CN111744008B - Use of a combination of NFIB monoclonal antibody and cisplatin for the preparation of a medicament and a cosmetic product - Google Patents

Abstract

The invention relates to an application of a combination containing NFIB monoclonal antibodies in preparation of medicines and cosmetics, and the antibodies and cisplatin can generate a synergistic effect when used together, so that the antibody has a better treatment effect and a better application prospect.

Description

Use of a combination of NFIB monoclonal antibody and cisplatin for the preparation of a medicament and a cosmetic product

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of a combination of an NFIB monoclonal antibody and cisplatin in preparation of medicines and cosmetics.

Background

Melanoma is also known as malignant melanoma. Is a skin tumor related to orthopedics and traumatology. Malignant melanoma (melanoma) is a malignant tumor of the skin that is derived from melanocytes of the neural crest and is caused by abnormal proliferation of such cells. Benign melanoma is also called pigmented nevus and mostly attracts attention. Melanoma may be initially malignant, but is usually malignant from a bordering nevus. This is a more malignant tumor, which is more common. The ratio of male to female is 2: 1. the hair part is the lower limb, the foot is the most, and the second is the upper limb, the head and neck, the trunk and the like. The cause of the disease is unknown, but some cases have a history of skin injury, burns and X-ray irradiation may be the cause, few cases are related to endocrine factors, and familial tendency is reported. The tumor is gray black, nodular, soft and ulcerated. The tumor cells under the microscope are mostly in the shape of fusiform, round or polygonal, the cytoplasm contains different numbers of melanin granules, the appearance is black, and the tumor cells are different in size and are arranged in a dispersed shape, a nested shape, a glandular shape or a trabecular shape. The medicine is good for skin, mucosa and pigment membrane, has extremely high malignancy degree, and is one of malignant tumors with extremely high mortality rate.

Melanoma morbidity and mortality continue to rise worldwide, with approximately 8 million new patients with malignant melanoma in the united states in 2014, with approximately 1 million patients dying from the disease. The statistics of disease incidence in 2000 years in China is only 0.2/10 ten thousand, the disease incidence in 2005-2007 year is about 1/10 ten thousand, the disease incidence in 2008 year is about 1.3/10 ten thousand, and the disease incidence accounts for 2% of malignant tumors, and the number of melanoma patients is doubled every 6-10 years. In 2018, the melanoma expert committee of the clinical oncology collaboration professional committee of the Beijing university tumor hospital and the China anticancer Association learned that the detection rate of melanoma in China increased, and about 2 ten thousand new cases are released each year.

Melanoma can be cured by early detection and primary focus excision, but has hidden onset, fast disease process and high recurrence rate and metastasis rate. For patients with malignant melanoma progressing to middle and advanced stages, combination treatment schemes are mainly adopted, including chemotherapy, radiotherapy, immunotherapy and biotherapy modes such as IFN-alpha 2b, interleukin-2, dacarbazine and temozolomide. Newly developed small molecule targeted drugs (BRAF inhibitors vemurafenib, dabrafenib and MAPK/MEK inhibitors trametinib, cobimetinib) bring new therapeutic eosinomas to patients with malignant melanoma carrying mutations in related genes such as BRAF, but few further treatment options are available compared to those patients without BRAF mutations or with progression of disease progression after treatment with BRAF mutations. Meanwhile, the CTLA-4 antibody and the PD-L1 antibody which are immune targeted therapeutic drugs relieve the condition of a part of patients with positive anti-PD-1 drugs, but are easy to generate drug resistance. Once the disease development of the drug-resistant malignant melanoma patient is explosive, almost no treatment means can effectively control the metastatic malignant melanin, and the survival rate in 5 years is only 16%. Melanoma has become one of the diseases harming the health of people in China, and the intensive research on the pathogenesis of melanoma has become a difficult problem all over the world.

With the rapid development of modern tumor immunology, immunotherapy for multiple links such as malignant melanoma development, development and metastasis has become a hot spot of current research. The purpose of tumor immunotherapy is to stimulate or mobilize the immune system of the body and enhance the anti-tumor immunity of the tumor microenvironment, thereby controlling and killing tumor cells. The antibody medicine is an important component of tumor immunotherapy, takes molecules closely related to tumor occurrence and development as targets, and realizes specific inhibition and killing of tumor cells or targeted interference on tumor growth microenvironment through different mechanisms including tumor cell growth inhibition, tumor cell apoptosis promotion, tumor angiogenesis interference and the like. Therefore, compared with the traditional treatment measures, the antibody medicament has more obvious effect and greatly reduced adverse reaction, has good application prospect for treating malignant tumors, and becomes an important research direction in the immunotherapy of malignant tumors including melanoma.

Currently anti-PD-L1 antibodies and several forms of PD-1 antibodies have been used to treat malignant melanoma, and US20190194328a1 discloses a method of treating melanoma comprising administering to a patient having a melanoma tumor an anti-PD-1 antibody or antigen-binding portion thereof that specifically binds to human PD-1 ("anti-PD-1 antibody") at a flat dose of 240mg every two weeks or 480mg every four weeks while also administering an anti-CTLA-4 antibody or antigen-binding portion thereof that specifically binds to human CTLA-4 ("anti-CTLA-4 antibody"). US10308721B2 discloses a method of treating melanoma in a subject, wherein the melanoma is characterized by DLL3 expression levels above a threshold index value, the method comprising the step of administering to the subject an anti-DLL 3 antibody drug conjugate. Antibodies directed against other targets for the treatment of melanoma are less relevant.

It has been shown that down-regulation of NFIB significantly inhibits melanoma cell proliferation, attenuates colony forming ability, and inhibits cell migration and invasion. Taken together, these results suggest that NFIB plays an important role in the development of melanoma development. However, monoclonal antibodies against NFIB have been studied very rarely.

The combination of PD-1 antibody and chemotherapeutic drugs for treating melanoma is reported, but the combination of NFIB antibody and other drugs for treating melanoma is not reported, and the invention provides another method for treating melanoma.

Disclosure of Invention

The invention provides an NFIB polypeptide with high antigenic determinant, and the amino acid sequence of the NFIB polypeptide is shown as SEQ ID NO: 1 is shown.

The invention provides an NFIB polypeptide with high antigenic determinant, and the nucleotide sequence of the NFIB polypeptide is shown as SEQ ID NO: 2, respectively.

The present invention also provides a method for preparing the NFIB monoclonal antibody, which is characterized in that the NFIB polypeptide is used as an immunogen.

Further, the present invention provides an NFIB monoclonal antibody, wherein the light chain variable region sequence of the NFIB monoclonal antibody is as shown in SEQ ID NO: 3, the heavy chain variable region sequence is shown as SEQ ID NO: 4, respectively.

The invention also provides an application of the NFIB monoclonal antibody in preparing a medicine or a cosmetic aiming at melanoma.

In addition, a light chain variable region containing sequence shown in SEQ ID NO: 3, the heavy chain variable region sequence is shown as SEQ ID NO: 4, or a fragment of the NFIB monoclonal antibody shown in figure 4.

Further, the invention also provides a pharmaceutical composition containing the NFIB monoclonal antibody.

In certain embodiments, the monoclonal antibodies can be formulated in unit dosage injectable forms, such as solutions, suspensions, or emulsions. Pharmaceutical formulations suitable for injection are generally sterile aqueous solutions and dispersions. The carrier for injectable preparations may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (for example, glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. The amount of cells and optional additives, vehicles (vehicles) and/or carriers in the compositions to be administered in the methods of the invention can be readily determined by one skilled in the art. Typically, any additives (other than the cells) are present in a solution such as phosphate buffered saline in an amount of 0.001 wt% to 50 wt%. The active ingredient is present in micrograms to milligrams, for example, from about 0.0001 wt% to about 5 wt%, preferably from about 0.0001 wt% to about 1 wt%, most preferably from about 0.0001 wt% to about 0.05 wt%, or from about 0.001 wt% to about 20 wt%, preferably from about 0.01 wt% to about 10 wt%, most preferably from 0.05 wt% to about 5 wt%.

The invention provides a pharmaceutical composition for treating melanoma, which comprises the monoclonal antibody and cisplatin.

The monoclonal antibody disclosed by the invention is combined with cisplatin to prepare a medicine for treating melanoma.

Advantageous effects

The invention carries out specific antigenic determinant analysis and corresponding codon optimization aiming at NFIB protein to obtain the amino acid and nucleotide sequence of specific NFIB antigen peptide, immunizes a mouse after recombining, expressing and purifying the antigen peptide, and prepares a corresponding hybridoma cell and a corresponding monoclonal antibody.

Drawings

FIG. 1 is a comparison between the nucleotide sequences of NFIB antigen peptides before and after codon optimization.

FIG. 2 is a SDS-PAGE image of recombinant expression and purification of NFIB antigenic peptide.

FIG. 3 is a graph showing the results of antibody titers.

FIG. 4 is a Western blot assay result chart, with blank controls on the left and detection results of NFIB antigen peptide and monoclonal antibody on the right.

FIG. 5 is a graph showing the results of potency assay of monoclonal antibodies.

FIG. 6 is a graph showing the results of the experiment for inhibiting melanoma cells.

Detailed Description

EXAMPLE 1 screening of epitopes for NFIB

TMHMM Server v.2.0 online software is used for Predicting the transmembrane structure of the NFIB protein, SOPMA online software is used for analyzing the secondary structure of the NFIB protein, and Predicting Antigenic Peptides online software is used for Predicting the Antigenic determinant of the NFIB protein. Optimizing and adjusting the structure of the epitope peptide sequence according to experience to finally obtain the optimized NFIB antigen peptide sequence shown as SEQ ID NO: 1 is shown.

The nucleotide sequence of the antigen peptide is shown as SEQ ID NO: 2, respectively.

Example 2 recombinant protein expression

Converting SEQ ID NO: 2 the Shanghai Biotech company was entrusted with the synthesis and ligation of the pMD-19-T vector. Plasmid pMD19-T-NFIB and pET-30a vector were double digested with restriction enzymes EcoRI and XhoI, and the desired fragment and the digested linear pET-30a vector were recovered with agarose gel recovery kit. Adding the recovered product into a 1.5mL centrifuge tube according to a molar ratio of 4:1, uniformly mixing, connecting in an ice bath at 4 ℃ overnight, transforming to escherichia coli BL21(DE3) competent cells, coating on a kanamycin-resistant plate containing 50mg/mL for culturing overnight, screening and identifying to obtain positive clones, inoculating the positive monoclone into 15mL LB liquid culture medium containing corresponding resistance, culturing at 37 ℃ and 200r/min until OD is 0.6-0.8, and performing induction culture for 2h by using 0.5mM IPTG at 37 ℃ and 200 r/min. Taking 1mL of induced bacterial liquid, carrying out 12000r/min, centrifuging for 1min, removing the supernatant, blowing off the precipitate by using 10mM Tris-HC1 pH8.0 solution, adding buffer solution and 2X loading buffer with the same volume into a 1.5mL centrifuge tube, boiling for 10min at 100 ℃, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection, inoculating the bacteria into a kanamycin-resistant LB (lysogeny broth) liquid culture medium according to the proportion of 1:50, and carrying out large-scale induced expression.

The nickel column was washed with deionized water to pH 7.0. The nickel was then suspended until pH 2-3, the column was washed with deionized water to pH 7.0, and 100mL of the nickel column was equilibrated with 10OmM Tris-HCl, pH8.0 solution. The nickel column was equilibrated to approximately 50mL with 1OmM Tris-HCl, pH8.0, solution containing 8M urea, 0.5M sodium chloride. Samples were diluted for loading with 0.5M NaCl, 8M Urea, 1OmM Tris-HCl (pH 8.0). After the end of the loading, the column was washed with 1OmM Tris-HC1(pH 8.0) containing 8M urea and 0.5M sodium chloride. Eluting with 10mM Tris-HCl (pH 8.0) solution containing 15mM imidazole, 60mM imidazole and 500mM imidazole, respectively, collecting protein peaks, and detecting the protein purification effect by electrophoresis.

As can be seen from FIG. 2, the SDS-PAGE shows that the target protein is highly expressed, and the effective purification of the target protein can be clearly seen by Ni column purification.

EXAMPLE 3 preparation of antibodies

1. Immunization of mice

The purified NFIB protein antigen peptide was used to immunize SPF Balb/c female mice (Beijing Wintolite laboratory animal technology, Inc.) subcutaneously in an amount of 40. mu.g protein/mouse. Three boosts were performed at 7 days each, with 30. mu.g/mouse per boost. Blood was collected from the orbit after boosting the immunity and the serum titer was measured. Coating with NFIB protein antigen peptide at 4 deg.C for overnight at 2 μ g/mL; 2 percent of skim milk, and sealing for 2 hours at 3 ℃; the serum was diluted in a 2-fold gradient from 200-fold, the blank control was PBS, and the negative control was negative serum diluted 200-fold.

After 3 mice are immunized, blood is collected by eyeballs, and the titer of serum is measured by performing 2-fold gradient dilution from 200-fold, so that the NFIB-2 mouse has the best titer effect. As a result of the antibody titer test shown in FIG. 3, the titer of the mice was significantly higher than that of the blank control and the negative control, which were used for hybridoma preparation (the blank control was PBS, and the negative control was a 200-fold dilution of negative serum).

2. Cell fusion and hybridoma cell selection

And taking the spleen cell of the NFIB-2 mouse and the SP2/0 cell, and fusing by a PEG method. After the culture, monoclonal cells are picked and coated with NFIB protein and His-tagged protein respectively, and hybridoma cells are screened. The hybridoma cells with the most significant positive effects obtained by screening 2 strains are named as NFIB-2-4D4 and NFIB-2-5F3, and the specific result of performing subspecies identification on the screened positive hybridoma cells is shown in Table 1, wherein a blank control is PBS, and a negative control is negative serum diluted by 200 times.

Identification results of the Positive hybridoma cells of Table 12

3. Preparation and collection of ascites fluid from mice

The ascites of the mice was prepared using the hybridoma NFIB-2-4D4 which gave the best positivity. Injecting 1mL of paraffin oil into Balb/c mice tested 7 days in advance, and injecting the mice intraperitoneally after culturing and counting hybridoma cells, wherein each mouse is injected with not less than 1X10⁶And (3) hybridoma cells. After the fifth day of hybridoma cell injection, the ascites can be collected and marked if the abdomen of the mouse is obviously enlarged.

4. Purification of monoclonal antibodies

1 part of ascites is centrifuged at 8000 r/min for 5min, the precipitate is discarded, 2 parts of 0.06mol/L (pH5.0) acetate buffer are added, and the pH is adjusted to 4.8 with 1.0M HC 1. Adding appropriate amount of octanoic acid, adding n-octanoic acid dropwise under stirring at room temperature, mixing at room temperature for 30min, standing at 4 deg.C for at least 2 hr, centrifuging at 12000r/min for 35min, and removing precipitate. If fine particles in the supernatant are still suspended after centrifugation, the supernatant needs to be centrifuged again for 15min at 12000r/min, and the precipitate is discarded. 0.01M PBS was added to 1/10 in the volume of the supernatant and the pH was adjusted to 7.2 with 1.0M NaOH. Adding sulfuric acid into ice water bath at a ratio of 0.277g/mL to 45% -50% saturation, mixing for 30min, standing at 4 deg.C for 2h, centrifuging at 12500r for 30min, and removing supernatant. Dissolving the precipitate in PBS, adding 5% volume glycerol, mixing, placing into dialysis bag, dialyzing in 2L0.01mol PBS buffer solution at 4 deg.C in refrigerator for at least 3 hr, and changing the buffer solution at least 3 times. Centrifuging the dialysate at 10000r/min for 10min, removing insoluble precipitate, adding 5% volume of glycerol, and packaging at-20 deg.C for freezing. After the mouse ascites is purified, SDS-PAGE detection is carried out, and a visible target band is found and has higher purity.

5. Protein content determination with BCA kit

(1) Adding 25 μ L of each diluted protein standard and protein eryA sample to be detected into a microporous plate (detection range of 20-2000Pg/mL)

(2) Add 200. mu.L of working solution to each well and shake on a shaker for 30s to mix well

(3) Sealing the microporous plate, and incubating at 37 deg.C for 30min

(4) The microplate was cooled to room temperature. The absorbance of the sample at 562nm was measured using a microplate reader.

(5) And subtracting the average absorbance value of the blank standard at 562nm from the absorbance value of each standard and the protein sample to be detected at 562 nm.

(6) The mean absorbance values of the BCA standard blank-corrected at 562nm were plotted against their concentration,

and drawing a standard curve. Measuring the content of the purified protein by using a BCA kit, and drawing a standard curve to obtain R²0.9948 shows a higher concentration of the antibodyAnd (4) degree. The purified antibody results are shown in table 2.

Table 2 antibody purification data

Example 4 detection of antibodies

The protein prepared in example 1 was subjected to SDS-PAGE and transferred to a PVDF membrane at a voltage of 14V for 50 min. The membrane was placed in a dish previously washed with PBST buffer, blocked with 1% skim milk overnight at 4 ℃ and the PVDF membrane was washed with PBST buffer 3 times for 15min each. The purified monoclonal antibody from example 3 was added at a 1:500 dilution, incubated for 4h at 37 ℃, the PVDF membrane was washed 3 times with PBST buffer, each for 15min, then horseradish catalase-labeled rabbit anti-sheep IgG was added at a 1:5000 dilution, incubated for 2h at 37 ℃, and the PVDF membrane was washed 3 times with PBST buffer, each for 15 min. Then, the color is developed by a color developing agent and photographed. As can be seen from FIG. 4, Western blot detection is used to find that the recombinant protein can be effectively combined with the monoclonal antibody, indicating that the antibody is successfully prepared.

EXAMPLE 5 potency assay of monoclonal antibodies

The monoclonal antibody purified from ascites fluid prepared in example 3 was diluted in equal fold using the purified recombinant protein of example 2 as an antigen and the mouse preimmune serum as a negative control, and detected by indirect ELISA. The results showed that the titer of the monoclonal antibody prepared in example 3 reached 1:1024000 (FIG. 5, wherein i represents absorbance values of sample wells/negative control wells >2.1 at different dilution times).

EXAMPLE 6 monoclonal antibody sequence identification

Downstream primers were designed based on conserved regions of the Fc fragment of murine heavy chain IgG1 and light chain Kappa using the SMARTER RACE kit (clonetech, 634859) instructions, and the heavy and light chain variable region genes of the antibody were amplified and sequenced using RACE PCR technology. The sequence of the variable region of the light chain of the obtained antibody is shown as SEQ ID NO: 3 and the heavy chain variable region sequence is shown as SEQ ID NO: 4, respectively.

Example 7 determination of the binding Capacity of anti-NFIB antibodies to NFIB protein

The antibody obtained in example 3 was diluted with a culture medium to a concentration gradient of 1000ng/ml, 250ng/ml, 62.5ng/ml, 15.63ng/ml, 3.91ng/ml, 0.98ng/ml, incubated at 4 ℃ for 2h, the medium was aspirated, glutaraldehyde was added and incubated at room temperature for 10min, after washing 3 times with PBS, 100ul of anti-human IgG antibody was added and incubated at room temperature for 2 h. Washing with PBS for 3 times, adding 50 μ l of color development solution, incubating at room temperature in dark place for 15min, adding stop solution to stop color development reaction, detecting absorbance value of sample at 450nm wavelength with microplate reader, and correcting with absorbance value at 620nm wavelength. The data was processed to finally determine the EC50 value for each sample, the results of which are shown in table 3 below.

TABLE 3 determination of the binding Capacity of anti-NFIB antibodies to NFIB

Name (R)	EC50(ng/ml)
		NFIB-2-4D4	14.3±1.2

Example 8 melanoma cell inhibition assay

Cell line used for the experiments human in situ melanoma cell line a375 was purchased from the center of the cell bank at the university of wuhan.

Melanocytes are cultured, cell passage cell counting is carried out when the cell fusion rate is about 80%, cells are evenly inoculated into 3 96-well plates according to the standard of 1000 cells per well, each well is kept as 100u1 culture medium, and simultaneously 6 multiple wells are designed for each experimental group (the antibody of the invention, the concentration is 10 ug/well) and the control group (PBS solution) and are marked. Preparing a CCK-8 dilution solution (l0ul/ml, adding an experimental group and a control group after uniformly mixing by blowing, setting a blank control group in the air without cells, placing the blank control group in an incubator for incubation for at least 3h, ensuring that the absorbance of the cells is detected at 490nm by using an enzyme labeling instrument every day at a fixed time, continuously detecting the data for 24h,48h and 72h, calibrating the blank data, taking the average value, and performing statistical analysis, drawing a melanoma cell growth curve by using time as an abscissa and an absorbance value as an ordinate, and finding that the proliferation of A375 cells expressing NFIB in a silent mode is remarkably inhibited as shown in a result shown in figure 6.

Example 9 growth inhibition of subcutaneous nude mouse transplantable tumors by NFIB antibody combination

Cell line used for experiments human in situ melanoma cell line a375 was purchased from the university of wuhan cell bank center in 2017. BALB/cA nude mice, female, 40-45 mouths old, weight 18 s 2g, purchased from Jinan Ono bioengineering Co., Ltd; animals were counted 6 per group.

The NFIB antibody of test agent example 3 was dissolved in PBS (pH6.0) at a dose of O.5mg/dose group; the cisplatin dose is 1mg/dose group; the temozolomide dosage is 1mg/dose group; the dose of the negative control PBS was 1 mg/dose.

1. Mouse inoculation of tumor cells

Collecting A375 cells cultured in vitro, adjusting the cell suspension concentration to 2.5 x10⁵Per ml; under the aseptic condition, 200ul of cell suspension is inoculated to the subcutaneous back of the nude mouse; measuring the diameter of the subcutaneous transplantation tumor of the nude mouse by using a vernier caliper; when the tumor grows to 100-200mm³Thereafter, animals were randomly grouped.

2. Group administration

Experimental group 1: the NFIB antibody of test agent example 3 was dissolved in PBS (pH6.0), and the dose was O.5mg/dose group + cisplatin dose was 1mg/dose group;

experimental group 2: the NFIB antibody of the test drug example 3 is dissolved by PBS (phosphate buffer solution) with pH6.0, and the dose is O.5mg/dose group and the temozolomide dose is 1mg/dose group;

experimental group 3: the NFIB antibody of test agent example 3 was dissolved in PBS (pH6.0) at a dose of O.5mg/dose group;

control group: the dose of the negative control PBS was 1 mg/dose.

Animals were randomly dosed in the 4 groups, i.e. twice weekly for 5 weeks.

3. Measuring and weighing

During the whole experiment, the diameter of the transplanted tumor is measured at the beginning and the end, and the weight of the mouse is weighed; the formula for calculating the Tumor Volume (TV) is 1/2XaXb2, wherein a and b respectively represent length and width; relative Tumor Volume (RTV) is calculated according to the measured result, and the calculation formula is that RTV is Vt/V0, wherein V0 is the measured tumor volume when the drug is administrated in a cage (i.e. do), and Vt is the tumor volume when the drug is finally measured.

4. Evaluation index

The evaluation index of the antitumor activity is relative tumor proliferation rate T/C (%), and the calculation formula is as follows:

T/C(％)＝(TRTV/CRTV)X100％

TRTV treatment group RTV; CRTV is negative control group RTV.

The evaluation standard of the curative effect is that T/C (%) > 40% is ineffective; T/C (%) <40, and p <0.05 was statistically valid. The results are as follows:

TABLE 3 growth inhibition results of NFIB antibody administered to melanoma nude mice graft tumor 40 days

The experimental results show that the T/C value of the experimental group 1 is 6.4% after 40 days of administration, and the better inhibition effect is shown. The weight of the nude mice did not drop significantly and did not die during the entire experiment. The experimental groups 2 and 3 can also effectively inhibit the growth of the tumor, but the effect is not obvious in the experimental group 1, which shows that the combination of the NFIB antibody and the cisplatin has better synergistic advantage and has unexpected technical effect.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

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Claims (3)

1. A combination, characterized by: the monoclonal antibody comprises a specific NFIB monoclonal antibody and cisplatin, wherein a light chain variable region of the antibody is shown as SEQ ID NO: 3, the heavy chain variable region is shown as SEQ ID NO: 4, respectively.

2. Use of a combination according to claim 1 in the manufacture of a medicament for inhibiting the proliferation of melanoma cells.

3. Use of a combination according to claim 1 for the preparation of a cosmetic product for inhibiting the proliferation of melanoma cells.

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