A polypeptide targeting folate receptor alpha and its application

Abstract

The present invention discloses a polypeptide targeting folate receptor α and its application, belonging to the field of biomedicine. The amino acid sequence of the polypeptide of the present invention is shown in any one of SEQ ID No. 1 to SEQ ID No. 4. The polypeptide targets and binds to folate receptor α overexpressed in tumor cells. The polypeptide has important application value in tumor molecular diagnosis and / or targeted therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polypeptide targeting folate receptor α and applications thereof. Background Art

[0002] Folate is an essential vitamin required for DNA synthesis, repair, and cell division. The folate receptor, a transmembrane single-chain glycoprotein linked to glycosylated phosphatidylinositol, has a high affinity for folate and transports folate across the cell membrane via endocytosis, where the folate receptor is internalized and then recycled back to the cell membrane. Four folate receptor subtypes have been identified: FRα, FRβ, FRγ, and FRδ, encoded by the genes FOLR1, FOLR2, FOLR3, and FOLR4, respectively.

[0003] Folate receptor α (FRα), encoded by the FOLR1 gene, is a cell surface glycoprotein with a molecular weight of approximately 40 kDa. Originally discovered as a folate-binding protein, it was later found to regulate tumor cell proliferation and metastasis in addition to transporting folate. FRα is not expressed or expressed at very low levels in normal tissues, but is widely expressed in solid tumors, such as mesothelioma (72-100%), triple-negative breast cancer (35-68%), ovarian cancer (76-89%), and non-small cell lung cancer (14-74%). A certain percentage of FRα is expressed in non-malignant tissues, such as the choroid plexus, and also in bronchial epithelial cells, thyroid, salivary glands, breast, colon, and bladder. The folate receptor is involved in tumor infiltration, metastasis, and progression, making it a promising target for cancer therapy.

[0004] Currently, there are many types of FRα-targeted drugs for tumor treatment, including monoclonal antibodies, ADC drugs, CAR-T therapy, and bispecific antibodies. Among them, ADC drugs have made the fastest progress. The first FRα ADC product, Elahere, was approved for accelerated marketing by the FDA in 2022. ImmunoGen screened the monoclonal antibody Mirvetuximab targeting human FRα based on a hybridoma platform, and then developed Elahere by connecting it to a cytotoxic drug DM4 through a cleavable linker. In addition, there are several ADC drugs under clinical development, including STRO-002 and CBP-1008, which have entered clinical phase II. Other drugs involving FRα-targeted drugs, such as bispecific antibodies and CAR-T drugs, are in clinical phase I, phase II, and preclinical stages. China Pharmaceutical University has disclosed a series of short peptides that specifically bind to folate receptor α and their applications in tumor diagnosis and targeted therapy (CN107353325A, CN107446020A, CN107674115A, CN107793471A, CN107446021A). The peptides specifically bind to folate receptor α overexpressed in tumor cells. The peptides can be effectively enriched in the tumor part and have tumor targeting in vivo, and have important application value in tumor molecular diagnosis and targeted therapy.

[0005] Folate receptors offer potential targets for cancer diagnosis and treatment. While certain anti-FRα antibodies have been developed, there remains a need for further development of FRα-targeted drugs based on existing technologies, particularly for active molecules with high specificity, high biological activity, enhanced endocytosis capacity, and improved in vivo safety. As one of the most important biological substances, peptides are widely present in living organisms, regulating the functional activities of various systems, organs, and cells. In recent years, peptide drugs have attracted considerable attention in the development of anti-tumor drugs due to their advantages such as high targeting, low immunogenicity, high tissue penetration, and safety.

[0006] In view of this, the development of a polypeptide that can target and bind to FRα has potential application value and provides new ideas for the targeted treatment and diagnosis of tumors. Summary of the Invention

[0007] The purpose of the present invention is to provide a polypeptide targeting folate receptor α and its application. The polypeptide targets and binds to folate receptor α overexpressed in tumor cells and has important application value in tumor molecular diagnosis and targeted therapy.

[0008] Furthermore, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a polypeptide targeting folate receptor α, wherein the amino acid sequence of the polypeptide is shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

[0010] WCWMPHIAMHTQFGCGQ(SEQ ID No.1)、

[0011] GCYEWCLWHATYKECQSMCG(SEQ ID No.2)、

[0012] GEALLYVYKAQSMDWYYG(SEQ ID No.3)、

[0013] GCYGTCPERDHWFFEYCAKICAG (SEQ ID No. 4).

[0014] In a second aspect, the present invention provides a polypeptide derivative, which is a modified product of the polypeptide described in the first aspect or a variant obtained by adding and / or replacing one or more amino acids.

[0015] Preferably, the polypeptide derivative is a variant obtained by adding and / or replacing one, two or three amino acids of the polypeptide shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

[0016] Further preferably, the polypeptide derivative is a variant obtained by adding and / or replacing one amino acid to the polypeptide shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

[0017] Preferably, the polypeptide derivative is an N-terminal or C-terminal modification product of the polypeptide shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, and the modification group is selected from one or more of an acetyl group, an amino group, an alkyl group, an aromatic group, a fatty acid group, a sugar group, a phosphate group, a sulfate group, a polyethylene glycol (PEG) group or a peptide linking group.

[0018] Further preferably, the modification is selected from: C-terminal amidation blocking and / or N-terminal modification with acetyl group.

[0019] In a third aspect, the present invention provides a polynucleotide encoding the above-mentioned polypeptide or polypeptide derivative.

[0020] In a fourth aspect, the present invention provides a vector containing the above-mentioned polynucleotide.

[0021] In a fifth aspect, the present invention provides a cell containing the above-mentioned vector.

[0022] In the sixth aspect, the present invention provides a composition comprising excipients and active ingredients, characterized in that the active ingredient comprises the polypeptide described in the first aspect or the polypeptide derivative described in the second aspect or the polynucleotide described in the third aspect or the vector described in the fourth aspect or the cell described in the fifth aspect; the excipients comprise at least one of a diluent, a filler, an adhesive, a wetting agent, an absorption enhancer, a surfactant, a lubricant and a stabilizer.

[0023] In the seventh aspect, the present invention provides the use of the polypeptide described in the first aspect, the polypeptide derivative described in the second aspect, the polynucleotide described in the third aspect, the vector described in the fourth aspect, the cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a drug for preventing and / or treating a disease associated with folate receptor α, wherein the disease associated with folate receptor α is a cancer in which folate receptor α is highly expressed.

[0024] Preferably, the cancer with high expression of folate receptor α is selected from at least one of ovarian cancer, non-small cell lung cancer, triple-negative breast cancer, mesothelioma, and lung cancer.

[0025] More preferably, the cancer with high expression of folate receptor α is ovarian cancer.

[0026] the term

[0027] As used herein, the term "amino acid" refers to a molecule containing an amino group and a carboxyl group. Suitable amino acids include, but are not limited to, the D- and L-isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. The term amino acid includes, but is not limited to, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

[0028] The term "naturally occurring amino acid" refers to any of the 20 L-amino acids commonly found in peptides synthesized in nature, i.e., the L-isomers of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0029] The polypeptides of the present invention can be prepared using methods familiar to those skilled in the art, including well-known chemical synthesis methods. Therefore, when a polypeptide or its derivatives contain one or more non-standard amino acids, it is very likely to be prepared by chemical synthesis. In addition to using chemical synthesis methods to prepare polypeptides or their derivatives, they can also be prepared by expressing encoding nucleic acids. This is particularly applicable to the preparation of polypeptides or their derivatives containing only natural amino acids. In this case, well-known methods for preparing nucleic acid encoding polypeptide sequences can be used (see Sambrook et al., Molecular Cloning: A Labour Manufacture, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). The polypeptide can be expressed in an organism and purified using well-known purification techniques.

[0030] Beneficial effects

[0031] The FRα binding peptide provided by the present invention can specifically bind to FRα recombinant protein. At the same time, through cell ELISA and flow cytometry experimental analysis, it was found that the screened phage-displayed short peptide can specifically bind to natural FRα on the cell surface, and has potential medical and pharmaceutical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The affinity between the polypeptide represented by SEQ ID No. 1 and FRα was detected by surface plasmon resonance in Example 2;

[0033] Figure 2 The affinity between the polypeptide represented by SEQ ID No. 2 and FRα was detected by surface plasmon resonance in Example 2;

[0034] Figure 3 This is a diagram of the cellular endocytosis of the polypeptide represented by SEQ ID No. 1 in Example 3;

[0035] Figure 4 This is a diagram of the cellular endocytosis of the polypeptide shown in SEQ ID No. 3 in Example 3;

[0036] Figure 5 This is a diagram of the cellular endocytosis of the polypeptide shown in SEQ ID No. 4 in Example 3. DETAILED DESCRIPTION

[0037] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work fall within the scope of protection of the present invention. In the following examples, unless otherwise specified, the raw materials used are commercially available.

[0038] Example 1 Synthesis of polypeptide

[0039] The polypeptide provided by the present invention is synthesized using a solid-phase synthesis method to obtain a linear precursor of the target polypeptide after cleavage. The solid-phase support is 2-Chlotrityl Resin resin. During the synthesis process, the 2-Chlotrityl Resin resin is first fully swollen in N,N-dimethylformamide (DMF). The swollen solid-phase support is then subjected to repeated condensation with an activated amino acid derivative, followed by washing, Fmoc deprotection, washing, and the next round of amino acid condensation to achieve the desired polypeptide chain length. Finally, a mixed solution of trifluoroacetic acid: water: triisopropylsilane: thioanisole (v:v:v:v = 90:2.5:2.5:5) is reacted with the solid-phase resin to cleave the polypeptide from the solid-phase support. The crude target polypeptide is then precipitated with chilled methyl tert-butyl ether to obtain the target polypeptide. The target polypeptide crude product is then purified and separated using a C18 reverse-phase preparative chromatography column in 0.1% trifluoroacetic acid in acetonitrile / water to obtain pure polypeptide and its derivatives.

[0040] Table 1 Experimental raw materials and their sources

[0041]

[0042]

[0043] (1) Solid-phase synthesis of the polypeptide represented by SEQ ID No. 1

[0044] SEQ ID No.1:WCWMPHIAMHTQFGCGQ

[0045] Step 1: Coupling of the first amino acid Fmoc-Gln-OH

[0046] 0.2 mmol of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. Fmoc-Gln-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin. The mixture was allowed to react at room temperature for 2 hours. After the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0047] Step 2: Linear peptide chain synthesis

[0048] The resin obtained in step 1 was fully swelled in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second amino acid at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0049] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0050] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0051] Dissolve 0.5 mmol of Fmoc-AA, 0.5 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol of 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0052] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0053] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0054] Step 3: Cleavage of the linear precursor peptide chain

[0055] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0056] Step 4: Peptide purification and preparation

[0057] After filtration through a 0.45 μm membrane, the product was separated using a reverse-phase high-performance liquid chromatography system using buffers A (0.1 wt% trifluoroacetic acid, aqueous solution) and B (0.1 wt% trifluoroacetic acid, acetonitrile). Product-related fractions were collected, and after HPLC analysis of purity, fractions with a purity >95% were combined and lyophilized to obtain the pure peptide.

[0058] Step 5: Detection and Characterization Methods

[0059] The purified peptide from step 4 was subjected to analytical HPLC and LC / MS to confirm its purity and compound identity.

[0060] (2) Solid-phase synthesis of the polypeptide represented by SEQ ID No. 3

[0061] SEQ ID No.3: GEALLYVYKAQSMDWYYG

[0062] Step 1: Coupling of the first amino acid Fmoc-Gly-OH

[0063] 0.2 mmol of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. Fmoc-Gly-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin. The mixture was allowed to react at room temperature for 2 hours. After the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0064] Step 2: Linear peptide chain synthesis

[0065] The resin obtained in step 1 was fully swelled in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second amino acid at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0066] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0067] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0068] Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0069] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0070] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0071] Step 3: Cleavage of the linear precursor peptide chain

[0072] Add a freshly prepared cleavage cocktail (10 mL) consisting of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate the crude product with 4 volumes of cold MTBE. Wash the crude product three times with MTBE and dry it in a vacuum.

[0073] Step 4: Peptide purification and preparation

[0074] After filtration through a 0.45 μm membrane, the product was separated using a reverse-phase high-performance liquid chromatography system using buffers A (0.1 wt% trifluoroacetic acid, aqueous solution) and B (0.1 wt% trifluoroacetic acid, acetonitrile). Product-related fractions were collected, and after HPLC analysis of purity, fractions with a purity >95% were combined and lyophilized to obtain the pure peptide.

[0075] Step 5: Detection and Characterization Methods

[0076] The purified peptide from step 4 was subjected to analytical HPLC and LC / MS to confirm its purity and compound identity.

[0077] The remaining polypeptides of the present invention can be prepared by referring to the above-mentioned Fmoc solid phase synthesis method. The purity (>90%) and identity of the polypeptides were determined by HPLC and mass spectrometry, respectively.

[0078] Example 2 Surface Plasmon Resonance (SPR) Detection of Peptide Binding Affinity

[0079] Experimental Materials:

[0080] (1) Main reagents

[0081]

[0082] (2) Main consumables

[0083] name Production brand Item No. Storage environment SA sensor chip Cytiva BR100531 4℃ 1.5ml centrifuge tube without cap Cytiva BR100287 RT 0.8ml centrifuge tube without cap Cytiva BR100212 RT

[0084] Experimental steps:

[0085] The FOLR1-Biotin protein was captured onto an SA chip, and the sample was diluted into a concentration gradient (3000, 1000, 333.3, 111.1, 37.04 nM). The chip was regenerated using Glycine 1.5 solution. The flow rate was set to 30 μL / min, the injection time for each cycle was set to 120 s, and the dissociation time was set to 600 s. Single-cycle kinetic testing was performed according to the Biacore instrument program flow. Finally, the binding-dissociation curves were analyzed and fitted using Biacore T200 Evaluation Software to calculate affinity-related parameters.

[0086] In the single concentration test, the peptide was used at concentrations of 500 nM and 100 nM.

[0087] Single-cycle kinetics: Set program parameters, place samples according to the plate layout, and perform single-cycle kinetics to determine KD.

[0088] Detection conditions (parameters): flow rate 30 μL / min, association time 120 s, dissociation time 600 s.

[0089] Buffer: 1×HBS-EP+.

[0090] Regeneration conditions: 10 mM Glycine-HCl, 30 s.

[0091] Blocking solution: 10 μg / mL biocytin.

[0092] Experimental results

[0093] The binding constants KD of SEQ ID No.1 and SEQ ID No.2 to FRα were 1.366 μM and 415.4 nM, respectively. Figure 1 、 Figure 2 As shown, Figure 1 is the detection result of the polypeptide shown in SEQ ID No.1, Figure 2 This is the detection result of the polypeptide shown in SEQ ID No.2.

[0094] Experimental results show that the polypeptides of the present invention have a strong affinity for FRα. In summary, the polypeptides of the present invention can effectively target cells with high FRα expression, and therefore have the potential to serve as pharmaceutical ingredients or drug carriers for the prevention, diagnosis, or treatment of cancers with high FRα expression, and can meet subsequent in vivo applications.

[0095] Example 3 Endocytosis Experiment

[0096] Experimental Materials:

[0097] Cells: Positive cells: FOLR1 (CHO-K1); Negative cells: CHO-K1.

[0098] (1) Main reagents

[0099] Reagent name factory Item No. FBS Excell FSP500 Puromycin Invivogen ant-pr-1 4% paraformaldehyde fixative Beyotime P0099 DAPI solution (10ug / ml, ready to use) Solarbio C0065 Streptavidin-Cy5(SA-Cy5) ApexBio K1080 Streptomycin sulfate Aladdin S105491 Penicillin G sodium Aladdin P105489)

[0100] (2) Main consumables

[0101] name Production brand Item No. Storage environment 96WellTC-TreatedBlackMicroplates Agilent 204626-100 RT

[0102] (3) Cell lines

[0103] cell lines culture medium CHO-K1 F12K+10% FBS FOLR1(CHO-K1) F12K+10%FBS+1%P / S+4μg / mlpuromycin

[0104] Experimental steps:

[0105] Prepare SA-Cy5 dilution: dilution ratio 1:50, diluent: cell growth medium.

[0106] Sample preparation: Dilute directly from the stock solution to the desired concentration (0.1-1 μM). Diluent: SA-Cy5 diluent prepared in step 1. Premix at room temperature in the dark for 1 hour.

[0107] Sample addition: Aspirate the culture medium in the wells and replace with samples of corresponding concentration (0.1-1 μM), incubate at 37°C for 2 h.

[0108] Fixation: Wash cells 3 times with DPBS, add 60 μL / well fixative, incubate at 4°C in the dark for 30 min.

[0109] Nuclear staining: Wash cells 3 times with DPBS, add 40-50 μL / well DAPI nuclear staining solution, and incubate at 37°C in the dark for 20 min.

[0110] Experimental results

[0111] The experimental results are as follows Figures 3 to 5 As shown, the polypeptide of the present invention can enter the FOLR1 (CHO-K1) cells that highly express FRα through endocytosis, especially the polypeptide shown in SEQ ID No. 3 can be endocytosed at 0.1 μM.

[0112] In summary, the above results demonstrate that the polypeptide of the present invention has FRα targeting and good penetration ability in highly expressed FRα cells, and can achieve high-sensitivity in vivo imaging of tiny tumors. Therefore, in practical applications, the polypeptide of the present invention can be used as a homing peptide in combination with anticancer drugs or imaging agents for targeted treatment and imaging of tumors.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A polypeptide targeting folate receptor α, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID No. 3, SEQ ID No. 3: GEALLYVYKAQSMDWYYG.

2. A polynucleotide encoding the polypeptide of claim 1.

3. A vector comprising the polynucleotide according to claim 2.

4. A cell containing the vector according to claim 3.

5. A composition comprising an excipient and an active ingredient, characterized in that: The active ingredient includes the polypeptide according to claim 1 or the polynucleotide according to claim 2 or the vector according to claim 3 or the cell according to claim 4; the excipient includes at least one of a diluent, a filler, a binder, a wetting agent, an absorption promoter, a surfactant, a lubricant and a stabilizer.

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