CN110964086A

CN110964086A - High-affinity polypeptide of integrin β 3 receptor and application

Info

Publication number: CN110964086A
Application number: CN201811165550.3A
Authority: CN
Inventors: 张蕾; 顾婷婷; 单雪; 张瑜; 陈建美; 宁兴海
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2018-09-28
Filing date: 2018-09-28
Publication date: 2020-04-07
Anticipated expiration: 2038-09-28
Also published as: CN110964086B

Abstract

The invention discloses a polypeptide of integrin β 3 receptor with high affinity, X is 1-5 amino acids, the amino acids are natural essential amino acids or non-essential amino acids, called RWRX for short, the invention exemplifies that a polypeptide RWRNR has strong affinity and selectivity to cells with high expression of integrin receptor β 3, the uptake in the cells with high expression of integrin receptor β 3 is higher than that of the cells with low expression, the invention exemplifies that the affinity of the polypeptide to recombinant protein of integrin receptor β 3 is higher, the developer obtained after the coupling of the exemplified polypeptide and molecular imaging probe can realize molecular imaging to tumors with over expression of integrin β 3, and has good application prospect to diagnosis of related diseases of integrin receptor β 3, the invention exemplifies that the drug delivery system modified by targeting polypeptide can selectively deliver chemotherapeutic drugs to the cells with high expression of integrin β 3, and can be placed in cell nucleus, thereby greatly improving the efficacy of chemotherapeutic drugs and reducing side effects, the polypeptide has good application prospect to treatment of related diseases of integrin β 3 in the invention.

Description

High-affinity polypeptide of integrin β 3 receptor and application

Technical Field

The invention relates to the technical field of bioengineering pharmacy, protein polypeptide drugs and biomedical engineering, in particular to a polypeptide which has high affinity for an integrin receptor β 3, is applied to targeted vaccines, diagnostic reagents, screening reagents, therapeutic drug molecules or targeted drug delivery systems and the like, and has important significance for disease diagnosis and treatment.

Background

Integrin β family, including integrin α v α and α IIb α, are cell adhesion molecules that mediate the interaction of the extracellular environment with the cytoplasm in some diseases, such as acute and chronic liver kidney disease, rheumatoid arthritis, adenomyosis of uterus, thrombosis, etc., and the abnormal expression of integrin α 23 in the foci, and is directly related to the malignancy of the disease, is an important target for diagnosis and treatment of these diseases.

Currently, these RGD-mimetic peptides, clinically used as drug molecules, have shown superior efficacy in treating brain gliomas, but have failed in The evaluation of phase III clinical trials due to their half-life and pharmacokinetic profiles limiting their anti-angiogenic properties (Stupp R, Picard M, Weller M. Does clinical drug delivery and drug delivery? -animals' reuse [ J ]. The Lancet Oncolog, 2014, 15 (13): 585-.

However, in the prior art, no specific ligand with high affinity for integrin β 3 has been found, which hinders the progress of preclinical and clinical applications of integrin β as a target in cancer therapy.

Disclosure of Invention

The invention aims to provide a targeting polypeptide related to integrin receptor β 3, which is applied to an imaging agent, a receptor antagonist, an anticoagulant, an anti-angiogenesis agent and a drug carrier of related diseases, and the application range is not limited by the examples.

The sequence of the polypeptide of the invention is arginine-tryptophan-arginine-X, X is 1-5 amino acids, the amino acids are natural amino acids or amino acid analogs, RWRX for short, in the integrin β 3 high affinity polypeptide in claim 1, when the motif of the pentapeptide is Arg-Trp-D-Arg-X₁-X₂When, X₁，X₂Is natural amino acid or amino acid analogue, called RwrX for short₁X₂. Wherein when X is₁When it is Asn, X₂Not simultaneously Met.

In order to detect the polypeptide-coupled targeted diagnosis or screening reagent of the invention for diagnosing diseases related to integrin β, taking arginine-tryptophan-arginine-aspartic acid-arginine (RWrNR) as an example, one of the polypeptides of the invention, the binding capacity of the polypeptide and integrin β 3 recombinant protein is detected by a microcalorimetric electrophoresis method, and the result shows that the polypeptide has better affinity.

The invention also provides a preparation method of the RWrNR modified DOX-entrapped liposome (RWrNR-LS-DOX) nano drug delivery system. The method comprises the following steps:

weighing phospholipid, cholesterol, DSPE-PEG and RWrNR-PEG-DSPE according to the prescription amount, wherein the mass ratio of the phospholipid, the DSPE-PEG and cholesterol is 5-20: 20-40: 0.1-2, the content of the RWrNR-PEG-DSPE in the prescription is 0.1-0.5 mol%, and the components are dissolved in a mixed solvent after being weighed. The mixed solvent is composed of chloroform and methanol, and is prepared according to the volume ratio of 2: 1. After the solution was evaporated to dryness at 30 ℃ under reduced pressure to form a film, it was hydrated with 5mL of an ammonium sulfate solution (250mM, pH 7.4), followed by sonication with an ultrasonic cell disrupter in an ice water bath (10min, 10%). After passing through a 0.22 μm microfiltration membrane, the liposome was dialyzed in 900mL of HEPES buffer (10mM, pH 7.4, 0.1% EDTA, 10% sucrose) for 24 hours to remove free ammonium sulfate from the solution, thereby obtaining RWRNR-modified liposomes. Mixing the obtained liposome with 4mg DOX solution, adjusting pH to 7.9, standing at room temperature for 1h, and then removing free doxorubicin hydrochloride by Sephadex G50 column to obtain drug-loaded liposome RWrNR-LS-DOX.

The invention also provides a DOX-entrapped liposome (RWrNR-LS-DOX) nano drug delivery system modified by RWrNR, which is applied to the treatment of tumors.

RWRNR-LS-DOX through integrin β 3 mediated active transport can deliver more DOX into MDA-MB-231 and PC-3 tumor cells, and in addition, since PC-3 cells express integrin β 3 in both cell membrane and nucleus, DOX can be further delivered into nucleus to improve drug efficacy, enhance its anti-tumor effect and reduce side effects.

The targeted polypeptide of the invention β 3 has the following beneficial effects that the targeted polypeptide has better selectivity and affinity to integrin β 3, and also shows better targeting in vivo, and provides a new strategy for diagnosis and treatment of diseases, and the related results of the targeted polypeptide in the invention prove that the targeted polypeptide modified drug delivery system of the invention can selectively deliver chemotherapeutic drugs to integrin β 3 high-expression tumor cells and further deliver the chemotherapeutic drugs to the cell nucleus of the tumor cells with perinuclear high-expression integrin β 3, thereby greatly improving the curative effect of the chemotherapeutic drugs and reducing side effects.

Drawings

FIG. 1 is a flow cytometer for detecting the affinity of rhodamine B labeled integrin β 3 targeting polypeptide for different tumor cells;

FIG. 2 is a diagram showing the uptake of rhodamine B labeled integrin β 3 targeting polypeptide in different tumor cells observed in a laser confocal cell uptake experiment;

FIG. 3 is a microcalorimetric electrophoresis method for detecting the affinity of integrin β 3 targeting polypeptide and integrin β 3 recombinant protein;

FIG. 4 shows the detection of tumor enrichment of integrin β 3-targeted polypeptide in vivo by near infrared fluorescence imaging;

FIG. 5 shows the distribution of integrin β 3 targeting polypeptide in visceral organs detected by near infrared fluorescence imaging technique;

FIG. 6 is a particle size distribution of RWRNR-LS-DOX nanoparticles and TEM image;

FIG. 7 is serum stability at 24 hours for RWrNR-LS-DOX;

FIG. 8 is a graph of the distribution of RWRNR-LS-DOX in MDA-MB-231 and PC-3 cells;

FIG. 9 is a graph of the in vitro anti-tumor activity of RWRNR-LS-DOX against MDA-MB-231 and PC-3 cells;

FIG. 10 is an in vivo profile of RWRNR-LS-DOX in MDA-MB-231 and PC-3 tumor-bearing mice;

FIG. 11 is a graph of the antitumor activity of RWRNR-LS-DOX in MDA-MB-231 and PC-3 tumor-bearing mice.

Detailed description of the preferred embodiments

The present invention will be described in further detail below with reference to specific embodiments and drawings, but the present invention is not limited thereto.

Example 1 flow cytometry detection of integrin β 3 targeting polypeptide for multiple cell affinities

Taking MDA-MB-231 cells, PC-3 cells, DU-145 cells, MCF-7 cells, NHBE cells and L02 cells in logarithmic growth phase according to the ratio of 10⁶Each cell is inoculated into a 6-well plate, 2mL of culture medium is added into each well, after 24 hours of culture, the upper layer of culture medium is discarded, 10 mu M of RhB-labeled integrin targeting polypeptide RhB-RWrNR is added, and the cells are cultured at 37 ℃. After 2h, the cells were washed three times with PBS, the polypeptides not taken up on the surface were washed off, after trypsinization, the cells were gently blown down and collected in a 1.5mL centrifuge tube at a speed of 800rmpAnd centrifuging for 3min, resuspending with PBS, repeating for three times, detecting the affinity of rhodamine-labeled polypeptide and six cells by a flow cytometer, selecting RhB-c-RGDyK as a control, and making 3 auxiliary holes for each experiment. All procedures need to be protected from light.

The fluorescence ratios of RhB-RWrNR and RhB-c-RGDyK in six strains of cells are shown in FIG. 1, and c-RGDyK is used as a positive control because its main binding sites are α v β 5 and α v β 3. in MDA-MB-231, PC-3 and DU-145 cells, the fluorescence ratio is greater than 1, and the result shows that the polypeptide RWrNR has higher affinity for cells with high integrin β 3 expression compared with c-RGDyK. compared with MCF-7, NHBE and L02 cell line, the ratio is less than 1, which further shows that RWrNR has lower affinity for cells with low integrin β 3 expression compared with c-RWrDGdyK.

Example 2 confocal laser investigation of integrin β 3 targeting peptide uptake in various cells

Taking MDA-MB-231 cells, PC-3 cells, DU-145 cells, MCF-7 cells, NHBE cells and L02 cells in logarithmic growth phase according to 10⁵Each well was inoculated into a NEST dish, and after 24h of culture, the supernatant was replaced with a blank medium containing RhB-RWrNR at a final concentration of 10. mu.M, and RhB-c-RGDyK was selected as a positive control and cultured at 37 ℃ for 2 h. After two washes with PBS, Hoechst was stained with nuclear staining solution for 15min, and excess staining solution was washed with PBS and observed using laser confocal.

As shown in FIG. 2, the uptake of RhB-RWrNR was higher in β 3-highly expressed MDA-MB-231, PC-3 and DU-145 cells than in RhB-c-RGDyK, and the uptake of RhB-RWrNR was lower in β 3-lowly expressed MCF-7, NHBE and L02 cells than in RhB-c-RGDyK. furthermore, in PC-3 cells, the fluorescence signal of RhB-RrNR was found to be concentrated in the nucleus as well as the cytoplasm, indicating that polypeptide RWrNR could further effectively target to integrin receptor α IIb β 3 of the nucleus after entering cells by binding integrin α v β 3 on the surface of PC-3 cells, and was concentrated in the nucleus in large quantities.

Example 3 microcalorimetric electrophoresis determination of the affinity of integrin targeting peptide to integrin β 3 recombinant protein

The interaction force of the target polypeptide RWrNR, c-RGDyK and human recombinant integrin β 3 protein is measured by using microcalorimetry electrophoresis, the NT-647 fluorescence labeled integrin β 3 protein is incubated with 13 concentration gradient polypeptides RWrNR and c-RGDyK for 0.5h at room temperature, the thermophoretic change of the polypeptide and the fluorescence labeled protein is detected by a thermophoresis method, and the K value is calculated by using a NanoTempersoftware system.

As shown in FIG. 3, the Kd values of RWrNR and c-RGDyK were calculated by curve fitting to be 0.21. + -. 0.02nM and 3.2. + -. 0.06nM, respectively, indicating that the affinity of RWrNR for integrin β 3 receptor was much higher than that of c-RGDyK.

Example 4 in vivo imaging experiments to investigate the in vivo targeting effects of integrin targeting polypeptides

Left axillary injection of male BALB/c nude mice 3X 10⁶One PC-3 tumor cell was used to construct PC-3 tumor model, while female BALB/c nude mice were injected with 1X 10 axilla right⁷Each MDA-MB-231 tumor cell was used to construct a tumor model for MDA-MB-231. The size of the tumor was measured using a vernier caliper, and the formula was: volume is 0.5 x long diameter x short diameter². When the tumor size reaches 400-³In time, each nude mouse was injected intravenously with 0.2mL of Cy5.5-labeled RWRNR (10mM) at the tail. Imaging was performed by a fluorescence imaging system at 1, 2, 4, 6, 8h, respectively. At 8h after injection, tumor-bearing mice were sacrificed by anesthesia, and tumors and major organs were dissected out, including: heart, liver, spleen, lung, and kidney for in vitro imaging.

As shown in figure 4, the fluorescence signal of Cy5.5-RWrNR reaches the tumor parts of MDA-MB-231 and PC-3 in 1h, the signal intensity of the tumor parts is increased along with the passage of time, and the fluorescence signal continues until 8h after injection, which shows better tumor targeting of RWrNR, 8h after injection, the tumor-bearing mice are sacrificed, and organs and tumor tissues are collected after dissection, as shown in figure 5, the tumor parts of MDA-MB-231 and PC-3 show obvious fluorescence signals after organ tissue imaging, which shows that RWrNR shows better in-vivo targeting to an integrin β 3 receptor, and can be applied to the diagnosis of tumors.

Example 5 preparation and characterization of RWrNR-LS-DOX NanoTayer delivery System

Weighing phospholipid, cholesterol, DSPE-PEG and RWrNR-PEG-DSPE according to the prescription amount, wherein the mass ratio of the phospholipid, the DSPE-PEG and the cholesterol is 5-20: 20-40: 0.1-2, the content of the RWrNR-PEG-DSPE in the prescription is 0.1-0.5 mol%, and the components are dissolved in a mixed solvent after being weighed. The mixed solvent is composed of chloroform and methanol, and is prepared according to the volume ratio of 2: 1. After the solution was evaporated to dryness at 30 ℃ under reduced pressure to form a film, it was hydrated with 5mL of an ammonium sulfate solution (250mM, pH 7.4), followed by sonication with an ultrasonic cell disrupter in an ice water bath (10min, 10%). After passing through a 0.22 μm microfiltration membrane, the liposome was dialyzed against 900mL of HEPES buffer (10mM, pH 7.4, 0.1% EDTA, 10% sucrose) for 24 hours to remove free ammonium sulfate from the solution. Mixing the obtained liposome with 4mg DOX solution, adjusting pH to 7.9, standing at room temperature for 1h, and then removing free doxorubicin hydrochloride by Sephadex G50 column to obtain drug-loaded liposome RWrNR-LS-DOX. The particle size and particle size distribution were measured by a malvern laser particle sizer and the morphology was observed by TEM.

The phospholipid is one of soybean phospholipid, lecithin, hydrogenated soybean phospholipid, dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE), but is not limited to the illustrative range. The molecular weight range of PEG in the DSPE-PEG is 500-5000 Da. The molecular weight range of EG in the RWrNR-PEG-DSPE is 500-5000 Da.

As shown in FIG. 6, the nano drug delivery system shown in 6- (a) is spherical and uniformly distributed, and has a liposome membrane structure shown in 6- (b). As shown in FIG. 6, the nanoparticle size was about 150nm, and the PDI was 0.166

Example 6 serum stability at 24 hours of RWrNR-LS-DOX

Mixing freshly prepared drug-loaded liposome RWrNR-LS-DOX and fetal calf serum in a volume ratio of 1: 1 in equal volume, performing parallel operation in three parts, and shaking in a shaker at 37 ℃ at 300 rmp. The particle size of the liposomes and the release of DOX were measured at 1, 2, 4, 6, 8 and 24h, respectively.

As shown in fig. 7, the stability of the nanoparticles is good, the particle size does not change significantly after being placed for 1 week, and DOX does not leak significantly in the liposome.

Example 7 distribution of RWrNR-LS-DOX in MDA-MB-231 and PC-3 cells

Taking MDA-MB-231 and PC-3 cells in logarithmic growth phase according to 10⁵Each cell was seeded in a confocal laser cuvette. After 24h of culture, RWRNR-DOX-LS liposome and c-RGDyK-DOX-LS liposome are respectively added, wherein the administration concentration of adriamycin is 1 mu g/mL, after 4h of culture at 37 ℃, the preparation which is not taken up in the supernatant is washed away by using a blank culture medium, then, after 2h and 4h of culture in a serum-free culture medium is continued, cell nuclei are stained by using a Hoechst dye, and the transportation condition of DOX in the cells is observed under a laser confocal microscope.

As shown in FIG. 8, the fluorescence of doxorubicin taken up by c-RGDyK-LS-DOX in MDA-MB-231 and PC-3 cells was much lower than that of RWrNR-LS-DOX, indicating that RWrNR-LS-DOX liposomes we prepared could mediate endocytosis into tumor cells through MDA-MB-231 and integrin β 3 receptor highly expressed on the surface of PC-3 cells, and further that the amount of DOX taken up was much higher than that of the control preparation c-RGDyK-LS-DOX due to its strong affinity for integrin β receptor, furthermore, after 4h uptake, the non-taken up preparation was washed away using serum-free blank medium, and incubation was continued for 2h or 4h in an incubator to continue to observe the transport of the preparation within the cells.

Example 8 in vitro anti-tumor Activity of RWrNR-LS-DOX against MDA-MB-231 and PC-3 cells

Collecting MDA-MB-231 and PC-3 cells in logarithmic phase, digesting the cells with trypsin, and adjusting the concentration of cell suspension to 5 × 10 after blowing uniformly⁴one/mL. And (2) adding 200 mu l of cell suspension into each well of a 96-well cell culture plate, culturing at 37 ℃ for 24h, co-culturing the cells and RWRNR-LS-DOX liposome with DOX concentration of 0.5, 1, 2, 5, 10 and 20 mu g/mL, washing out the liposome which is not taken up in the supernatant by using a blank culture medium after 8h, and continuously culturing in an incubator for 24 h. 2 per wellAfter 0. mu.l of MTT solution was cultured for 4 hours, the culture medium in the wells was aspirated out using a line gun, 150. mu.l of DMSO was added, and the absorbance of each well at 490nm was measured using a microplate reader.

As shown in FIG. 9, each of the preparations showed concentration-dependent resistance to tumor cell toxicity in MDA-MB-231 and PC-3 cells, whereas at DOX concentrations of 0.5-20. mu.g/mL, the RWRNR-LS-DOX group showed good antitumor activity compared to the c-RGDyK-LS-DOX group, and furthermore, when DOX was administered at concentrations greater than 5. mu.g/mL, RWRNR-LS-DOX mediated by α IIb β 3 overexpressed on the PC-3 nuclear pericyte, actively delivered DOX into the PC-3 nucleus, thus greatly increasing the antitumor effect of DOX.

Example 9 in vivo distribution of RWrNR-LS-DOX in MDA-MB-231 and PC-3 tumor mice

Left axillary injection of male BALB/c nude mice 3X 10⁶One PC-3 tumor cell was used to construct PC-3 tumor model, while female BALB/c nude mice were injected with 1X 10 axilla right⁷Each MDA-MB-231 tumor cell was used to construct a tumor model for MDA-MB-231. The size of the tumor was measured using a vernier caliper, and the formula was: volume is 0.5 x long diameter x short diameter². When the tumor size reaches 400-³In this case, 200. mu.l of DiR-labeled liposomes (DiR-LS, DiR-c-RGDyK-LS and DiR-RWrNR-LS) were injected intravenously into the tail of each nude mouse, and the final concentration of DiR was 0.2 mg/kg. Imaging was performed by a fluorescence imaging system at 1, 2, 4, 8, 12 and 24h, respectively. 24h after injection, mice were sacrificed by anesthesia to dissect out tumors and major organs including: heart, liver, spleen, lung, and kidney for in vitro imaging.

Monitoring The biodistribution of DiR-labeled rwrrr-LS (DiR/rwrrr-LS) intravenously injected into MDA-MD-231 and PC-3 tumor-bearing mice with The IVIS spectroscopic imaging system as shown in fig. 10, DiR signal was observed at The tumor site 4h after MDA-MB-231 tumor-bearing mice injection 4h faster than DiR/C-RGDyK-LS (fig. 10a1 and fig. 10b1), furthermore DiR/rwrrr-LS also showed significant tumor targeting in PC-3 tumor-bearing mice (fig. 10a2 and fig. 10b2) due to a significant 25-fold increase in 64 3 mRNA Expression in PC-3 xenografts (Taylor M, severs V, Brown D C, protein. promoter targeting motifs: Expression of α V63, Expression of tumor 3, tissue 671, and 5 g tumor-bearing tissue uptake, and further evidence of strong tumor targeting in these tumor cells, tumor-3 tissue targeting, tumor cells, tumor tissue uptake, tumor-expressing high tumor targeting effects after pcr-3 transplantation [ rwor5J 3, tumor tissue uptake in vivo ] 3, tumor-mice).

Example 10 antitumor Activity of RWrNR-LS-DOX in MDA-MB-231 and PC-3 tumor mice

To investigate the antitumor effect of RWRNR-LS-DOX liposomes, 30 nude mice were randomly divided into 5 groups when the tumor volume reached 100mm³In time, nude mice bearing MDA-MB-231 and PC-3 tumors were injected with the following 5 groups of preparations 1) PBS, respectively; 2) DOX; 3) DOX-LS; 4) c-RGDyK-DOX-LS, and 5) RWrNR-DOX-LS, wherein DOX was administered at a dose of 3mg/kg 1 time every two days for 5 times, and after 14 days, tumors from each group were weighed, photographed, and immersed in formalin for HE staining and apoptosis analysis.

To evaluate the antitumor effect of rwrrnr-LS-DOX on integrin β overexpressing tumors, MDA-MB-231 and PC-3 tumor-bearing mice were used as animal models (fig. 11a) as shown in fig. 11a1 and fig. 11a2, different DOX formulations (dose of DOX administered at 3mg/kg) had significant effects on reducing tumor volume compared to saline group, furthermore, rwrrnr-LS-DOX showed significantly stronger inhibitory effects on MDA-MB-231 and PC-3 tumor growth than c-rgbyk-LS-DOX and LS-DOX at 18 days of treatment, indicating that rwrrnr-LS-DOX modified nano drug delivery system could achieve effective targeting and intracellular delivery to integrin β, having important significance for inhibiting tumor growth compared to c-rgryk-LS-DOX, rwrrnr-DOX also showed significant inhibitory effects on PC-3 tumors at 1.67mg/kg dose (fig. 11a) and showed significantly higher tumor-3-tumor-resistant efficacy than other rwrrnr-LS-DOX-3 tumor-resistant nanoparticles, and their clinical results showed a high tumor-resistant tumor-3 staining potential in rwrrnr-3-tumor-resistant tumor treatment and tumor-cancer treatment.

Claims

1. The motif of the targeting polypeptide with high affinity of the integrin β 3 is Arg-Trp-D-Arg-X, X represents 1-5 amino acids, and the amino acids are natural amino acids or amino acid analogues, which are called RWrX for short.

2. The integrin β 3 high affinity polypeptide of claim 1, wherein when the pentapeptide has the motif Arg-Trp-D-Arg-X₁-X₂When, X₁，X₂Is natural amino acid or amino acid analogue, called RwrX for short₁X₂. Wherein when X is₁When it is Asn, X₂Not simultaneously Met.

3. A reagent for targeted diagnosis or screening of diseases, comprising an imaging agent, wherein the imaging agent is coupled with the integrin β 3 high affinity targeting polypeptide of claim 1.

4. A targeted vaccine for diseases, a targeted therapeutic drug or a targeted drug delivery system is characterized in that the vaccine, the therapeutic drug or the drug delivery system is modified with targeted polypeptide with high affinity of integrin β 3 in claim 1, the vaccine comprises inactivated vaccine, attenuated live vaccine, component vaccine and the like, the targeted drug comprises receptor antagonist, anticoagulant, anti-angiogenesis agent, anti-tumor drug, anti-inflammatory drug and the like, the targeted drug delivery system comprises liposome, micelle, nanosphere, nanoemulsion, nanocrystal, nanogel, carbon nanotube and the like, and the contents are not limited in the range of examples.

5. The use of the diagnostic reagent, screening reagent, targeted vaccine, therapeutic drug or targeted agent of claims 1-4 for diagnosis or treatment of acute and chronic liver and kidney diseases, rheumatoid arthritis, adenomyosis, thrombosis and cancers with high integrin β 3 receptor expression, including breast cancer, cervical cancer, liver cancer, brain glioma, prostate cancer, ovarian cancer, gastric cancer, melanoma, and the like, without limitation to the scope of examples.