The method according to claim 10, wherein step (a) comprises the sub-steps of: (a.sub.1) providing several test substances; (a.sub.2) screening the test substances with regard to their inhibitory effect on (i) the expression of FKBP1A; (a.sub.3) selecting at least one screened test substance, the inhibitory effect of which is stronger than the inhibitory effect of at least one other screened test substance, and providing this selected test substance as an inhibitor of FKBP1A.

12. (canceled)

13. The method according to claim 1, wherein the cancer cell line is a cancer cell line of triple-negative mammary carcinoma of the mesenchymal stem-like sub-type.

14. (canceled)

15. The method according to claim 13, wherein the cancer cell line is selected from MDA-MB-231 and MDA-MB-436.

16. (canceled)

17. The method according to claim 3, wherein the characteristic (A) is determined in step (b) by quantifying mesenchymal markers and the characteristic (A) is cellular differentiation status.

18. (canceled)

19. The method according to claim 3, wherein the characteristic (C) is determined in step (b) by quantifying immunomodulating proteins and the characteristic (C) is cellular immunogenicity.

20. (canceled)

21. The method according to claim 3, wherein the characteristic (E) is determined in step (b) by quantifying (tumor) stem cell markers, and the characteristic (E) is cellular stem cell character.

22. The method according to claim 3, wherein the characteristic (F) is determined in step (b) the characteristic is determined by quantifying (tumor) stem cell markers and/or behavior, and the characteristic (F) is cellular metastasis and invasion.

23. The method according to claim 1, wherein the characteristic determined in step (b) is the quantifying the expression of FKBP1A.

24. The method according to claim 1, comprising the steps of: (a) providing the inhibitor of (i) the expression of FKBP1A; (b) incubating cancer cells of a cancer cell line of triple-negative mammary carcinoma, of the mesenchymal stem-like sub-type; with the inhibitor of FKBP1A, and determining a characteristic of the cancer cells, wherein the characteristic is the cellular differentiation status, by quantifying mesenchymal markers; (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); (d) comparing the determined characteristic of the cancer cells according to steps (b) and (c).

25. The method according to claim 1, comprising the steps of: (a) providing the inhibitor of (i) the expression of FKBP1A; (b) incubating cancer cells of a cancer cell line of triple-negative mammary carcinoma, of the mesenchymal stem-like sub-type with the inhibitor of FKBP1A, and determining a characteristic of the cancer cells, wherein the characteristic is cellular immunogenicity, by quantifying immunomodulating proteins; (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); (d) comparing the determined characteristic of the cancer cells according to steps (b) and (c).

26. The method according to claim 1, comprising the steps of: (a) providing an inhibitor of (i) the expression of FKBP1A; (b) incubating cancer cells of a cancer cell line of triple-negative mammary carcinoma, of the mesenchymal stem-like sub-type with the inhibitor of FKBP1A, and determining a characteristic of the cancer cells, wherein the characteristic is cellular stem cell character, by quantifying (tumor) stem cell markers; (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); (d) comparing the determined characteristic of the cancer cells according to steps (b) and (c).

27. The method according to claim 1, comprising the steps of: (a) providing an inhibitor of (i) the expression of FKBP1A; (b) incubating cancer cells of a cancer cell line of triple-negative mammary carcinoma, of the mesenchymal stem-like sub-type with the inhibitor of FKBP1A, and quantifying the expression of FKBP1A; (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); (d) comparing the determined characteristic of the cancer cells according to steps (b) and (c).

28-49. (canceled)

Description

[0040] FIG. 1 shows a western blot analysis of the protein expression of FKBP1A in different cells.

[0041] FIG. 2 shows the downregulation of FKBP1A protein expression by specific siRNA as a result of western blot analysis.

[0042] FIG. 3 shows the result of an expression analysis of (tumor) stem cell and mesenchymal markers after FKBP1A knock-down.

[0043] FIG. 4 shows downregulation of FKBP1A protein expression by inhibitor (I) as a result of western blot analysis of protein expression.

[0044] FIG. 5 shows the result of an expression analysis of (tumor) stem cell and epithelial differentiation markers and PD-L1 after expression reduction of FKBP1A using inhibitor (I).

[0045] FIG. 6 shows known interaction partners of FKBP1A (source: canSAR Black (https://cansarblack.icr.ac.uk/?type=protein,compound,cellline,disease).

[0046] FIG. 7 shows an example of the theoretical structure of a dual-targeting agent against FKBP1A and PIN1.

[0047] FIG. 8 shows examples, based on FIG. 7, of dual-targeting agents composed of rapamycin and KPT-6556.

[0048] FIG. 9 shows examples, based on FIG. 7, of dual-targeting agents composed of rapamycin and ATRA.

[0049] FIG. 10 shows a co-expression analysis of FKBP1A and TGF-beta receptor II in primary breast cancer tissue.

[0050] One aspect of the invention relates to a method for finding inhibitors of the peptidyl prolyl cis-trans isomerase FKBP1A, which are suitable for the treatment of diseases; including neurodegenerative diseases such as, for example, Alzheimer's disease; in particular cancers; preferably triple-negative mammary carcinoma; Her2-positive hormone receptor-negative mammary carcinoma or malignant melanoma; particularly preferably triple-negative mammary carcinoma, comprising the steps of [0051] (a) providing an inhibitor of [0052] (i) the expression of FKBP1A; and/or [0053] (ii) the enzymatic activity of FKBP1A; and/or [0054] (iii) the interaction(s) of FKBP1A with interaction partner(s); [0055] (b) incubating cancer cells of a cancer cell line with the inhibitor of FKBP1A, and determining a characteristic of the cancer cells; [0056] (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); [0057] (d) comparing the determined characteristic of the cancer cells according to steps (b) and (c).

[0058] Step (a) of the method according to the invention preferably comprises the sub-steps of [0059] (a.sub.1) providing several test substances; [0060] (a.sub.2) screening the test substances with regard to their inhibitory effect on [0061] (i) the expression of FKBP1A; and/or [0062] (ii) the enzymatic activity of FKBP1A; and/or [0063] (iii) interaction(s) of FKBP1A with interaction partner(s); [0064] (a.sub.3) selecting at least one screened test substance, the inhibitory effect of which is stronger than the inhibitory effect of at least one other screened test substance, and providing this selected test substance as an inhibitor of FKBP1A.

[0065] Typically, the cancer cell line is characteristic of triple-negative mammary carcinoma or is a cancer cell line of triple-negative mammary carcinoma, preferably selected from the group consisting of MDA-MB-231, MDA-MB-436 and MDA-MB-468, especially MD-MB-231. Suitable cancer cell lines are available, for example, from the American Type Culture Collection.

[0066] The characteristic of the cancer cells determined in step (b) is preferably selected from the group consisting of (A) cellular differentiation status, (B) epithelial characteristics, (C) cellular immunogenicity, (D) apoptosis induction or apoptosis ability, and (E) cellular stem cell character.

[0067] In preferred embodiments of the method according to the invention, the inhibitor provided in step (a) inhibits the interaction(s) of FKBP1A with interaction partner(s), with at least one interaction partner being selected from the group consisting of interaction partners which effect a reduction or arrest of (A) the cellular differentiation status, (B) the epithelial characteristics, (C) the cellular immunogenicity, (D) the induction of apoptosis or apoptosis ability, and/or (E) the cellular stem cell character.

[0068] In preferred embodiments of the method according to the invention, the inhibitor provided in step (a) inhibits the interaction(s) of FKBP1A with interaction partner(s), at least one interaction partner being selected from the group consisting of interacting proteins, interacting peptides, and interacting nucleic acids.

[0069] Numerous interaction partners of FKBP1A and their interactions are known to a person skilled in the art. According to the invention, preferred interaction partners of FKBP1A are selected from the group consisting of AHSP, cyclophilin-type peptidyl prolyl isomerases, FKBP-type peptidyl prolyl isomerases, inositol triphosphate receptors, MDM2/MDM4, phosphoprotein phosphatases, ryanodine receptors, SF3B4, TGF-beta, TKL Ser/Thr protein kinases and type B carboxylesterases/lipases. Interaction partners of FKBP1A that are preferred according to the invention are summarized in the following table (cf. FIG. 6):

TABLE-US-00001 Surname Uniprot ID Family ACVR1B P36896 TKL Ser/Thr protein kinases (tyrosine kinase like) ACVRL1 P37023 TKL Ser/Thr protein kinases (tyrosine kinase like) AHSP Q9NZD4 AHSP (alpha hemoglobin stabilizing protein) BARD1 Q99728 BMPR1A P36894 TKL Ser/Thr protein kinases (tyrosine kinase like) FKBP1A P62942 FKBP-type peptidyl prolyl isomerases (FK506 binding protein) FKBP4 Q02790 GLMN Q92990 ITPR1 Q14643 Inositol triphosphate (InsP3) receptors MDM2 Q00987 MDM2/MDM4 (murine double minute 2/ human homologue) NLGN3 Q9NZ94 Type B carboxylesterases/lipases PPIA P62937 Cyclophilin-type peptidyl prolyl isomerases PPP3CA Q08209 Phosphoprotein phosphatases RYR3 Q15413 Ryanodine receptors (TC 1.A.3.1) SF3B4 Q15427 SF3B4 (splicing factor 3B subunit 4) TGFB1 P01137 TGF-beta (transforming growth factor beta) TGFBR1 P36897 TKL Ser/Thr protein kinases (tyrosine kinase like) TRPC3 Q13507 TRP receptors (TC 1.A.4) (tryptophan transient receptor)

[0070] In preferred embodiments of the method according to the invention, the characteristic of the cancer cells is determined in step (b) by quantifying differentiation markers (cell type markers).

[0071] Suitable differentiation markers and methods for their respective quantification are known to a person skilled in the art. In this context, for example, full reference can be made to: Akrap, N. et al, Identification of Distinct Breast Cancer Stem Cell Populations Based on Single-Cell Analyzes of Functionally Enriched Stem and Progenitor Pools. Stem Cell Reports (2016). doi:10.1016/j.stemcr.2015.12.006; Prabhakaran P., Hassiotou F., Blancafort P. & Filgueira L. Cisplatin induces differentiation of breast cancer cells. Front. Oncol. (2013). doi:10.3389/fonc.2013.00134.

[0072] Suitable differentiation markers are, for example, CD3 on T lymphocytes, CD14 on monocytes, CD16 and CD56 on NK cells and granulocytes, CD19 and CD20 on B lymphocytes. Differentiation markers for mesenchymal stem cells are, for example, CD13, CD29, CD44, CD49e, CD54, CD71, CD73, CD90, CD105, CD106, CD166 and HLA-ABC. The differentiation markers are preferably those of epithelial cells.

[0073] In preferred embodiments of the method according to the invention, the characteristic of the cancer cells is determined in step (b) by quantifying epithelial markers.

[0074] Suitable epithelial markers and methods for their respective quantification are known to a person skilled in the art. In this context, for example, full reference can be made to Frixen, U. H. et al. E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol (1991). doi:10.1083/jcb.113.1.173.

[0075] Epithelial markers (epithelial cell markers) preferred according to the invention are selected from the group consisting of cytokeratins, cell adhesion molecules and cell surface proteins. A preferred cell adhesion molecule according to the invention, which can be used as an epithelial marker, is E-cadherin.

[0076] In preferred embodiments of the method according to the invention, the characteristic of the cancer cells is determined in step (b) by quantifying mesenchymal markers.

[0077] Suitable mesenchymal markers and methods for their respective quantification are known to a person skilled in the art. In this context, for example, full reference can be made to Yamashita, N. et at Vimentin as a poor prognostic factor for triple-negative mammary carcinoma. J. Cancer Res, Can. Oncol. (201), doi:10.1007/s00432-013-1376-6.

[0078] Mesenchymal markers preferred according to the invention are vimentin, fibronectin and N-cadherin (Ogunbolude, Y. et al. FRK inhibits breast cancer cell migration and invasion by suppressing Epithelial-mesenchymal transition. Oncotarget (2017). doi:10.18632/oncotarget.22958):

[0079] Mesenchymal markers and markers for EMT preferred according to the invention are vimentin, fibronectin. N-cadherin, SNAI1, TWIST1. TWIST2, ZEB1 and ZEB2 (Ogunbolude, Y. et al. FRK inhibits breast cancer cell migration and invasion by suppressing epithelial-mesenchymal transition: Oncotarget (2017). doi:10.18632/oncotarget.22958):

[0080] In preferred embodiments of the method according to the invention, the characteristic of the cancer cells is determined in step (b) by quantifying (tumor) stem cell markers.

[0081] Suitable (tumor) stem cell markers and methods for their respective quantification are known to a person skilled in the art. In this context, for example, full reference can be made to Li, W. et al. Unraveling the roles of CD44/CD24 and ALDH1 as cancer stem cell markers in tumorigenesis and metastasis. Sci. Rep. (2017). doi:10.1038/s41598-017-14364-2.

[0082] (Tumor) stem cell markers preferred according to the invention are CD44/CD24 and ALDH1.

[0083] In preferred embodiments of the method according to the invention, the characteristic of the cancer cells is determined in step (b) by quantifying immunomodulating proteins.

[0084] Suitable immunomodulating proteins and methods for their respective quantification are known to a person skilled in the art. In this context, full reference can be made, for example, to Bu, X., Yao, Y. & Li, X. Immune checkpoint blockade in breast cancer therapy; in Advances in Experimental Medicine and Biology (2017). doi:10.1007/978-981-10-6020-518.

[0085] Immunomodulating proteins preferred according to the invention are check point proteins, e. g. PD-L1, PD-L2; (possibly in co-culture with T cells): PD-1 and CTLA-4,

[0086] In preferred embodiments of the method according to the invention, the characteristic of the cancer cells is determined in step (b) by quantifying immune-mediating proteins.

[0087] Suitable immune-mediating proteins and methods for their respective quantification are known to a person skilled in the art. In this context, for example, full reference can be made to Vertuani, S. et al. Retinoids Act as Multistep Modulators of the Major Histocompatibility Class I Presentation Pathway and Sensitize Neuroblastomas to Cytotoxic Lymphocytes. Cancer Res. 63:8006-8013 (2003).

[0088] An immune-mediating protein preferred according to the invention is the MHC class I protein complex.

[0089] In preferred embodiments of the method according to the invention, step (b) comprises incubating a mixture of cancer cells of a cancer cell line and immune cells of an immune cell line with the inhibitor of FKBP1A, the cellular immunogenicity of which is determined as a characteristic of the cancer cells by quantifying the immune cell activation and/or by quantifying the immune cell-mediated cytotoxic effect on cancer cells.

[0090] Suitable methods for quantifying the immune cell activation and for quantifying the immune cell-mediated cytotoxic effect on the cancer cells are known to a person skilled in the art.

[0091] According to this preferred embodiment, step (c) of the method according to the invention comprises incubating a mixture of cancer cells of the same cancer cell line and immune cells of the same immune cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b).

[0092] In preferred embodiments of the method according to the invention, the inhibitor provided in step (a) inhibits (i) the expression of FKBP1A and at the same time does not inhibit (ii) the enzymatic activity of FKB1A.

[0093] In other preferred embodiments of the method according to the invention, the inhibitor provided in step (a) inhibits (ii) the enzymatic activity of FKB1A and at the same time does not inhibit (i) the expression of FKBP1A.

[0094] In preferred embodiments, the method according to the invention comprises the steps of [0095] (a) providing an inhibitor of (i) the expression of FKBP1A; [0096] (b) incubating cancer cells of a cancer cell line, preferably a cancer cell line which is characteristic of triple-negative mammary carcinoma, with the inhibitor of FKBP1A and determining a characteristic of the cancer cells; [0097] (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); and [0098] (d) comparing the determined characteristic of the cancer cells according to steps (b) and (C).

[0099] According to this preferred embodiment, step (a) comprises the sub-steps [0100] (a.sub.1) providing several test substances; [0101] (a.sub.2) screening the test substances with regard to their inhibitory effect on (i) the expression of FKBP1A; and [0102] (a.sub.3) selecting at least one screened test substance, the inhibitory effect of which is stronger than the inhibitory effect of at least one other screened test substance, and providing this selected test substance as an inhibitor of FKBP1A.

[0103] In preferred embodiments of the method according to the invention, the cancer cell line is characteristic of triple-negative mammary carcinoma; the cancer cell line is preferably a cancer cell line of the triple-negative mammary carcinoma.

[0104] In preferred embodiments of the method according to the invention, the cancer cell line is a cancer cell line of the triple-negative mammary carcinoma of the mesenchymal stem-like sub-type. The cancer cell line of the triple-negative mammary carcinoma of the mesenchymal stem-like sub-type is preferably selected from MDA-MB-231 and MDA-MB-436; preferably MDA-MB-231.

[0105] In preferred embodiments of the method according to the invention, the cancer cell line is a cancer cell line of the triple-negative mammary carcinoma of the basal-like sub-type. The cancer cell line MDA-MB-468 of the triple-negative mammary carcinoma of the sub-type basal-like is preferred.

[0106] In preferred embodiments of the method according to the invention, the characteristic (A) determined in step (b) is cellular differentiation status. According to this preferred embodiment of the method according to the invention, the characteristic is preferably determined in step (b) by quantifying mesenchymal markers: preferably by quantifying vimentin.

[0107] In further preferred embodiments of the method according to the invention, the characteristic (C) determined in step (b) is cellular immunogenicity. According to this preferred embodiment of the method according to the invention, the characteristic is preferably determined in step (b) by quantifying immunomodulating proteins; preferably by quantifying PD-L1.

[0108] In further preferred embodiments of the method according to the invention, the characteristic (E) determined in step (b) is cellular stem cell character. According to this preferred embodiment of the method according to the invention, the characteristic is preferably determined in step (b) by quantifying (tumor) stem cell markers; preferably by quantifying CD44/CD24 and ALDH1.

[0109] In further preferred embodiments of the method according to the invention, the characteristic determined in step (b) is the quantifying the expression of FKBP1A. Suitable methods for quantifying the expression of FKBP1A are known to a person skilled in the art, e. g. western blot analysis.

[0110] In preferred embodiments, the method according to the invention comprises the steps of [0111] (a) providing an inhibitor of (i) the expression of FKBP1A; [0112] (b) incubating cancer cells of a cancer cell line of triple-negative mammary carcinoma, preferably of the mesenchymal stem-like sub-type, more preferably the cancer cell line MDA-MB-231; with the inhibitor of FKBP1A, and determining a characteristic of the cancer cells, preferably the cellular differentiation status, more preferably by quantifying mesenchymal markers; [0113] (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); and [0114] (d) comparing the determined characteristic of the cancer cells according to steps (b) and (c).

[0115] In preferred embodiments, the method according to the invention comprises the steps of [0116] (a) providing an inhibitor of (i) the expression of FKBP1A; [0117] (b) incubating cancer cells of a cancer cell line of triple-negative mammary carcinoma, preferably of the mesenchymal stem-like sub-type, more preferably the cancer cell line MDA-MB-231; with the inhibitor of FKBP1A, and determining a characteristic of the cancer cells, preferably cellular immunogenicity, more preferably by quantifying immunomodulating proteins; [0118] (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); and [0119] (d) comparing the determined characteristic of the cancer cells according to steps (b) and (c).

[0120] In preferred embodiments, the method according to the invention comprises the steps of [0121] (a) providing an inhibitor of (i) the expression of FKBP1A; [0122] (b) incubating cancer cells of a cancer cell line of triple-negative mammary carcinoma, preferably of the mesenchymal stem-like sub-type, more preferably the cancer cell line MDA-MB-231; with the inhibitor of FKBP1A, and determining a characteristic of the cancer cells; preferably cellular stem cell character; more preferably by quantifying (tumor) stem cell markers; [0123] (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); and [0124] (d) comparing the determined characteristic of the cancer cells according to steps (b) and (c).

[0125] In preferred embodiments, the method according to the invention comprises the steps of [0126] (a) providing an inhibitor of (i) the expression of FKBP1A; [0127] (b) incubating cancer cells of a cancer cell line of triple-negative mammary carcinoma; preferably of the mesenchymal stem-like sub-type, more preferably the cancer cell line MDA-MB-231; with the inhibitor of FKBP1A, and determining a characteristic of the cancer cells, preferably quantifying the expression of FKBP1A; [0128] (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of FKBP1A and determining the same characteristic of the cancer cells as in step (b) under the same conditions as step (b); and [0129] (d) comparing the determined characteristic of the cancer cells according to steps (b) and (c).

[0130] According to this preferred embodiment; step (a) comprises the sub-steps of [0131] (a.sub.1) providing several test substances; [0132] (a.sub.2) screening the test substances with regard to their inhibitory effect on (i) the expression of FKBP1A, [0133] (a.sub.3) selecting at least one screened test substance, the inhibitory effect of which is stronger than the inhibitory effect of at least one other screened test substance, and providing this selected test substance as an inhibitor of FKBP1A.

[0134] Another aspect of the invention relates to the use of an [0135] (A) inhibitor of [0136] (i) the expression of FKBP1A; and/or [0137] (ii) the enzymatic activity of FKBP1A; and/or [0138] (iii) the interaction(s) of FKBP1A with interaction partner(s);

[0139] and/or [0140] (B) PROTACs (PROteolysis TArgeting Chimeras), which specifically induces the cellular degradation of FKBP1A, preferably FKBP12 PROTAC RC32 (CAS No.: 2375555-66-9),

[0141] for the production of a drug for the treatment of a disease; in particular cancer; preferably triple-negative mammary carcinoma, Her2-positive hormone receptor-negative mammary carcinoma, pancreatic ductal adenocarcinoma (PDAC) or malignant melanoma; particularly preferably from so-called tumor stem cells; particularly preferably the triple-negative mammary carcinoma, preferably the mesenchymal stem-like sub-type.

[0142] In a preferred embodiment, the inhibitor inhibits (i) the expression of FKBP1A and at the same time does not inhibit (ii) the enzymatic activity of FKB1A.

[0143] In a preferred embodiment, the inhibitor inhibits (ii) the enzymatic activity of FKB1A and at the same time does not inhibit (i) the expression of FKBP1A.

[0144] Therapies according to the invention are thus based on different approaches, in particular they intervene in different stages of the formation and effect of FKBP1A in tumor cells. The aim of inhibiting the expression of FKBP1A is that FKBP1A is not formed in the first place or that its concentration is at least reduced. The aim of inhibiting the enzymatic function of FKBP1A is to prevent expressed FKBP1A from developing the enzymatically catalyzed effect. The inhibition of the interaction(s) of FKBP1A with interaction partner(s) aims to suppress secondary processes.

[0145] In a preferred embodiment, an inhibitor of the expression of FKBP1A is used in the therapeutic treatment of the disease.

[0146] In preferred embodiments of the invention, the inhibitor of the expression of FKBP1A is an FKBP1A-specific siRNA.

[0147] Suitable siRNA which is specific for FKBP1A is known to a person skilled in the art and is commercially available, for example siRNA 55205 (ThermoFisher Scientific, Waltham, USA).

[0148] In a preferred embodiment, an inhibitor of FKBP1A enzymatic activity is used in the therapeutic treatment of the disease.

[0149] In preferred embodiments of the invention, the inhibitor of FKBP1A enzymatic activity is a macrolide, FK506 or a specific non-immunosuppressive inhibitor.

[0150] In preferred embodiments of the invention, the inhibitor of FKBP1A enzymatic activity is a specific, non-immunosuppressive, isolated enzyme-targeting inhibitor. The inhibitor is preferably a specific inhibitor of FKBP1A without binding to mTOR or calcineurin and having an immunosuppressive effect.

[0151] In a preferred embodiment, an inhibitor of the interaction(s) of FKBP1A with interaction partner(s) is used for the therapeutic treatment of the disease.

[0152] Interaction partners of FKBP1A preferred according to the invention are preferably selected with regard to this aspect of the invention from the group consisting of AHSP, cyclophilin-type peptidyl prolyl isomerases, FKBP-type peptidyl prolyl isomerases, inositol triphosphate receptors, MDM2/MDM4, phosphoprotein phosphatases, ryanodine receptors, SF3B4, TGF-beta, TKL Ser/Thr protein kinases and type B carboxylesterases/lipases.

[0153] The therapeutic treatment according to the invention preferably takes place as an adjuvant treatment of a cancer. The therapeutic treatment according to the invention is preferably carried out as an adjuvant treatment in an immunotherapy of the cancer and/or to avoid and/or specifically address invading tumor cells, to avoid tumor cell invasion and metastasis formation.

[0154] A special form of therapy for the targeted treatment of triple-negative mammary carcinoma is the simultaneous addressing of FKBP1A and the parvulin PIN1 or alternatively the retinoic acid receptors RAR or RXR. For the first time, a strong synergistic effect of inhibition of FKBP1A and inhibition of PIN1 (here using FKBP1A-specific siRNA (or alternatively an FKBP1A-specific inhibitor) and co-incubation with the PIN1-specific inhibitor all-trans retinoic acid, ATRA) was observed in triple-negative mammary carcinoma on, inter alia, the reduction of the tumor stem cell character and the ability to induce apoptosis. On the one hand, such a therapy can be achieved via the combined application of individual specific agents. In this context, on the other hand, a dual-targeting inhibitor can be developed via the concept of pioneering polypharmacology, which as a single agent specifically addresses both peptidyl prolyl cis-trans isomerases. This agent could be composed of, for example, specific inhibitors such as, for example, ElteN378, V-10367 or GPI-1046 against FKBP1A on the one hand and, for example, KPT-6566, ATRA (all-trans retinoic acid) or an ATRA derivative against PIN1, RAR or RXR (Chen, Y. et al. Prolyl isomerase Pin1: a promoter of cancer and a target for therapy, Cell Death and Disease (2018), doi:10.1038/s41419-018-0844-y) on the other, both being either fused or connected via a linker, or in which functional substructures of the inhibitors are “merged” (Ramsay, R. R., Popovic-Nikolic, M. R., Nikolic, K., Uliassi, E. & Bolognesi, M. L. A perspective on multi-target drug discovery and design for complex diseases, Clin. Transl. Med. (2018), doi:10.1186/s40169-017-0181-2). The agents RapaLink-01 (CAS No.: 1887095-82-0) or FKBP12 PROTAC R032 (CAS No.: 2375555-66-9) serve as a chemical example in which an FKBP1A ligand was coupled to a second inhibitor/ligand. A comparable agent can be developed and synthesized by a skilled person with a PIN1 inhibitor such as, for example, KPT-6566 or ATRA (all-trans retinoic acid) or an ATRA derivative. Other linker structures of FKBP1A ligands are known (Kolos, J. M., Voll, A. M., Bauder, M. & Hausch, F. FKBP Ligands—Where We Are and Where to Go? Front. Pharmacol. (2018), doi:10.3389/fphar.2016.01425) and can also be developed by a skilled person with a PIN1 inhibitor such as KPT-6566 or ATRA (all-trans retinoic acid) or an ATRA derivative to form a dual-targeting agent. Exemplary structural formulas for the dual-targeting agents mentioned are shown in FIGS. 7 to 9. The agent according to dependent claim 13 preferably corresponds to one of the agents listed in FIGS. 7 to 9.

[0155] FIG. 7 shows an example of the theoretical structure of a dual-targeting agent against FKBP1A and PIM The left box shows the structural component corresponding to rapamycin, the middle box shows an exemplary linker structure and the right box shows an exemplary PIN1 inhibitor. As an alternative to the linker structure given in the middle box, the alternative linker structure can also be used, wherein R.sub.1 represents the rapamycin structure and R.sub.2 represents the PIN1 inhibitor structure. The value for n.sub.1 or n.sub.2 is any positive value including the value 0.

[0156] FIG. 8 shows examples, based on FIG. 7, of dual-targeting agents composed of rapamycin and KPT-6556, The chemical bonding of the linker to KPT-6556 or rapamycin is also conceivable to all other possible atoms of the respective partial structures and can be carried out by a skilled person. The value for n.sub.1 or n.sub.2 is any positive value including the value 0.

[0157] FIG. 9 shows examples, based on FIG. 7, of dual-targeting agents composed of rapamycin and ATRA. The chemical bonding of the linker to ATRA or rapamycin is also conceivable to all other possible atoms of the respective partial structures and can be carried out by a skilled person. The value for n.sub.1 or n.sub.2 is any positive value including the value 0.

[0158] The chemical bonding of a PIN1-specific agent as a dual-targeting agent to a high-affinity ligand of FKBP1A such as rapamycin, as described in [0062] to [0065], and the intracellular bonding of the PIN1-specific agent to FKBP1A effected thereby can also take place for any other agents. Coupling to the cytosolic protein FKBP1A has significant changes to the pharmacokinetics and dynamics of any agent: in tumor cells, especially tumor stem cells, various transporters, inter alia, ABC transporters, are highly overexpressed (Choi, Y. & Yu, A.-M. ABC Transporters in Multidrug Resistance and Pharmacokinetics, and Strategies for Drug Development Curr. Pharm. Des. (2014) doi:10.2174/138161282005140214165212. These transporters ensure rapid removal of the agents from the cell via increased efflux of the agents, so that they can only have a limited and short-term intracellular effect. In addition, there is the metabolism of agents in the cell, which also leads to a loss of effectiveness. A chemical coupling as described in [0062] to [0065] would bind any agent to the cytosolic protein FKBP1A diffusing freely in the cell. However, inter alia due to its size, in contrast to the individual agent, the complex formed is not transported out of the cell, or to a much lesser extent, via the transporters mentioned or is metabolized by cellular processes, which means that a significantly better intracellular effect of the agent can be achieved. The chemical bonding of any agent to a high-affinity ligand of FKBP1A or another cytosolic protein can be introduced in the future as a general novel pharmacological principle with the aim, inter alia, of increasing the effect of any agent in the cell, especially in tumor stem cells or resistant tumor cells.

[0159] The therapeutic treatment according to the invention takes place as a treatment of diseases which are preferably selected from cancers and neurodegenerative diseases such as, for example, Alzheimer's disease.

[0160] The therapeutic treatment according to the invention preferably takes place as a treatment of cancers which are selected from FKBP1A-positive tumors. The cancer is particularly preferably selected from triple-negative mammary carcinoma, Her2-positive hormone receptor-negative mammary carcinoma and malignant melanoma; in particular triple-negative mammary carcinoma, especially the TNBC mesenchymal stem-like sub-type.

[0161] A further aspect of the invention relates to the use of FKBP1A as a prognostic or diagnostic marker for diseases, in particular cancers or neurodegenerative diseases; especially FKBP1A-positive tumors; in particular triple-negative mammary carcinoma, Her2-positive hormone receptor-negative mammary carcinoma or malignant melanoma; in particular triple-negative mammary carcinoma, especially the TNBC mesenchymal stem-like sub-type.

[0162] A further aspect of the invention relates to a method for the quantitative detection of FKBP1A in human material for the prognostic and diagnostic assessment of diseases; in particular cancers; preferably cancers with high metastasis potential including for the analysis and diagnosis of tumor invasion and for the prediction of a metastasis tendency; in particular triple-negative mammary carcinoma, Her2-positive hormone receptor-negative mammary carcinoma or malignant melanoma; in particular triple-negative mammary carcinoma, especially the TNBC mesenchymal stem-like sub-type; including the steps of:

[0163] (a) providing human material, preferably serum, plasma or tissue; and

[0164] (b) quantifying FKBP1A, preferably by determining the concentration.

[0165] Step (a) of the method according to the invention for the quantitative detection of FKBP1A in human material preferably includes providing the human material, but not the removal of the material per se. The human material, preferably serum, plasma or tissue, has therefore preferably already been removed beforehand and the human body from which the material was removed does not have to be present in itself for the implementation of the method according to the invention. Thus, step (a) of the method according to the invention includes providing the human material that has already been removed, whereas the removal of the human material per se is preferably not part of the method according to the invention.

[0166] Suitable methods for quantifying FKBP1A, in particular for determining the concentration of FKBP1A, are known to a person skilled in the art. In preferred embodiments, step (b) is carried out immunologically by FKBP1A-specific ELISA (serum, plasma) or immunohistologically (tissue).

[0167] A further aspect of the invention relates to a method for finding antibodies, proteins or molecules having a specific affinity to FKBP1A, which are suitable for the treatment of diseases, in particular cancers or neurodegenerative diseases, comprising the steps of [0168] (a) providing a library of antibodies, antibody fragments, proteins or protein fragments; [0169] (b) selecting from said library specific FKBP1A-binding antibodies, antibody fragments, proteins or protein fragments; [0170] (c) isolating and sequencing said specific FKBP1A-binding antibodies, antibody fragments, proteins, protein fragments or their encoding genes from step (a); and [0171] (d) expressing the specific FKBP1A-binding antibodies, antibody fragments, proteins or protein fragments in suitable host cells.

[0172] Suitable methods for providing antibodies, antibody fragments, proteins or protein fragments, for selecting specific FKBP1A-binding antibodies, antibody fragments, proteins or protein fragments, for isolating and sequencing and for expression are known to a person skilled in the art.

[0173] In preferred embodiments; step (b) comprises selecting FKBP1A-binding phages in phage display or selecting by biopanning of libraries of antibodies; antibody fragments; proteins and/or protein fragments.

[0174] Another aspect of the invention relates to the use of an antibody, antibody fragment, protein, protein fragment or molecule having specific affinity to FKBP1A for the production of a drug for the treatment of diseases: in particular cancers; in particular FKBP1A-positive tumors; in particular triple-negative mammary carcinoma, Her2-positive hormone receptor-negative mammary carcinoma or malignant melanoma; especially triple-negative mammary carcinoma, especially the TNBCmesenchymal stem-like sub-type.

[0175] Another aspect of the invention relates to the use of an antibody; antibody fragment, protein, protein fragment or molecule having specific affinity to FKBP1A for the production of a drug comprising a conjugate of the antibody, protein or molecule with an agent or a special vesicle containing an agent for targeted transport of the agent.

[0176] A further aspect of the invention relates to such a conjugate made of antibody, protein or molecule having specific affinity to FKBP1A and agent or agent-containing vesicle.

[0177] Another aspect of the invention relates to the use of an antibody; antibody fragment, protein, protein fragment or molecule having specific affinity to FKBP1A for the production of a drug for the specific isolation and characterization of circulating tumor cells; especially FKBP1A-positive tumors; in particular triple-negative mammary carcinoma, Her2-positive hormone receptor-negative mammary carcinoma or malignant melanoma; in particular triple-negative mammary carcinoma, especially the TNBC mesenchymal stem-like sub-type; especially for diagnosis by liquid biopsy.

[0178] The following examples serve to illustrate the invention, but are not to be interpreted as limiting.

[0179] The following commercial cell lines \, ere used in the experiments below:

TABLE-US-00002 Her2 HR MDA-MB- Triple-negative mammary carcinoma negative negative 231 mesenchymal stem-like MDA-MB- Triple-negative mammary carcinoma negative negative 436 mesenchymal stem-like MDA-MB- Triple-negative mammary carcinoma (positive) negative 453 androgen receptor-positive MDA-MB- Triple-negative mammary carcinoma negative negative 468 basal-like MCF-7 luminal breast cell line negative positive MCF-10A benign breast cell line, i.e. non- positive positive malignant SKBR3 mammary carcinoma Her2-positive positive negative A375 malignant melanoma cells

EXAMPLE 1

[0180] MDA-MB-231 (mesenchymal stem-like), MDA-MB-436 (mesenchymal stem-like), MDA-MB-468 (basal-like), MCF-7: (luminal, HR-positive) and MCF-10A (benign breast cell line) (see FIG. 1A) and MDA-MB-453, SKBR3 (Her2-positive) and A375 (melanoma) (see FIG. 1B) were cultivated in uniform growth medium (RPMI-1640 medium, 10% FOS), washed with PBS, lysed and analyzed by western blot.

[0181] FIG. 1 shows a western blot analysis of the protein expression of FKBP1A. FIG. 1A shows the result of the western blot analysis for MDA-MB-231 and MDA-MB-436: mesenchymal stem-like; MDA-MB-468: basal-like; MCF-7: luminal, HR-positive; MCF-10A: benign breast cell line. FIG. 1A shows that FKBP1A is highly expressed in triple-negative mammary carcinoma cells but, in contrast, is not detectable in hormone receptor-positive mammary carcinoma cells or in non-malignant cells. FIG. 1A shows that FKBP1A is more highly expressed in “mesenchymal stem-like” triple-negative mammary carcinoma cells (MDA-MB-231 and MDA-MB-436) than in basal-like triple-negative mammary carcinoma cells (MDA-MB-468). FIG. 1B shows the result of western blot analysis for Her2-positive, HR-negative mammary carcinoma cells. FIG. 1B shows the high expression of FKBP1A also in cells of Her2-positive, hormone-receptor-negative mammary carcinoma and malignant melanoma.

EXAMPLE 2

[0182] A culture of MDA-MB-231 cells was incubated with siRNA specific to FKBP1A (35202, Thermo Fisher) for three days, the cells washed with PBS and incubated for another three days without the presence of the siRNA. The cells were then washed again with PBS, lysed, and the expression of FKBP1A was analyzed by western blotting.

[0183] FIG. 2 shows the downregulation of FKBP1A protein expression by specific siRNA as a result of western blot analysis of FKBP1A protein expression in MDA-MB-231 cells after incubation with FKBP1A-specific siRNA (S5202, Thermo Fisher).

EXAMPLE 3

[0184] MDA-MB-231 cells were incubated with siRNA specific to FKBP1A (S5202, Thermo Fisher) for three days, the cells washed with PBS and incubated for another three days without the presence of the siRNA. The cells were then washed again with PBS, lysed, the RNA was extracted, cDNA was synthesized, and the expression of different (tumor) stem cell and mesenchymal markers was analyzed by qPCR.

[0185] FIG. 3 shows the result of an expression analysis of (tumor) stem cell and mesenchymal markers after FKBP1A knock-down. Expression analysis was performed using quantitative RT-PCR of described tumor stem cell markers and the mesenchymal marker vimentin relative to mock (=1) after FKBP1A knock down using siRNA in MDA-MB-231 cells after 6 days. FIG. 3A shows the result normalized to GAPDH. FIG. 3B shows the result normalized to HRPT1.

EXAMPLE 4

[0186] A culture of MDA-MB-231 cells a incubated with Inhibitor I for six days, changing the medium after three days in the presence of the inhibitor. The cells were then washed with PBS, lysed and the expression of FKBP1A was analyzed by western blotting.

[0187] FIG. 4 shows the downregulation of FKBP1A protein expression by inhibitor (I) as a result of a western blot analysis of FKBP1A protein expression in MDA-MB-231 cells after incubation with 10 μM inhibitor (I). FIG. 4 shows that agents can induce a reduction in expression of FKBP1A in triple-negative mammary carcinoma.

EXAMPLE 5

[0188] A culture of MDA-MB-231 cells was incubated with Inhibitor I for six days, changing the medium after three days in the presence of the inhibitor. The cells were then washed again with PBS, lysed, the RNA was extracted, cDNA was synthesized and the expression of different (tumor) stem cell and mesenchymal markers as well as PD-L1 analyzed by qPCR.

[0189] FIG. 5 shows the result of an expression analysis of (tumor) stem cell and epithelial differentiation markers after expression reduction of FKBP1A using inhibitor (I). The expression analysis was carried out using quantitative RT-PCR of described (A) tumor stem cell and (B) differentiation markers as well as the (C) immune checkpoint PD-L1 after 6 h incubation of MDA-MB-231 cells with inhibitor (I). FIG. 5 shows that agents in triple-negative mammary carcinoma can bring about a reduction in the expression of (tumor) stem cell markers.