CN111303300A - Polypeptide inhibitor for degrading CREPT (cell-specific oncogene receptor potential) and application of polypeptide inhibitor in inhibiting pancreatic cancer cell proliferation and tumorigenesis - Google Patents
Polypeptide inhibitor for degrading CREPT (cell-specific oncogene receptor potential) and application of polypeptide inhibitor in inhibiting pancreatic cancer cell proliferation and tumorigenesis Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/82—Translation products from oncogenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
Abstract
The invention discloses a CREPT polypeptide inhibitor and application thereof in inhibiting pancreatic cancer cell proliferation and tumorigenesis. The CREPT polypeptide inhibitor PRTC provided by the invention comprises a targeting module TA, a degradation module DA, a connector and a transmembrane peptide; the amino acid sequence of the targeting module TA is the protein shown in 266-286 site of the amino acid sequence of the CREPT protein; the amino acid sequence of the degradation module DA is protein shown as a sequence 3; the linker is 6-aminocaproic acid; the amino acid sequence of the transmembrane peptide is a protein shown as a sequence 4. The invention designs and prepares PRTC capable of degrading CREPT protein by proteasome approach by using a targeted protein degradation chimera technology. Experiments prove that: PRTC can inhibit the proliferation and migration of pancreatic cancer cells, reduce the tumorigenic capacity of the pancreatic cancer cells, and provide a new idea for treating pancreatic cancer.
Description
Technical Field
The invention relates to the field of molecular targeted drugs, in particular to design and application of a polypeptide inhibitor for targeted degradation of CREPT (cell-specific oncogene receptor potential) protein, and particularly relates to design and application of the polypeptide inhibitor for targeted degradation of the CREPT protein in the process of inhibiting pancreatic cancer cell proliferation and tumorigenesis.
Background
Cancer is one of the most lethal diseases, and seriously harms human health. Traditional therapies, including chemotherapy and radiation therapy, produce a series of side effects because they not only kill cancer cells but also harm normal cells near the cancer. To address this problem, molecular targeted therapies that can target specific pathways have emerged in recent years, which is the basis of precision medicine.
In 2001, targeted protein degradation chimera (PROTACs) technology was beginning to attract attention, combining the advantages of many of the existing traditional targeted cancer therapies. The PROTAC technology not only has cell permeability and the ability of targeted degradation of protein, but also can overcome the problem of drug resistance of cells. The PROTAC molecule consists of a targeting module, a degradation module and a connecting body. Existing PROTAC molecules are classified into polypeptide PROTAC and small molecule PROTAC. The targeting module can specifically recognize intracellular specific proteins using small molecules or polypeptide molecules, and once the targeting module binds to the target, the degradation module can pull the target protein onto E3 ubiquitin ligase and degrade it via the proteasome pathway. This technique allows the selective degradation of intracellular proteins rather than merely inhibiting the activity of the target protein. Currently, ProTAC technology can target a range of oncoproteins including nuclear receptors, protein kinases, etc., intracellularly.
CREPT (RPRD1B) is the oncogene that the applicant of the present patent first cloned. Research shows that CREPT plays an important role in promoting cell proliferation and tumor formation. Immunohistochemistry results show that CREPT is expressed at high levels in various cancer tissues compared to paracancerous tissues. Mechanistically, CREPT not only regulates the cell cycle, it also plays an important role in DNA damage repair.
Disclosure of Invention
It is a first object of the invention to provide a protein degradation chimera (PROTAC) that targets CREPT.
The protein degradation chimera targeting CREPT provided by the invention comprises a degradation module, a targeting module and a connector for connecting the targeting module and the degradation module;
the amino acid sequence of the targeting module is the protein shown in 266-286 of the amino acid sequence of the CREPT protein (SEQ ID NO: 1).
Furthermore, the amino acid sequence of the targeting module is specifically a protein (KDVLSEKEKKLEEYKQKLARV) shown in sequence 2, and is a ligand combined with the target protein CREPT.
The amino acid sequence of the degradation module is a protein (IYP (OH) AL) shown as a sequence 3, and is reacted with E3Ligase knot A ligand of the complex. Wherein P (OH) represents hydroxyproline.
The linker is specifically 6-aminocaproic Acid (AHX).
Furthermore, the protein degradation chimera further includes a transmembrane peptide for increasing cell permeability, and the transmembrane peptide, the degradation module, the linker, the targeting module and the transmembrane peptide are sequentially comprised.
The amino acid sequence of the transmembrane peptide is specifically protein (RRRRRRK) shown in sequence 4.
The CREPT-targeted protein degradation chimera is polypeptide PROTAC, and the structural formula is as follows: IYP (OH) AL-AHX-KDVLSEKEKKLEEYKQKLARV-RRRRK. The protein degradation chimera can be synthesized by itself or by related companies or units. In a specific embodiment of the invention, the CREPT-targeted protein degradation chimeras are synthesized by shanghai intense biotechnology limited.
The CREPT-targeting protein degradation chimera has the function of degrading CREPT, and the action mechanism is as follows: the targeting module can specifically recognize and bind to the target protein CREPT in a cell (such as a cancer cell, more specifically a pancreatic cancer cell), and once the targeting module binds to the target protein CREPT, the degradation module can pull the target protein CREPT onto E3 ubiquitin ligase and degrade it via the proteasome pathway.
The second purpose of the invention is to provide a new application of the protein degradation chimera targeting CREPT.
The invention provides an application of the protein degradation chimera targeting CREPT in any one of the following B1) -B10):
B1) degrading CREPT protein;
B2) preparing a product for degrading CREPT protein;
B3) preventing and/or treating cancer or tumor;
B4) preparing a product for preventing and/or treating cancer or tumor;
B5) inhibiting cancer cell or tumor cell proliferation;
B6) preparing a product for inhibiting the proliferation of cancer cells or tumor cells;
B7) inhibiting cancer cell or tumor cell migration;
B8) preparing a product for inhibiting the migration of cancer cells or tumor cells;
B9) inhibiting cancer cell or tumor cell neoplasia;
B10) preparing the product for inhibiting cancer cell or tumor cell from forming tumor.
A third object of the invention is to provide a product.
The active ingredient of the product provided by the invention is the protein degradation chimera of the targeted CREPT;
the product has the functions of any one of the following C1) -C5):
C1) degrading CREPT protein;
C2) preventing and/or treating cancer or tumor;
C3) inhibiting cancer cell or tumor cell proliferation;
C4) inhibiting cancer cell or tumor cell migration;
C5) inhibiting cancer cell or tumor cell tumor formation.
In the above application or product, the inhibition of cancer cell or tumor cell neoplasia is embodied in reducing tumor volume and/or reducing tumor mass.
The cancer may in particular be pancreatic cancer.
The cancer cell may specifically be a pancreatic cancer cell.
In the protein degradation chimera or the application or the product, the amino acid sequence of the CREPT protein is a sequence 1.
The present invention provides a cell-permeable polypeptide-type ProTAC molecule. Experiments prove that: the polypeptide type PROTAC molecule can accurately degrade CREPT in pancreatic cancer cells, thereby realizing the inhibition of pancreatic cancer cell proliferation and tumor formation.
Drawings
Figure 1 shows PRTC degrades CREPT protein. FIG. 1A shows the results of measuring the expression level of CREPT protein after the treatment of Panc-1 cells with PRTC at different concentrations. FIG. 1B shows the results of measuring the CREPT protein expression level of Panc-1 cells treated with PRTC at different times.
FIG. 2 shows the effect of PRTC on the ability of tumor cells to migrate and proliferate. FIG. 2A shows the identification of CREPT knockout Panc-1 cells. FIG. 2B is a graph of the effect of PRTC on the ability of tumor cells to migrate. FIG. 2C is a graph of the effect of PRTC on the proliferative capacity of tumor cells. Wherein Ctrl is ddH for Panc-1 cells2O treatment for 24 hours; CREPT-/-CREPT knockout Panc-1 cells Using ddH2O treatment; PRTC for Panc-1 cells treated with 10. mu.M PRTC for 24 hours.
FIG. 3 is a graph of the effect of PRTC on the tumorigenic capacity of cancer cells. Figure 3A is a comparison of tumor sizes for each mouse model. FIG. 3B is a comparison of tumor weights for various mouse models. Figure 3C is a comparison of tumor volume size for each mouse model. Wherein Ctrl is 5 × 10 injected subcutaneously in nude mice6Injecting 0.9% normal saline into the abdominal cavity of each Panc-1 cell for 1 time and 14 times every other day; CREPT-/-injection of 5X 10 subcutaneously in nude mice6Intraperitoneal injection of 0.9% physiological saline is performed on each CREPT knockout Panc-1 cell for 1 time and 14 times; PRTC was injected subcutaneously into nude mice at 5X 106Each Panc-1 cell was injected intraperitoneally with 10mg/kg PRTC 1 times every other day for 14 times.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 design and Synthesis of CREPT degrading PROTAC molecules
PROTAC molecule design for degrading CREPT
The CREPT degrading PROTAC molecule designed by the invention comprises the following four parts: a targeting module, a degradation module, a linker, and a transmembrane peptide. The specific selection conditions of the components are as follows:
1. selection of targeting modules
(1) The sequence shown in the 266-286 site of the amino acid sequence of the CREPT protein shown in the sequence 1: KDVLSEKEKKLEEYKQKLARV as a targeting module for the ProTAC molecule.
(2) Three leucines (L) critical for forming a dimer in the targeting module PRTC sequence (KDVLSEKEKKLEEYKQKLARV) designed in step (1) were mutated to proline (P) to obtain the following sequence: KDVPSEKEKKPEEYKQKPARV, and using the sequence as a mutant of the targeting module of the ProTAC molecule.
2. Selection of degradation modules
Sequence to be bound to E3 ligase: IYP (OH) AL acts as a degradation module for the PROTAC molecule. Wherein "P (OH)" represents hydroxyproline.
3. Selection of connectors
6-aminocaproic Acid (AHX) is used as a linker of the PROTAC molecule, and the targeting module and the degradation module are connected through two acid-amine condensation reactions.
4. Selection of transmembrane peptides
To increase the cell permeability of the PROTAC molecule, the following transmembrane peptide sequences are ligated at the C-terminus of the PROTAC molecule: RRRRK.
Secondly, synthesizing PROTAC molecule for degrading CREPT
And (3) entrusting Shanghai Qiang Biotechnology Limited to synthesize the following PROTAC molecules according to the degradation module, the connecting body, the targeting module and the transmembrane peptide which are designed in the step one:
PRTC: the PROTAC molecule, having iyp (oh) AL as the degradation module, 6-aminocaproic Acid (AHX) as the linker, KDVLSEKEKKLEEYKQKLARV as the targeting module, and RRRRK as the transmembrane peptide, is designated PRTC, which has the following specific structural formula: IYP (OH) AL-AHX-KDVLSEKEKKLEEYKQKLARV-RRRRK.
PRTC-m: the PROTAC molecule with IYP (OH) AL as the degradation module, 6-aminocaproic Acid (AHX) as the linker, KDVPSEKEKKPEEYKQKPARV as the mutant of the targeting module, and RRRRRRK as the transmembrane peptide is designated PRTC-m, which has the following specific structural formula: IYP (OH) AL-AHX-KDVPSEKEKKPEEYKQKPARV-RRRRK.
Example 2 binding ability of PRTC to CREPT
The binding ability of PRTC designed in example 1 to CREPT was tested using a microcalorimetric thermophoresis experiment (MST). The method comprises the following specific steps:
1. it was determined by preliminary experiments that 100nM of FITC-labeled PRTC could produce the optimum fluorescence.
2. And (3) taking FITC marked PRTC as a detection ligand, diluting the purified CREPT protein solution with the concentration of 4 mu M for 15 times in a 2-fold dilution manner to obtain 16 CREPT protein solutions with concentration gradients, and taking the CREPT protein solutions as detection targets.
3. The dissociation constant of PRTC and CREPT is 0.34+/-0.11 mu M by selecting a binding constant detection program in a microcalorimetric electrophoresis apparatus.
The binding ability of PRTC-m designed in example 1 to CREPT was tested as described above.
The results show that: PRTC-m was unable to bind to CREPT.
Example 3 application of PRTC in degradation of CREPT protein
First, PRTC treatment of Panc-1 cells at various concentrations
Mix 8X 105A Panc-1 cell is inoculated on a 6cm culture dish and cultured by a DMEM medium containing 10% (volume fraction) FBS to obtain a Panc-1 cell culture system. Adding PRTC to the Panc-1 cell culture system to make the final concentration in the cell culture system be 0. mu.M, 5. mu.M, 10. mu.M and 20. mu.M respectively, treating for 24 hours, and passing through Western blotthe expression level of CREPT in cells is detected by t experiment.
The results are shown in FIG. 1A. The results show that: when the PRTC concentration reaches 10 mu M, the CREPT protein expression level is obviously reduced. Indicating that PRTC can degrade CREPT protein.
Two, PRTC treatment of Panc-1 cells at different times
Mix 8X 105A Panc-1 cell is inoculated on a 6cm culture dish and cultured by a DMEM medium containing 10% (volume fraction) FBS to obtain a Panc-1 cell culture system. Adding PRTC into the Panc-1 cell culture system to enable the final concentration of the PRTC in the cell culture system to be 10 mu M, and detecting the expression level of CREPT in cells by a Western blot experiment after treating for 0 hour, 12 hours, 24 hours and 48 hours respectively.
The results are shown in FIG. 1B. The results show that: CREPT expression levels began to decline after 12 hours of treatment with 10 μ M PRTC until CREPT was almost completely degraded after 48 hours of treatment.
Example 4 application of PRTC in inhibition of tumor cell proliferation, migration and neoplasia
Preparation of CREPT knockout Panc-1 cells
1. Construction of CREPT knockout Cas9 vector
The CREPT-knocked out Cas9 vector (containing GFP) in this example is The "The CRISPR/Cas9(clustered interleaved short palindromic repeat/CRISPR-associated 9) -mediated CRISPR deletion plasmid" in The "CREPT/RPRD 1 bases with Aurora B to regulated Cyclin B1expression for accessing crystalline G2/M translation in targeted plasmid", and The specific construction procedure is as in The "CREPT/RPRD 1B organized with Aurora B to regulated Cyclin B1expression for accessing The" CREPT 2/M translation in targeted plasmid "method of" CREPT/RPRD1 linked with CRISPR/modified protein-9 (clustered CRISPR-linked CRISPR-9 ".
2. Acquisition of CREPT knockout Panc-1 cells
Laying human pancreatic cancer cells Panc-1 to be transfected one day in advance, transfecting the CREPT knockout Cas9 vector in the step 1 into the human pancreatic cancer cells Panc-1 by using Viofect, and screening cells containing GFP by a flow cytometer after transfecting for 48 hours to obtain CREPT knockout Panc-1 cells.
3. Identification of CREPT knockout Panc-1 cells
The CREPT knockout Panc-1 cells were identified using Western blot technique (primary antibodies used in Western blot are described in the literature: fragmentation of monoclonal antibodies against CREPT, alpha novel proteinaceous expressed in tumors, secondary antibodies are goat anti-mouse secondary antibodies purchased from Jackson ImmunoResearch) with wild type Panc-1 cells as controls.
The Western blot detection results are shown in FIG. 2A. The results show that: CREPT knockdown successful CREPT knockdown in Panc-1 cells.
Application of PRTC (platelet-rich protoxin-like) in regulation of proliferation and migration capacity of tumor cells
1. Effect of PRTC on the migratory Capacity of tumor cells
(1) Panc-1 cells (ctrl, containing wild-type CREPT) and CREPT knockout Panc-1 cells (CREPT-/-, containing no CREPT) were seeded in 6-well plates, respectively, and cultured to a density of 95% using DMEM medium containing 10% (volume fraction) FBS. The cells were then washed 3 times with PBS using a pipette tip to draw a straight line and the suspended cells were washed away.
(2) And (2) adding DMEM (DMEM) medium without FBS (fetal bovine serum) into the 6-well plate obtained in the step (1) to obtain a Panc-1 cell culture system and a CREPT knockout Panc-1 cell culture system.
Treatment 1 (Ctrl): adding equal volume of ddH into the Panc-1 cell culture system2O。
Treatment group 2 (CREPT-/-): adding equal volume of ddH into CREPT knockout Panc-1 cell culture system2O。
Treatment group 3(PRTC (10 μ M)): adding PRTC solution (ddH as solvent) into the Panc-1 cell culture system2O) so that the final concentration of PRTC in the cell culture system was 10 μ M.
Treatment group 4(PRTC-M (10. mu.M)): adding PRTC-m solution (ddH as solvent) into the Panc-1 cell culture system2O), the final concentration of PRTC-M in the cell culture system was 10. mu.M.
(3) After 24 hours of treatment, the migration distance was observed with a microscope and photographed, the migration distance was measured with image J software, and prism was counted for each treatment group. Three replicates per treatment group were performed.
The results are shown in FIG. 2B and Table 1. The results show that: migrating PRTC can inhibit the ability of tumor cells to migrate.
TABLE 1
Note: the values in the table represent the distance of the 24h scratch divided by the distance of the 0 hour scratch; a larger number indicates a smaller cell migration ability.
2. Effect of PRTC on tumor cell proliferation Capacity
(1) Panc-1 cells (Ctrl, containing wild-type CREPT) and CREPT knockout Panc-1 cells (CREPT-/-, containing no CREPT) were inoculated into 6-well plates, 500 cells were inoculated per well, and cultured using DMEM medium containing 10% (volume fraction) FBS, to obtain a Panc-1 cell culture system and a CREPT knockout Panc-1 cell culture system, respectively.
(2) The cell treatment was performed according to each of the following treatment groups:
treatment group 5 (Ctrl): adding equal volume of ddH into the Panc-1 cell culture system2O。
Treatment group 6 (CREPT-/-): adding equal volume of ddH into CREPT knockout Panc-1 cell culture system2O。
Treatment group 7(PRTC (10. mu.M) adding PRTC solution (ddH as solvent) to the Panc-1 cell culture System2O) so that the concentration of PRTC in the cell culture system was 10 μ M.
Treatment group 8(PRTC-M (10. mu.M) adding PRTC-M solution (ddH as solvent) to Panc-1 cell culture System2O), the concentration of PRTC-M in the cell culture system was set to 10. mu.M.
(3) After treatment, the cells were co-cultured for 10 days, washed with PBS, and stained with 0.1% crystal violet. The number of colonies was counted by software Image J, and the colony area (colony formation area) was counted for each treatment group by prism. Three replicates per treatment group were performed.
The results are shown in FIG. 2C and Table 2. The results show that: PRTC can inhibit tumor cell proliferation.
TABLE 2
Application of PRTC (platelet-rich protoxin-transferase) in regulation of cancer cell tumorigenicity
The influence of PRTC on the tumor forming capability of cancer cells is detected by adopting a nude mouse tumor forming experiment. The method comprises the following specific steps:
1. respectively mixing 5 × 106A Panc-1 cell (Ctrl containing wild type CREPT) and a CREPT knockout Panc-1 cell (CREPT-/-, containing no CREPT) are injected subcutaneously under the armpit of a nude mouse (Balb/c nude, four-week old female mouse, a product of Beijing Wintolite laboratory animal technologies Co., Ltd.) to respectively obtain a CREPT wild type cell subcutaneous tumorigenic mouse model and a CREPT knockout cell subcutaneous tumorigenic mouse model.
2. The mouse models were treated as follows:
treatment group 9 (Ctrl): 100 μ l of 0.9% physiological saline was intraperitoneally injected 1 time every other day for 14 times in a CREPT wild type cell subcutaneous tumorigenic mouse model.
Treatment group 10 (CREPT-/-): 100 μ l of 0.9% physiological saline was intraperitoneally injected 1 time every other day for 14 times in a CREPT knockout cell subcutaneous tumorigenic mouse model.
Treatment group 11 (PRTC): to CREPT wild type cell subcutaneous tumor mouse model every other day, 100 μ l PRTC solution (solvent is 0.9% physiological saline) is injected intraperitoneally 1 time, the PRTC injection dose per kg mouse is 10mg, and the total injection time is 14 times.
Tumors were taken from 5 mice per treatment group for 4 weeks, on day 28 of treatment, from each treatment group, tumor length and width, and tumor mass were recorded, and tumor volume was calculated.
The results are shown in FIG. 3 and Table 3. The results show that: indicating that PRTC can inhibit the tumorigenicity of cancer cells.
TABLE 3
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> CREPT (cell-specific oncogene receptor) degrading polypeptide inhibitor and application thereof in inhibiting pancreatic cancer cell proliferation and tumorigenesis
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Claims (10)
1. A CREPT-targeted protein degradation chimera comprising a targeting module, a degradation module, a linker for connecting the targeting module and the degradation module;
the amino acid sequence of the targeting module is the protein shown at position 266-286 of the amino acid sequence of the CREPT protein.
2. The protein degradation chimera of claim 1, wherein: the amino acid sequence of the targeting module is a protein shown in a sequence 2.
3. The protein degradation chimera of claim 1 or 2, wherein: the amino acid sequence of the degradation module is protein shown in a sequence 3.
4. The protein degradation chimera of any one of claims 1-3, wherein: the linker is 6-aminocaproic acid.
5. The protein degradation chimera of any one of claims 1-4, wherein: the protein degradation chimera further includes a transmembrane peptide;
or the amino acid sequence of the transmembrane peptide is a protein shown as a sequence 4.
6. The protein degradation chimera of any one of claims 1-5 in any one of the following B1) -B10):
B1) degrading CREPT protein;
B2) preparing a product for degrading CREPT protein;
B3) preventing and/or treating cancer or tumor;
B4) preparing a product for preventing and/or treating cancer or tumor;
B5) inhibiting cancer cell or tumor cell proliferation;
B6) preparing a product for inhibiting the proliferation of cancer cells or tumor cells;
B7) inhibiting cancer cell or tumor cell migration;
B8) preparing a product for inhibiting the migration of cancer cells or tumor cells;
B9) inhibiting cancer cell or tumor cell neoplasia;
B10) preparing the product for inhibiting cancer cell or tumor cell from forming tumor.
7. A product comprising as an active ingredient the protein degradation chimera of any one of claims 1-5;
the product has the functions of any one of the following C1) -C5):
C1) degrading CREPT protein;
C2) preventing and/or treating cancer or tumor;
C3) inhibiting cancer cell or tumor cell proliferation;
C4) inhibiting cancer cell or tumor cell migration;
C5) inhibiting cancer cell or tumor cell tumor formation.
8. Use according to claim 6 or product according to claim 7, characterized in that: the inhibition of cancer cell or tumor cell neoplasia is embodied in a reduction of tumor volume and/or a reduction of tumor mass.
9. Use or product according to any of claims 6 to 8, characterized in that: the cancer is pancreatic cancer.
10. Use or product according to any of claims 6 to 8, characterized in that: the cancer cell is a pancreatic cancer cell.
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