RETINOIC ACID MODIFIED LYSOSOME TARGETING CHIMERA MOLECULE, PREPARATION METHOD AND APPLICATIONS THEREOF

Abstract

A retinoic acid modified lysosome targeting chimera (LYTAC) molecule, a preparation method and applications thereof are provided. A structural formula of the retinoic acid modified LYTAC molecule is shown as following Formula I, in which, n represents a molecular weight of polyethylene glycol (PEG), n is in a range of 60 to 5000, R is one of an antibody, a polypeptide and a micromolecule compound targeting a targeted protein. The retinoic acid modified LYTAC molecule can be applied to targeted protein degradation and preparation of tumor cell activity inhibitors. Retinoic acid has high biosafety, is easy to obtain and modify, can be coupled with any antibody, polypeptide or micromolecule that targets the binding protein receptor to realize lysosomal degradation of extracellular or membrane proteins, can be used to prepare tumor cell activity inhibitors and to treat tumors, inflammation and other diseases, and has a broad application prospect.

##STR00001##

Claims

1. A retinoic acid modified lysosome targeting chimera (LYTAC) molecule, wherein a structural formula of the retinoic acid modified LYTAC molecule is shown as following Formula I: ##STR00007## where n represents a molecular weight of polyethylene glycol (PEG), n is in a range of 60 to 5000, and R is one of an antibody, a polypeptide and a micromolecule compound targeting a targeted protein.

2. The retinoic acid modified LYTAC molecule according to claim 1, wherein the R is one selected from the group consisting of anti-epidermal growth factor receptor (anti-EGFR) antibody cetuximab, anti-programmed cell death 1 ligand 1 (anti-PDL-1) antibody atezolizumab, EGFR-blinding polypeptide GE11, anti-human epidermal growthfactor receptor 2 (anti-HER2) antibody HERCEPTIN, anti-G protein-coupled receptor (anti-GPCR) related antibody, anti-fibroblast growth factor receptor (anti-FGFR) antibody, anti-vascular endothelial growth factor receptor (anti-VEGFR) antibody, anti-cytotoxic t-lymphocyte-associated protein 4 (anti-CTLA4) antibody and anti-interleukin-5 receptor α-subunit (anti-IL-5Ra) antibody.

3. A preparation method of the retinoic acid modified LYTAC molecule according to claim 1, comprising: reacting a retinoic acid (RA) with NH.sub.2-PEG.sub.n-Mal, and then obtaining an intermediate after separation and purification; and reacting the intermediate with the one of the antibody, the polypeptide and the micromolecule compound with a sulfhydryl group targeting the targeted protein, and then obtaining the retinoic acid modified LYTAC molecule after a first post-treatment.

4. The preparation method according to claim 3, wherein the reacting a RA with NH.sub.2-PEG.sub.n-Mal, and then obtaining an intermediate after separation and purification, comprises: dissolving the RA into a solvent, and then adding 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCL) and N-hydroxysuccinimide (NHS) to perform an ice bath reaction for 30 minutes (min) to 60 min to obtain an initial reaction mixture; adding the NH.sub.2-PEG.sub.n-Mal into the initial reaction mixture for reaction at room temperature for 10 hours (h) to 15 h to obtain a reaction mixture, and performing a second post-treatment on the reaction mixture to obtain the intermediate.

5. The preparation method according to claim 4, wherein the second post-treatment comprises: removing the solvent in the reaction mixture to obtain an oily concentrate; dissolving the oily concentrate in a mixed solvent of ultra-pure water and methanol with a volume ratio of 4:1, and then performing the separation and the purification by using preparative high performance liquid chromatography; wherein a mobile phase is a mixed solvent of ultra-pure water and methanol with a volume ratio of 3:7; an elution rate is 4 mL/min; a column temperature is 30° C.; and a retention time is 14 min.

6. The preparation method according to claim 3, wherein a molar ratio of the retinoic acid to the NH.sub.2-PEG.sub.n-Mal is 1:0.5 to 2.

7. The preparation method according to claim 3, wherein the reacting the intermediate with the one of the antibody, the polypeptide and the micromolecule compound with a sulfhydryl group targeting the targeted protein, and then obtaining the retinoic acid modified LYTAC molecule after a first post-treatment, comprises: dissolving the intermediate and the one of the antibody, the polypeptide and the micromolecule compound with the sulfhydryl group targeting the targeted protein in a phosphate buffered saline (PBS) buffer for reaction at room temperature for 1 h to 5 h, removing unreacted raw materials after the reaction, and then drying to obtain the retinoic acid modified LYTAC molecule.

8. The preparation method according to claim 3, wherein a molar ratio of the intermediate to the one of the antibody, the polypeptide and the micromolecule compound with the sulfhydryl group targeting the targeted protein is 8 to 10:1.

9. A method of an application of the retinoic acid modified LYTAC molecule according to claim 1 in performing targeted degradation on one of a membrane protein and an extracellular protein, or treating a disease caused by an expression of one of a membrane protein and an extracellular protein.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0030] FIG. 1 shows mass spectrum test results of RA-PEG2K-Ctx, a product of retinoic acid coupled with anti-EGFR antibody cetuximab prepared in an embodiment 2.

[0031] FIG. 2 shows mass spectrum test results of RA-PEG2K-Ate, a product of retinoic acid coupled with anti-PDL-1 antibody atezolizumab prepared in an embodiment 3.

[0032] FIG. 3A shows an evaluation of a degradation effect of RA-PEG2K-Ctx on an EGFR receptor protein of a lung cancer cell H1975 (after incubation for 24 hours) in an embodiment 4.

[0033] FIG. 3B shows an evaluation of a degradation effect of RA-PEG2K-Ctx on the EGFR receptor protein of a lung cancer cell PC9 (after incubation for 24 hours) in the embodiment 4.

[0034] FIG. 4A shows an evaluation of a degradation effect of RA-PEG2K-Ctx on an EGFR receptor protein of a lung cancer cell H1975 (after incubation for 48 hours) in an embodiment 5.

[0035] FIG. 4B shows an evaluation of a degradation effect of RA-PEG2K-Ctx on the EGFR receptor protein of a lung cancer cell PC9 (after incubation for 48 hours) in the embodiment 5.

[0036] FIG. 5 shows an evaluation of a degradation effect of RA-PEG2K-Ate on a PDL-1 protein of a tumor cell MDA-MB-231 (after incubation for 24 hours) in an embodiment 6.

DETAILED DESCRIPTION OF EMBODIMENTS

[0037] The disclosure is further described below in combination with specific embodiments, but the scope of protection of the disclosure is not limited to this.

[0038] The room temperature of the disclosure is in a range of 25° C. to 30° C.

[0039] Embodiment 1: Synthesis of an Intermediate RA-PEG2K-Mal

##STR00003##

[0040] Adding the retinoic acid (10.2 mg, 34 μmop to dichloromethane (shorted as DCM) to dissolve, then adding 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (shorted as EDC.HCL) (17.1 mg, 90 μmop and N-Hydroxysuccinimide (shorted as NHS) (10.3 mg, 90 μmop to perform an ice bath reaction for half an hour to obtain an initial reaction mixture, and adding NH2-PEG2K-Mal (MW:2000) (34 μmol, 68 mg) to the initial reaction mixture for reaction at room temperature for 12 h to obtain a reaction mixture, rotating and evaporating the reaction mixture to remove dichloromethane to obtain an oily concentrate; after the oily concentrate is dissolved in a mixed solvent of ultra-pure water and methanol with a volume ratio of 4:1, performing separation and purification by using preparative high performance liquid chromatography (a model of a chromatographic column is COSMOSIL 5C18-MS-II 20 mm*250 mm), and eluting with a mixed solvent of ultra-pure water and methanol with a volume ratio of 3:7 as a mobile phase, an elution rate is 4 mL/min, a detection wavelength is 254 nm, a column temperature is 30° C., and a retention time is 14 min, a product RA-PEG2K-Mal is collected, after rotating and evaporating the methanol, freeze-drying was performed to obtain the intermediate RA-PEG2K-Mal with 0.071 g.

[0041] Embodiment 2: Synthesis of RA-PEG2K-Ctx, a Product of the Retinoic Acid Coupled with Anti-EGFR Antibody Cetuximab

##STR00004##

[0042] Incubating the anti-EGFR antibody cetuximab (14.5 mg, 0.1 μmop with tris (2-carboxyethyl) phosphine hydrochloride (shorted as TCEP) (0.28 mg, 1 μmop in 2 ml phosphate buffered saline (PBS) buffer for half an hour, then adding the intermediate RA-PEG2K-Mal (2.4 mg, 1 μmop prepared by the embodiment 1 into the above solution for reaction at room temperature for 3 h, after the reaction, unreacted raw materials were removed by centrifugation with an ultrafiltration centrifuge tube (MWCO 50KD). After freeze-drying, 10.2 mg of LYTAC drug (recorded as RA-PEG2K-Ctx) shown as Formula I was obtained. The mass spectrum is shown in FIG. 1. A molecular weight of cetuximab (shorted as Ctx) is 145779.44, and a molecular weight of the product RA-PEG2K-Ctx is 158465.73, so each antibody is connected with 5 molecules of the retinoic acid.

##STR00005##

[0043] Embodiment 3: Synthesis of RA-PEG2K-Ate, a Product of the Retinoic Acid Coupled with Anti-PDL-1 Antibody Atezolizumab

##STR00006##

[0044] Incubating anti-PDL-1 antibody atezolizumab (14.4 mg, 0.1 μmol) with TCEP (0.28 mg, 1 μmop in 2 ml PBS buffer for half an hour, then adding the intermediate RA-PEG2K-Mal (2.4 mg, 1 μmop prepared by the embodiment 1 into the above solution for reaction at room temperature for 3 h, after the reaction, unreacted raw materials were removed by centrifugation with an ultrafiltration centrifuge tube (MWCO 50KD). After freeze-drying, 10.1 mg of a LYTAC drug (recorded as RA-PEG2K-Ate) shown as Formula I was obtained. The mass spectrum is shown in FIG. 2. A molecular weight of atezolizumab (shorted as Ate) is 144590.5, and a molecular weight of the product RA-PEG2K-Ate is 148797.335, so each antibody is connected with 2 molecules of the retinoic acid.

[0045] Embodiment 4: Evaluations of Degradation Effects of RA-PEG2K-Ctx on EGFR Proteins of Two Tumor Cells (after 24 Hours of Incubation)

[0046] Anti-EGFR antibody cetuximab (10 nM) and RA-PEG2K-Ctx (10 nM) are respectively added into the tumor cells: H1975 cells and PC9 cells. After incubation for 24 hours, the proteins were collected and the expressions of EGFR were detected by western blot (shorted as WB). The results are shown in FIG. 3A and FIG. 3B. EGFR proteins were expressed in both blank group and Ctx treatment group. Compared with the blank group, the EGFR protein in the RA-PEG-Ctx treatment group was degraded by more than 50%. The results showed that the RA-PEG-Ctx has a good degradation effect on the EGFR proteins of the two tumor cells.

[0047] Embodiment 5: Evaluations of Degradation Effects of RA-PEG2K-Ctx on EGFR Proteins of Two Tumor Cells (After 48 Hours of Incubation)

[0048] Anti-EGFR antibody cetuximab (10 nM) and RA-PEG2K-Ctx (10 nM) are respectively added into the tumor cells: H1975 cells and PC9 cells. After incubation for 48 hours, the proteins were collected and the expressions of EGFR were detected by WB. The results are shown in FIG. 4A and FIG. 4B. EGFR proteins were expressed in both blank group and Ctx treatment group. Compared with the blank group, the EGFR protein in the RA-PEG-Ctx treatment group was degraded by more than 80%. The results showed that the RA-PEG-Ctx has a good degradation effect on the EGFR proteins of the two tumor cells.

[0049] Embodiment 6: Evaluation of a Degradation Effect of RA-PEG2K-Ate on PDL-1 Protein of a Tumor Cell MDA-MB-231 (After 24 hours of Incubation)

[0050] Anti-PDL-1 antibody atezolizumab (Ate) (25 nM) and RA-PEG2K-Ate (25 nM) were added to breast cancer cells, i.e., MDA-MB-231 cells respectively. After incubation for 24 hours, the proteins were collected and the expression of PDL-1 were detected by WB. The results are shown in FIG. 5. The expressions of the PDL-1 proteins in the blank group and the Ate treatment group are basically the same. Compared with the blank group, the PDL-1 protein in the RA-PEG-Ate treatment group was degraded by more than 50%. The results showed that the RA-PEG2K-Ate has a good degradation effect on the PDL-1 protein.

[0051] The above-mentioned embodiments have described in detail the technical schemes and beneficial effects of the disclosure. It should be understood that the above-mentioned embodiments are only specific embodiments of the disclosure and are not used to limit the disclosure. Any amendments, supplements and equivalent replacements made within the scope of the principles of the disclosure should be included in the scope of protection of the disclosure.