LYPD3 specific binding polypeptide and polypeptide conjugate

Abstract

The invention discloses an LYPD3 specific binding polypeptide, and the amino acid sequence of the polypeptide is shown as SEQ ID NO.4-6. The polypeptide has excellent specificity and can be used for diagnosis and treatment of diseases with positive expression of LYPD3. In addition, the invention also discloses a polypeptide conjugate (PDC, Peptide Tug Conjugate) based on the polypeptide, the polypeptide conjugate (PDC) has a good treatment effect on various cancers positively expressed by LYPD3, and in addition, the polypeptide conjugate can be used for treatment of refractory tumors, especially tumors resistant to immunotherapy and / or chemotherapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine technology, and particularly relates to a LYPD3 specific binding polypeptide and a polypeptide conjugate thereof. BACKGROUND

[0002] LYPD3 (Ly6 / PLAUR Domain-Containing Protein 3), also known as C4.4A, is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein as a member of the LY6 / uPAR family. It is a metastasis-associated protein and is closely related to the occurrence and development of various cancers, and is abnormally highly expressed in lung cancer, melanoma, head and neck squamous cell carcinoma and other tumors, and plays a key role in driving tumor malignant progression by regulating cell adhesion and migration, especially in the metastasis process and regulation of cell stemness. However, the specific molecular mechanism network of LYPD3 in regulating tumor progression has not been fully elucidated, and its regulatory role as a core molecule in the immune microenvironment is still unclear.

[0003] Due to the expression difference of LYPD3 in tumor cells and normal tissues, it is considered as a potential therapeutic target. For example, in non-small cell lung cancer and esophageal cancer, high expression of LYPD3 is associated with poor prognosis of patients and as a potential therapeutic target. Currently, researchers have developed an antibody against LYPD3, which can specifically recognize tumor-associated LYPD3 glycosylation epitopes (Neumann, Theresa, et al. "Targeting a cancer-specific LYPD3 glycoform for tumor therapy." Frontiers in Drug Discovery 3 (2023): 1298916.). The development of antibody conjugate drugs (ADC) based on LYPD3 has shown antitumor activity against non-small cell lung cancer (NSCLC) in preclinical studies (Willuda, et al. "Preclinical antitumor efficacy of BAY 1129980—a novel auristatin-based anti-C4.4A (LYPD3) antibody–drug conjugate for the treatment of non–small cell lung cancer." Molecular cancer therapeutics 16.5 (2017): 893-904.).

[0004] ​Although the prior art has developed antibodies or ADC drugs for LYPD3, the current research on LYPD3 target is still in the preliminary stage, and there is no drug on the market or even in clinical research, and the current LYPD3 targeted drugs are mainly developed for specific tumors, and it is necessary to further strengthen the research and development of LYPD3 drugs. SUMMARY

[0005] The purpose of the present application is to provide a LYPD3 specific binding polypeptide which can be bound to LYPD3 with high specificity for the diagnosis and treatment of diseases with high expression of LYPD3.

[0006] Another purpose of the present application is to provide a LYPD3 specific polypeptide conjugate which has a better therapeutic effect on LYPD3 high expression diseases.

[0007] Another purpose of the present application is to provide a LYPD3 specific polypeptide conjugate which has a good therapeutic effect on refractory cancer.

[0008] The above purposes of the present application are achieved by the following technical means:

[0009] The present application provides a LYPD3 specific binding polypeptide, and the amino acid sequence of the LYPD3 specific binding polypeptide is shown in SEQ ID NO. 4-6.

[0010] Further, the nucleotide sequence corresponding to the LYPD3 specific binding polypeptide is shown in SEQ ID NO. 1-3.

[0011] In some preferred embodiments, the LYPD3 specific binding polypeptide is a cyclic peptide.

[0012] The LYPD3 specific binding polypeptide has the following effects:

[0013] (1) Targeted drug delivery: LYPD3 specific binding polypeptide can be used as a carrier to accurately deliver chemotherapeutic drugs, toxins or nanoparticles and other effective payloads to LYPD3 positive tumor cells.

[0014] (2) Blocking LYPD3 from playing a biological function: LYPD3 specific binding polypeptide can block the interaction between LYPD3 and CD300E+ macrophages in tumor-derived exosomes, ultimately reducing T cell exhaustion.

[0015] (3) Enhancing drug penetration: LYPD3 specific binding polypeptide can increase the penetration of drugs in LYPD3 positive tumor tissues, thereby improving the therapeutic effect.

[0016] (4) Imaging diagnosis: LYPD3 specific binding polypeptides can be used as targeting ligands combined with imaging agents such as radioisotopes, fluorescent dyes or magnetic nanoparticles, for positron emission tomography (PET), computed tomography (CT) or magnetic resonance imaging (MRI) and other imaging examinations.

[0017] (5) Early detection: Using the high affinity of polypeptides to tumor marker LYPD3 protein, diagnostic tools for early detection of tumors can be developed.

[0018] (6) Targeted immunotherapy: LYPD3 specific binding polypeptides can be combined with immune checkpoint inhibitors to enhance the recognition and attack of the immune system on tumors.

[0019] (7) Combination therapy: Polypeptides can be used in combination with other treatment methods such as chemotherapy, radiotherapy or targeted therapy to improve treatment efficacy. For example, combining chemotherapy drugs with tumor targeting polypeptides can reduce drug toxicity to normal tissues while improving tumor efficacy.

[0020] The LYPD3 specific binding polypeptides of the present application have broad application prospects in the diagnosis and treatment of LYPD3 positive tumors. They not only can be used as drug delivery carriers, but also can directly exert anti-tumor effects, and also have important value in imaging diagnosis and early detection.

[0021] In another aspect, the present application provides a polypeptide-drug conjugate (PDC) based on the above-mentioned LYPD3 specific binding polypeptide, referred to as LYPD3 specific polypeptide conjugate in this application, which has the structure shown in formula (1):

[0022]

[0023] Wherein:

[0024] P is a LYPD3 specific binding polypeptide, the amino acid sequence of which is shown in any one of SEQ ID NO. 4-6;

[0025] D is an active substance, which is a substance capable of playing a diagnostic and therapeutic role on diseases;

[0026] L is any linker capable of connecting the LYPD3 specific binding polypeptide and the active substance.

[0027] n is an integer from 1 to 100.

[0028] In some preferred embodiments, n is an integer from 1 to 8, for example 1, 2, 3, 4, 5, 6, 7 and 8.

[0029] In some embodiments, the active substance D is selected from an antitumor drug or a tracer.

[0030] In some embodiments, the antitumor drug is selected from one or more of a tubulin inhibitor, a topoisomerase inhibitor, an alkylating agent, an antitumor antibiotic, a metal platinum agent, an antimetabolite, an antitumor plant drug, a tyrosine kinase receptor, a terpene, a tyrosine kinase receptor, a radionuclide, and an NF-KB inhibitor.

[0031] In some specific embodiments, the tubulin inhibitor is selected from one or more of monomethyl auristatin E (MMAE), vinblastine, vinorelbine, and docetaxel.

[0032] In some specific embodiments, the topoisomerase inhibitor is selected from one or more of topotecan, irinotecan, and etoposide.

[0033] In some specific embodiments, the alkylating agent is selected from one or more of carmustine, chlorambucil, cyclophosphamide, and melphalan.

[0034] In some specific embodiments, the antitumor antibiotic is selected from one or more of mitomycin, eprubicin, doxorubicin, bleomycin, and daunorubicin.

[0035] In some specific embodiments, the antitumor hormone is selected from one or more of tamoxifen, toremifene, letrozole, aminoglutethimide, leuprorelin, and gonadorelin.

[0036] In some specific embodiments, the metal platinum agent is selected from one or more of cisplatin, carboplatin, and oxaliplatin.

[0037] In some embodiments, the anti-metabolite agent is selected from one or more of 5-Fluorouracil, Methotrexate, Gemcitabine, Cytarabine, and the like.

[0038] In some embodiments, the anti-tumor plant drug is selected from one or more of Camptothecins, Vinca alkaloids, Paclitaxel, Docetaxel, and the like.

[0039] In some embodiments, the tyrosine kinase receptor is selected from one or more of Lapatinib, Imatinib, Gefitinib, Erlotinib, and the like.

[0040] In some embodiments, the radionuclide agent is selected from one or more of 177 Lu, 131 I or 90 Y.

[0041] In some embodiments, the NF-KB inhibitor is Bortezomib.

[0042] In some embodiments, the tracer is one or more of a PET tracer, a CT tracer, and an MRI tracer.

[0043] In some embodiments, the PET tracer is selected from one or more of 18 F, 68 Ga, 64 Cu, 99 mTc and 89 Zr, and the like.

[0044] In some embodiments, the CT tracer is selected from one or more of iodine nanocomtrast agents, gold nanocomtrast agents, tantalum oxide nanocomtrast agents, bismuth nanocomtrast agents, and lanthanide nanocomtrast agents; in some preferred embodiments, the CT tracer is selected from one or more of Iohexol, Ioversol, Iodixanol, Iopromide, Iobitridol, Iomeprol, Iopamidol, Acetrizoic acid, Ioglycamic acid, Diatrizoic acid, Sodium Iothalamic acid, Iotalamic acid, Metrizoate, Meglumine, Metrizoate, and Metrizamide.

[0045] In some preferred embodiments, the MRI tracer is selected from the group consisting of longitudinal relaxivity contrast agents and / or transverse relaxivity contrast agents; more preferably, the MRI tracer is selected from the group consisting of paramagnetic contrast agents, ferromagnetic contrast agents or superparamagnetic contrast agents; further preferably, the MRI tracer is selected from one or more of the following: Gd-DTPA and its linear, cyclic polyamine polycarboxylate chelates and porphyrin chelates of manganese, macromolecules.

[0046] In some embodiments, the linker L is selected from the group consisting of cleavable linkers, non-cleavable linkers, or a combination thereof.

[0047] In some preferred embodiments, the linker L is selected from the group consisting of cleavable linkers including acid cleavable linkers, reduction condition cleavable linkers, enzyme cleavable linkers, cathepsin B cleavable linkers, and other cleavable linkers such as GSH cleavable reduction linkers.

[0048] As exemplary examples, the cleavable linkers include hydrazone bond, valine-citrulline (Val-Cit), disulfide bond, etc.

[0049] As exemplary examples, the non-cleavable linkers include poly-sarcosine (Sar), thioether bond, and / or amide bond.

[0050] In some preferred embodiments, the linker L is selected from the group consisting of a combination of cleavable linkers and non-cleavable linkers, such as a combination of cleavable linkers hydrazone bond, Val-Cit dipeptide, disulfide bond, etc., and non-cleavable linkers poly-sarcosine, amide bond, or thioether bond, etc.

[0051] As an exemplary example, the linker is selected from the group consisting of a combination of 10 units of poly-sarcosine (Sar) and valine-citrulline (Val-Cit) dipeptide.

[0052] In some embodiments, the LYPD3 specific multi-conjugate drug of the present application has the following structural formula:

[0053]

[0054] In another aspect, the present application also provides a composition comprising (1) any of the above polypeptides, or polypeptide conjugates; and (2) a pharmaceutically acceptable carrier or excipient.

[0055] In some embodiments, the pharmaceutically acceptable excipient is selected from one or more of the following: diluents, binders, lubricants, wetting agents, antioxidants, buffers, bacteriostatic agents, suspending agents, solubilizers, thickening agents, stabilizers, preservatives.

[0056] In some embodiments, the composition of the present application is an oral dosage, an injection dosage or an infusion dosage.

[0057] In some preferred embodiments, the pharmaceutical composition of the present application is an injection dosage, the dosage used is 0.01-5 ug / g of polypeptide conjugate PDC; preferably 0.1-2 ug / g, the administration frequency is once a day, continuously for 10-30 days, such as continuously for 21 days.

[0058] In some preferred embodiments, the pharmaceutical composition further comprises an immunotherapeutic agent and / or a chemotherapeutic agent.

[0059] In some embodiments, the immunotherapeutic agent is selected from a PD-1 / PD-L1 inhibitor, a CTLA-4 inhibitor,

[0060] a LAG-3 inhibitor or a TIGIT inhibitor.

[0061] In some embodiments, the PD-1 / PD-L1 inhibitor is selected from one or more of Nivolumab, Pembrolizumab, Sintilimab, Tislelizumab, Camrelizumab, Atezolizumab, Durvalumab and Envafolimab.

[0062] In some embodiments, the CTLA-4 inhibitor is selected from one or more of Ipilimumab and Tremelimumab.

[0063] In some embodiments, the LAG-3 inhibitor is selected from one or more of Relatlimab and Ivosidenib.

[0064] In some embodiments, the TIGIT inhibitor is selected from one or more of Vibostolimab and Tiragolumab.

[0065] In some preferred embodiments, the immunotherapeutic agent is selected from one or more of Tislelizumab, Camrelizumab and Nivolumab.

[0066] In some embodiments, the chemotherapeutic agent is selected from one or more of oxaliplatin, cisplatin, carboplatin, nedaplatin, dicycloplatin, lobaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, miriplatin, satraplatin, gemcitabine, capecitabine, ancitabine, fluorouracil, bifuridine, doxifluridine, tegafur, carmofur, trifluridine, paclitaxel, albumin-bound paclitaxel, and docetaxel, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, 7-ethylcamptothecin, irinotecan, topotecan, vinorelbine, vinblastine, vincristine, vindesine, vindesine sulfate, epirubicin, doxorubicin, daunorubicin, pirarubicin, aminoglutethimide, idarubicin, mitoxantrone, epirubicin, valrubicin, zorubicin, pixantrone, pemetrexed, carmustine, melphalan, etoposide, teniposide, mytomycin, ifosfamide, cyclophosphamide, azacitidine, methotrexate, bendamustine, liposomal doxorubicin, dactinomycin, bleomycin, pepleomycin, temozolomide, dacarbazine, pepleomycin, eribulin, plinabulin, Sapacitabine, triaziquone, 153Sm-EDTMP, and tegafur, and encequidar.

[0067] In another aspect, the present application provides use of the polypeptide, or the polypeptide conjugate, or the composition in the preparation of a medicament for treating and / or preventing a tumor.

[0068] Further, the tumor is selected from a tumor that positively expresses LYPD3.

[0069] In some embodiments, the tumor is selected from bladder cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, cervical squamous cell carcinoma and cervical adenocarcinoma, cutaneous melanoma, breast cancer, prostate cancer, hepatocellular carcinoma, pancreatic cancer, renal cancer, ovarian cancer, endometrial cancer, or thyroid cancer, etc.

[0070] In some embodiments, the bladder cancer includes urothelial carcinoma, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, mixed carcinoma, undifferentiated carcinoma, and carcinosarcoma.

[0071] In some preferred embodiments, the tumor is selected from a refractory tumor. A refractory tumor refers to a malignant tumor that lacks effective response to existing standard treatment regimens (such as surgery, radiotherapy, chemotherapy, targeted therapy, etc.), or relapses or progresses within a short period of time (such as within 3-6 months) after treatment. Such tumors usually have high invasiveness, drug resistance or complex biological characteristics, leading to difficulty in controlling the disease with conventional treatment.

[0072] In some more preferred embodiments, the tumor is a refractory bladder cancer, such as a bladder cancer that is resistant to chemotherapy, immunotherapy, and combinations thereof.

[0073] The beneficial technical effects of the present application are as follows:

[0074] (1) In one embodiment, the LYPD3-targeting polypeptide of the present application can be distributed in tumor tissues with high specificity in vivo, and has high specific binding capacity to cells with high LYPD3 expression and tumors.

[0075] (2) In another embodiment, the LYPD3-targeting polypeptide conjugate of the present application can effectively kill tumor cells, and has significant killing effect on various tumors with high LYPD3 expression, such as bladder cancer and breast cancer tumors with high LYPD3 expression. More significantly, the LYPD3-targeting polypeptide conjugate of the present application has better tumor killing effect than immunotherapy, chemotherapy, or even immunotherapy combined with chemotherapy, and can be used alone or in combination with other existing therapies such as immunotherapy anti-PD-1 to effectively reverse the treatment resistance of existing therapies.

[0076] (3) In another embodiment, the LYPD3-targeting polypeptide and polypeptide conjugate of the present application have higher tumor tissue permeability and lower non-specific uptake in liver and bone marrow.

[0077] (4) In another embodiment, the polypeptide conjugate of the present application has low immunogenicity compared to antibody drug conjugates, and is less likely to trigger the body's immune system to produce antibodies or immune responses against the drug, thereby prolonging the drug's efficacy and improving safety.

[0078] (5) The polypeptide and polypeptide conjugate of the present application can be quickly prepared by chemical synthesis, and the process is simple. BRIEF DESCRIPTION OF DRAWINGS

[0079] Figure 1 is the ELISA result of LYPD3-specific polypeptide binding to human LYPD3.

[0080] Figure 2 is the ELISA result of LYPD3-specific polypeptide binding to murine LYPD3.

[0081] Figure 3 is the flow detection of LYPD3-specific polypeptide FITC conjugate binding to different LYPD3 expression cells.

[0082] Figure 4 is the distribution and organ metabolic pathway of LYPD3-specific polypeptide FITC conjugate in mice in vivo.

[0083] Figure 5 is the result graph of LYPD3-specific polypeptide suppressing LYPD3 to play biological function and reducing T cell exhaustion.

[0084] Figure 6is the mass spectrum of polypeptide conjugate LPDC-1 of the present application.

[0085] Figure 7 is the ultra-high liquid chromatogram of polypeptide conjugate LPDC-1 of the present application.

[0086] Figure 8 is the therapeutic effect of LPDC-1 on different LYPD3-expressed human breast cancer and bladder cancer tumors.

[0087] Figure 9 is the therapeutic effect of LPDC-1 and other treatment methods on bladder cancer resistance. DETAILED DESCRIPTION

[0088] The technical solutions of the present application are further illustrated below through specific examples, which do not represent a limitation on the scope of protection of the present application. Some non-essential modifications and adjustments made by others according to the concept of the present application still fall within the scope of protection of the present application.

[0089] It should be noted that the terms "comprise", "contain" or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method, product or equipment comprising a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, product or equipment. Without more limitations, the element defined by the statement "comprises a" does not exclude the presence of another identical element in the process, method, product or equipment comprising the element.

[0090] The structure of the polypeptide described in the present application can be linear or cyclic. In some embodiments, the polypeptide is linear, and in other embodiments, the polypeptide is cyclic. In some preferred embodiments, the polypeptide is selected from a cyclic peptide.

[0091] In the present application, the cleavable linker is a chemical structure that can be broken and release drugs under specific conditions, which is applied in the design of antibody drug conjugates (ADC) and polypeptide conjugates (PDC). According to the different mechanisms of cleavage, the cleavable linker can be mainly divided into the following categories:

[0092] 1. Chemical cleavable linker, which mainly relies on specific chemical environment (such as acidic or reducing conditions) to achieve drug release. Common types include:

[0093] Acid-cleavable linker: uses the acidic environment (pH 4.5-5.0) of lysosomes in cells to cleave the linker and release the drug. For example, Pfizer's and Acid-cleavable linker

[0094] Reduction-cleavable linker: usually relies on intracellular reducing agents (such as glutathione) to break the linker. For example, some ADC drugs use disulfide bond linkers, which are reduced and broken in the high concentration of glutathione environment in cells.

[0095] 2. Enzyme-cleavable linker, which relies on the catalytic action of specific enzymes to achieve drug release. Such linkers are usually designed to be sensitive to enzymes highly expressed in tumor cells. For example:

[0096] Cathepsin B-cleavable linker: Cathepsin B is an enzyme highly expressed in tumor cells and can specifically cleave certain peptide linkers (such as valine-citrulline Val-Cit).

[0097] 3. Other cleavable linkers

[0098] GSH-cleavable reduction linker: such linker can be cleaved in the presence of GSH, thereby releasing the drug. For example, the linkers of Mylotarg and Besponsa belong to the combination of acid-cleavable and GSH-cleavable reduction.

[0099] In the present application, the non-cleavable linker refers to a linker group that remains chemically stable under physiological conditions or specific target conditions, and its structure does not actively break due to the body environment (such as pH, enzymes, reducing agents, etc.). The release of the drug depends on the complete degradation of the carrier molecule (such as antibody), and the active molecule is free to play a role. The non-cleavable linker mainly realizes the persistent connection of the toxin and the antibody through stable chemical bonds, and its core types include: thioether bond and stable amide bond. In addition, hydrophilic spacers such as polyethylene glycol (PEG) or poly sarcosine (Poly Sarcosine) are sometimes used with non-cleavable bonds to adjust the hydrophilic or hydrophobic properties of the drug or prolong the circulation half-life.

[0100] In some embodiments, the tumor highly expresses LYPD3 protein. Wherein "high expression" refers to the expression level of a certain gene or protein in cells is significantly higher than that in normal cells or tissues.

[0101] Example 1 Phage panning of LYPD3-targeting peptides

[0102] Using LYPD3 human protein, NEB's random peptide library: Ph.D.-C7C TMPhage ELISA detection: 1) coating: coating LYPD3 human protein, 0.5 ug / mL, coating, 4°C overnight; 2) blocking: 2% skim milk powder blocking, 200 uL / well, 37°C 1 h; 3) primary antibody: Phage supernatant, 100 uL / well, 37°C 1 h; 4) secondary antibody: anti-M13 antibody (HRP), 1:20000, 100 uL / well, 37°C 1 h; 5) color development: color developing solution 100 uL / well, 37°C, 15 min; 6) termination: termination solution 100 uL / well; 7) plate reading: Antibody titer.

[0103] Blocking condition: 1% BSA or 0.1% skim milk-PBS: for blocking non-specific binding sites.

[0104] Washing condition: 0.1% skim milk-PBS washing 10 times (the second round increased to 20 times), strictly remove weakly bound phage

[0105] Elution system: 0.2M glycine-HCl (pH2.2): dissociate phage-protein binding in acidic environment, 1mg / mL BSA: stabilize phage in elution solution, 1M Tris-HCl: quickly neutralize acidic environment, maintain phage activity.

[0106] Example 2 Phage ELISA identification

[0107] Preparation of phage supernatant: 1) 1:100 dilution of overnight culture of E. coli engineering strain ER2738, 1mL per centrifuge tube; 2) picking single clones into centrifuge tubes (Note: the phage plaques on the plate cannot be more than 3 days, and are stored at 4°C, and the single clones on each plate are not more than 100, to ensure that the picked ones are single clones); 3) 37°C, 220rpm culture for 4.5-5h (Note: the culture time should not exceed 5h); 4) 14000rpm centrifugation for 30s, and the supernatant is taken into a new centrifuge tube; centrifugation at 14000rpm for 30s, and 80% of the supernatant is taken into a new centrifuge tube, which is the phage supernatant.

[0108] Phage ELISA detection: 1) coating: coating LYPD3 human protein, 0.5 ug / mL, coating, 4°C overnight; 2) blocking: 2% skim milk powder blocking, 200 uL / well, 37°C 1 h; 3) primary antibody: Phage supernatant, 100 uL / well, 37°C 1 h; 4) secondary antibody: anti-M13 antibody (HRP), 1:20000, 100 uL / well, 37°C 1 h; 5) color development: color developing solution 100 uL / well, 37°C, 15 min; 6) termination: termination solution 100 uL / well; 7) plate reading: Antibody titer.

[0109] Picking 49 positive clones obtained by screening the library Ph.D.-C7C, and performing PBS and multi-tag protein (containing His tag) phage ELISA detection. A total of 18 positive clones that do not react with multi-tag protein and empty plate are obtained by retesting, and are tested for mouse protein (the coating concentration is changed to 2ug / mL).

[0110] Single-stranded DNA extraction and sequencing

[0111] Single-stranded DNA extraction and sequencing: 1) Dilute 1:100 the overnight culture of E. coli engineering strain ER2738, 1 mL per centrifuge tube; 2) pick single colonies into centrifuge tubes: the phage plaques on the plate cannot be more than 3 days, and are stored at 4°C, and the single colonies on each plate are not more than 100, to ensure that the picked ones are single colonies; 3) culture at 37°C, 220 rpm for 4.5-5 h (note: the culture time should not exceed 5 h); 4) centrifuge at 14000 rpm for 30 s, and take the supernatant into a new centrifuge tube; centrifuge at 14000 rpm for 30 s, and take 500 μL of the supernatant into a new centrifuge tube. 5) add 200 μL of 20% PEG / 2.5 M NaCl, mix well by inverting, and stand at room temperature for 20 min; 6) centrifuge at 4°C, 14000 rpm for 10 min, and discard the supernatant; 7) centrifuge briefly, and discard the supernatant; 8) add 100 μL of Iodide Buffer, resuspend and mix well. Add 250 μL of ethanol, and incubate at room temperature for 20 min (note: short-time incubation at room temperature can effectively precipitate single-stranded phage DNA, so that most of the phage proteins remain in the solution); 9) centrifuge at 4°C, 14000 rpm for 10 min, and discard the supernatant. Wash twice with 0.5 mL of 70% ice ethanol, centrifuge at 4°C, 14000 rpm for 10 min, and discard the supernatant. Dry for 3-5 min; 10) resuspend in 30 μL of TE, and store at -20°C.

[0112] Sequencing verification of the single-stranded DNA was performed using 96 sequencing primers. The single-stranded DNA of the positive clones obtained by screening was subjected to sequencing analysis, and 3 loop peptides were successfully sequenced. The 3 loop peptides can simultaneously and efficiently bind to human and murine LYPD3 proteins. The base sequences and amino acid sequences of the 3 loop peptides are shown in the following table.

[0113]

[0114] Example 4: Polypeptide synthesis and ELISA retesting

[0115] 1) According to the DNA sequence to get the amino acid sequence and ring peptide synthesis; 2) coating: coated with human and mouse LYPD3 protein, 0.1 ug / mL, coated, 4°C overnight; 3) blocking: 2% skim milk powder blocking, 200 uL / well, 37°C 1h; 4) primary antibody (biotin-labeled polypeptide): 5ug / ml start, 2-fold dilution, add 11 wells, the 12th well negative control, 100 uL / well, 37°C 1h; 5) secondary antibody: SA-HRP, 1:100000, 100 uL / well, 37°C 1h; 6) color development: color developing solution 100 uL / well, 37°C, 15min; 7) termination: termination solution 100 uL / well; 8) plate reading: Antibody titer.

[0116] ELISA results are shown in Figure 1 and Figure 2 , the 3 ring peptides can effectively bind to human Figure 1 ) and mouse Figure 2 ) LYPD3 recombinant protein, the 3 ring peptides of the present application have high specificity for LYPD3.

[0117] Example 5 Polypeptide for the diagnosis of LYPD3 positive disease

[0118] In vitro experiment: further test the specific binding ability of polypeptide to LYPD3 and the diagnosis ability of LYPD3 positive disease, take polypeptide 1 / Petide 1 (SEQ ID NO. 4) as an example, we couple FITC group on LYPD3 targeting polypeptide, and overexpress LYPD3 in human bladder cancer cells T24 and UM-UC-3, and mouse bladder cancer cells MB49. As shown in Figure 3 , by adding LYPD3 specific polypeptide FITC conjugate to different LYPD3 expressing tumor cells, we found that LYPD3 targeting polypeptide FITC conjugate showed significant specific binding ability in LYPD3 highly expressed human (T24, UM-UC-3) and mouse (MB49) bladder cancer cells, and the binding strength was dependent on the drug concentration.

[0119] In vivo experiment: mouse LYPD3 overexpressing MB49 bladder cancer cell line was cultured with DMEM medium containing 10% fetal bovine serum, and 5x10 5 cells were inoculated subcutaneously in C57BL / 6 mice. Every 3-4 days, the health status of the mice was observed. After tumor formation, when the subcutaneous tumor volume of the mice reached 50-100mm 3Afterwards, random grouping: Vector+Peptide 1, MB49 oeLYPD3+Peptide 1, Vector+Peptide 2, MB49 oeLYPD3+Peptide 2. After grouping, tail vein injection of LYPD3 specific polypeptide FITC conjugate 1 and 2 was performed respectively, and before injection, 0H, 2H, 4H after injection, mouse fluorescence imaging was performed, and the results are shown in Figure 4 As shown, LYPD3 specific polypeptide FITC conjugate 1 and 2 were significantly enriched in tumor tissues with high expression of LYPD3 (significantly higher than the control group), and were mainly metabolized through the kidney.

[0120] The data show that LYPD3 targeting polypeptide exhibits specific binding capacity in vitro and in vivo, which can be used as a diagnostic tool for LYPD3 positive diseases, especially tumors.

[0121] Example 6 LYPD3 specific binding polypeptide can block the interaction of LYPD3 with CD300E+ macrophages in tumor-derived exosomes, ultimately reducing T cell exhaustion

[0122] Exosome extraction: First, T24 and UM-UC-3 cells overexpressing LYPD3 were cultured in exosome-free serum medium for 24-48 hours, and the cell supernatant was collected; then the cell debris was removed by centrifugation at 300xg for 10 minutes, and the microvesicles were removed by centrifugation at 2000xg for 10 minutes, and finally the non-exosomal impurities were removed by centrifugation at 10000xg for 30 minutes. Then the exosomes were precipitated by ultracentrifugation (100,000xg for 70-90 minutes), resuspended the precipitate with pre-cooled PBS buffer after discarding the supernatant, and washed the residual impurities by centrifugation at the same centrifugal force for the second time. Finally, the exosome precipitate was resuspended with appropriate PBS, aliquoted and stored at -80°C. The particle size (40-160nm) was detected by nanoparticle tracking analysis (NTA), and the exosome markers (CD63 / CD9) and LYPD3 overexpression were verified by Western Blot. The whole experiment should be operated at 4°C, and RNase-free consumables should be used to avoid contamination.

[0123] CD300E + macrophages and CD8 + T cell extraction: First, fresh bladder cancer surgical specimens were prepared into single cell suspension by enzymatic digestion (such as collagenase / hyaluronidase) combined with mechanical grinding, and the cell density was adjusted to 1x10 6 / mL after filtration through a 70μm filter. Then labeled with fluorescent antibodies: CD300E +Macrophages were labeled with Ghost Dyed / CD45 / CD11b / CD300E combination (e.g. Ghost Dyed-BV421, CD45-APC-Cy7, CD11b-FITC, CD300E-PE), and CD8 + T cells were labeled with Ghost Dyed-BV421 / CD45-APC-Cy7 / CD3-APC / CD8-PE-Cy7 combination, incubated at room temperature for 15 minutes in the dark, and resuspended in PBS containing 2% FBS after washing with 1x PBS for 3 times. When loaded onto the flow cytometer, the cells were gated with Ghost Dyed-BV421 / CD45-APC-Cy7 / CD3-APC / CD8-PE-Cy7 combination, and then analyzed with CD3 - CD45 + Gated immune cell population. Sorted CD300E + macrophages by CD11b + / CD300E + double positive gate, and CD8 + T cells by CD3 + / CD8 + double positive gate, sorted with purity mode, and collected in a tube preloaded with 200 μL RPMI 1640 medium containing 10% FBS. After sorting, part of the cells were taken for purity verification (CD300E + macrophages were >95%, and CD8 + T cells were >98%), and the remaining cells were centrifuged (300g x 5 min) for co-culture or functional analysis. The whole experiment was operated at 4°C and used nuclease-free consumables to avoid contamination.

[0124] Cell co-culture: CD300E + macrophages were seeded at a density of 1x10 6 / mL with an equal amount of CD8 + T cells at a ratio of 1:1 in RPMI 1640 medium containing 10% exosome-free serum, and tumor-derived exosomes (100 μg / mL, purified by ultracentrifugation and verified for uptake efficiency by DiD labeling) or target peptides (2.5 ug / ml) were added to the experimental groups. The co-culture system was incubated at 37°C, 5% CO2 for 24-48 hours, and the Transwell separation system was used to distinguish direct contact and paracrine effect. After collecting the cells, T cells were labeled with anti-CD3-APC and CD8-PE-Cy7, and PD-1-FITC and TIM-3-PE antibodies were added and incubated at room temperature for 15 minutes in the dark, and then resuspended in a buffer containing 2% FBS after washing with PBS for 3 times. Flow cytometry was used for detection, and the proportion of PD-1 and TIM-3 co-expression in the CD3 + / CD8 + double positive gate was analyzed, and isotype antibodies were used as negative controls. The results are shown in Figure 5As shown, LYPD3 specific binding polypeptides can block LYPD3 binding to CD300E in tumor-derived exosomes + Macrophage interaction, ultimately reducing T cell exhaustion, can significantly enhance anti-tumor immune response.

[0125] Example 7 LYPD3 polypeptide conjugate (LPDC-1)

[0126] LYPD3 polypeptide conjugate

[0127] Wherein P is Petide 1 / polypeptide 1, the polypeptide ACLSSTKKTCGGG shown in SEQ ID NO. 4

[0128] D is an active substance, monomethyl auristatin E (MMAE): a potent anti-mitotic agent, by inhibiting tubulin polymerization to prevent cell division, thereby exerting anti-tumor effect;

[0129] L is a valine-citrulline (Valine-Citrulline, Vc) dipeptide linker: used to link the active substance and polypeptide.

[0130] The specific structural formula is as follows:

[0131]

[0132] Example 8 LYPD3 polypeptide conjugate (LPDC-2)

[0133]

[0134] The structure of LPDC-2 is basically the same as that of LPDC-1, and the difference is that the polypeptide P of LPDC-2 is Petide 2 / polypeptide 2, the polypeptide ACVIGTERECGGG shown in SEQ ID NO. 5.

[0135] Example 9 Preparation method of LYPD3 polypeptide conjugate (PDC) (take LPDC-1 as an example)

[0136] I. Synthesis of MMAE linker

[0137] 1) Synthesis of compound 1

[0138] Compound VCMMAE (Valine-Citrulline-p-aminobenzyloxycarbonyl- monomethyl auristatin E) (3 g, 2.67 mmol) was dissolved in DMF solution (30 mL), and glutaric anhydride (534.2 mg, 5.34 mmol), DIEA (690.4 mg, 5.34 mmol) and DMAP (325.7 mg, 2.67 mmol) were added to the above solution in order. The above reaction solution was stirred at room temperature for 3 hours, and after the reaction was completed, the above reaction solution was slowly added to a mixed solution of petroleum ether: ethyl acetate (200 mL, 5: 1), and after the solid was precipitated, it was centrifuged to obtain a white solid crude product. The crude product was purified by reverse phase Flash (H2O / ACN, 0.1% TFA, 50-95%), and lyophilized to obtain a white powder (2.6 g, yield: 78.8%).

[0139]

[0140] 2) Synthesis of compound 2

[0141] Compound 1 (2.6 g, 2.1 mmol) and HOSu (362.2 mg, 3.1 mmol) were dissolved in DMF (15 ml), and then DMAP (24.4 mg, 0.2 mmol) and EDCI (804.3 mg, 4.2 mmol) were added in order. The above reaction solution was stirred at room temperature for 3 hours, and after the reaction was completed, the above reaction solution was slowly added to a mixed solution of petroleum ether: ethyl acetate (200 mL, 5: 1), and after the solid was precipitated, it was centrifuged to obtain a white solid crude product. The crude product was purified by reverse phase Flash (H2O / ACN, 0.1% TFA, 50-95%), and lyophilized to obtain a white powder (2.3 g, yield: 82.1%).

[0142]

[0143] II. Polypeptide synthesis

[0144] Solid-phase synthesis of polypeptide 1 was synthesized as follows:

[0145] The synthesis of the polypeptide was performed using Fmoc SPPS synthesis with the following specific steps: 1) Dichloromethane (30 ml) was added to a polypeptide reaction tube containing CTC resin (0.74 mmol, 1 g, 0.74 mmol / g), followed by the addition of Fmoc-Lys(Alloc)-OH (401.8 mg, 0.88 mmol, 1.2 eq) and bubbling with nitrogen; 2) DIEA (4.0 eq) was added and the reaction was bubbled for 2 h; 3) Methanol (1 mL) was added and the reaction was bubbled for 30 min; 4) The solvent was drawn off and the resin was washed 5 times with DMF; 5) A 20% piperidine / DMF solution was added and the reaction was bubbled for 30 min; 6) The solvent was drawn off and the resin was washed 5 times with DMF; 7) The appropriate Fmoc-amino acid was added according to the following scheme and the mixture was bubbled for 30 s, followed by the addition of the activating reagent in the following scheme and bubbling with nitrogen for approximately 1 h; 8) The above steps 4 to 7 were repeated for the condensation of the next amino acid.

[0146]

[0147] III. Cleavage and purification of the polypeptide:

[0148] 1) Cleavage solution (92.5% TFA / 2.5% EDT / 2.5% TIS / 2.5% H2O) was added to the resin and the reaction was shaken on a shaker for 2 h; 2) The cleavage solution was added to cold methyl tert-butyl ether and a white solid was precipitated, which was centrifuged (200 mL) using a centrifuge (3 min, 3000 rpm). The solid was washed twice more with methyl tert-butyl ether (200 mL) and the resulting crude polypeptide was dried using a vacuum pump; 3) The resulting crude polypeptide was purified using semi-preparative HPLC and lyophilized to obtain polypeptide 1 (122.4 mg).

[0149] IV. Synthesis of disulfide bond

[0150] Synthesis of polypeptide 1 - disulfide bond:

[0151] Polypeptide 1 (122.4 mg, 0.05 mmol) was dissolved in a solution of water and acetonitrile (3 / 1, 122 mL) and the pH of the solution was adjusted to approximately 11-12 using NH4HCO3, and the reaction was oxidized overnight in air. After the reaction was complete, the pH was adjusted to approximately 5 using 1 N aqueous HC1, and then lyophilized. The resulting crude product was dissolved in a small amount of water and acetonitrile and purified using semi-preparative HPLC to obtain polypeptide 1 - disulfide bond as a white powder (72.5 mg, 58.8%).

[0152]

[0153] V. Chemical ligation of VCMMAE

[0154] The synthesis of polypeptide 1 - VCMMAE was performed as follows:

[0155] Compound 2 (30 mg, 22.4 umol) and polypeptide 1-disulfide (53.69 mg, 22.4 umol) were dissolved in H2O / ACN (2 mL) solution, and NaHCO3 (11.3 mg, 134.4 umol) was added. The above reaction solution was stirred at room temperature for 4 hours, and LCMS was monitored. After the reaction was completed, 1N HCl was added to adjust the pH to about 5. Directly purified by semi-preparative HPLC, and freeze-dried to obtain 36.3 mg of white powder.

[0156] Six, removal of Alloc:

[0157] Synthesis of LPDC-1:

[0158] Polypeptide 1-VCMMAE (36.3 mg, 10.06 umol) was dissolved in H2O / ACN=1:1 (2 mL) solution, followed by the addition of Pd(PPh3)4 (3.45 mg, 3.0 umol) and morpholine (8.7 mg, 0.1 mmol). The above reaction solution was stirred at room temperature for 2 hours, and LCMS was monitored. If not completely reacted, Pd(PPh3)4 (3.45 mg, 3.0 umol) was added. After the reaction was completed, the reaction solution was directly purified by semi-preparative HPLC, and freeze-dried to obtain white powder LPDC-1 (9.3 mg, 27.6%).

[0159] The prepared LPDC-1 was subjected to mass spectrometry and ultra-high performance liquid chromatography, and the results are shown in Figure 6 and Figure 7 .

[0160] Example 10 Anti-tumor effect of LYPD3 polypeptide conjugate

[0161] 1. Fresh surgical specimens were obtained from human breast cancer high LYPD3 tumor, bladder cancer high LYPD3 tumor, and bladder cancer low LYPD3 tumor. After the tissue was washed with PBS, it was cut into 3-4 mm tissue blocks with tissue scissors and inoculated in NCG mice. After successful inoculation, when the volume of human-derived tumor subcutaneously inoculated in NCG mice reached 50-100 mm 3 , the mice were randomly divided into Vehicle (normal saline) group and LPDC-1 group.

[0162] 2. LPDC-1 was resuspended in normal saline to a concentration of 0.1 ug / ul. The LPDC-1 drug volume was calculated at 0.5 ug / g, and the drug was administered by intraperitoneal injection. The administration frequency was once a day for LPDC-1 and Vehicle, and the administration lasted for 21 days. The size of the tumor was observed and recorded every 3-4 days by volume measurement, and the results are shown in Figure 8As shown, the LYPD3 polypeptide conjugate showed significant anti-tumor therapeutic effect in breast cancer and bladder cancer human tumors with high LYPD3 expression, while there was no statistical difference in tumor size and weight between the LYPD3 polypeptide conjugate treatment group and the control group in bladder cancer human tumors with low LYPD3 expression, which indicates the specificity of LYPD3 polypeptide conjugate treatment and effectiveness on various tumors.

[0163] Example 11 Treatment of refractory tumors based on LYPD3 polypeptide conjugate

[0164] 1. Mouse LYPD3 overexpressing MB49 bladder cancer cell lines were cultured in DMEM medium containing 10% fetal bovine serum, and 5x10 5 were inoculated subcutaneously in C57BL / 6 mice. Every 3-4 days, the health status of the mice was observed. After tumor formation, when the subcutaneous tumor volume of the mice reached 50-100mm 3 Vehicle group (normal saline group), Peptide group (polypeptide 1), GC+PD-1 group (G: gemcitabine (Sellck, S1714), C: cisplatin (Selleck, S1166), PD-1: Anti-mouse PD-1 (CD279)-InVivo (Selleck, A2122), LPDC-1 group, LPDC-1+PD-1 group (PD-1: Anti-mouse PD-1 (CD279)-InVivo (Selleck, A2122)) were randomly divided.

[0165] 2. LPDC-1 and Peptide were resuspended in normal saline to a concentration of 0.1 ug / ul, and the volume of LPDC-1 and Peptide was calculated at 0.5 ug / g, the volume of PD-1 was calculated at 10 ug / g, the volume of gemcitabine (G) was calculated at 50 ug / g, and the volume of cisplatin (C) was calculated at 2 ug / g. Intraperitoneal injection was used for administration, with LPDC-1 and Peptide once a day, PD-1 once every 3 days, gemcitabine and cisplatin administered sequentially, twice a week. The control group was perfused with the same volume of normal saline. The change in tumor size of the mice was observed and counted every 3-4 days by volume measurement, and the results are shown in Figure 8 As shown, the change in tumor size in the Vehicle group, Peptide group and GC+PD-1 group was not obvious, and the tumor size in the LPDC-1 group was significantly reduced compared with the normal saline group, Peptide group and GC+PD-1 group. Non-obviously, the therapeutic effect of LPDC-1 is better than that of the combination of chemotherapy + immune target inhibitor (GC+PD-1).

[0166] In addition, the LPDC-1+PD-1 group is further reduced compared with the LPDC-1 group. Therefore, the polypeptide of the present application and the conjugate PDC based on the polypeptide can be used for the treatment of refractory tumors. Compared with the single immune checkpoint treatment, the LYPD3-targeting polypeptide conjugate PDC of the present application, or the combination of the immune checkpoint treatment, significantly enhances the killing effect on LYPD3-high-expressing tumor load mice, while having no toxic side effects, good safety, low cost and other advantages.

[0167] Although the present application is described with reference to the current preferred embodiments, it should be understood by those skilled in the art that the above preferred embodiments are only used to illustrate the present application, and are not used to limit the protection scope of the present application, and any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A LYPD3 specific binding polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown as SEQ ID NO. 4-6.

2. The polypeptide according to claim 1, the corresponding nucleotide sequence of which is shown as SEQ ID NO. 1-3.

3. A LYPD3 specific polypeptide conjugate, characterized in that, having a structure shown as formula (1): wherein: P is an LYPD3 specific binding polypeptide, the amino acid sequence of which is shown as any one of SEQ ID NO. 4-6; D is an active substance, which is a substance capable of playing a diagnostic and therapeutic role on diseases; L is a linker capable of connecting the LYPD3 specific binding polypeptide and the active substance; n is an integer from 1 to 100; preferably, the n is an integer from 1 to 8.

4. The polypeptide conjugate of claim 3, wherein, The active substance is selected from an anti-tumor drug or a tracer.

5. The polypeptide conjugate of claim 4, wherein, The anti-tumor drug is selected from one or more of a tubulin inhibitor, a topoisomerase inhibitor, an alkylating agent, an anti-tumor antibiotic, a metal platinum drug, an anti-metabolite, an anti-tumor plant drug, a tyrosine kinase receptor, a nuclide, and an NF-KB inhibitor; Preferably, the tubulin inhibitor is selected from one or more of monomethyl auristatin, vincristine, vinorelbine and docetaxel; the topoisomerase inhibitor is selected from one or more of topotecan, irinotecan and etoposide; the alkylating agent is selected from one or more of carmustine, mechlorethamine hydrochloride, cyclophosphamide and milphalan; the antitumor antibiotic is selected from one or more of mitomycin, epirubicin, doxorubicin, bleomycin and doxorubicin; the antitumor hormone is selected from one or more of tamoxifen, droloxifene, letrozole, aminoglutethimide, leuprolide and goserelin; the metal platinum is selected from one or more of cisplatin, carboplatin and oxaliplatin; the antimetabolite is selected from one or more of 5-fluorouracil, methotrexate, gemcitabine and cytarabine; the antitumor plant drug is selected from one or more of camptothecins, vinblastines, paclitaxel and docetaxel; the tyrosine kinase receptor is selected from one or more of lapatinib, imatinib, gefitinib and erlotinib; the radionuclide is selected from 177 Lu, 131 I or 90 Y; the NF-KB inhibitor is selected from bortezomib.

6. The polypeptide conjugate of claim 4, wherein, The tracer is selected from one or more of a PET tracer, a CT tracer, and an MRI tracer; Preferably, the PET tracer is selected from one or more of 18 F, 68 Ga, 64 Cu, 99 mTc and 89 Zr. Preferably, the CT tracer is selected from one or more of an iodine nano-contrast agent, a gold nano-contrast agent, a tantalum oxide nano-contrast agent, a bismuth nano-contrast agent, and a lanthanide nano-contrast agent; More preferably, the CT tracer is selected from one or more of iohexol, ioversol, iodixanol, iopromide, iobitridol, iomeprol, iopamidol, acetrizoate, metrizamide, diatrizoic acid, sodium itothalamate, itothalamate, metrizamide, metrizoic acid, and meglucamine. Preferably, the MRI tracer is selected from a longitudinal relaxation contrast agent and / or a transverse relaxation contrast agent; More preferably, the MRI tracer is selected from a paramagnetic contrast agent, a ferromagnetic contrast agent, or a superparamagnetic contrast agent; More preferably, the MRI tracer is selected from one or more of Gd-DTPA and its linear, cyclic polyamine polycarboxyl chelates and manganese porphyrin chelates, and macromolecules.

7. The polypeptide conjugate of claim 3, wherein, The linker is selected from a cleavable linker, a non-cleavable linker, or a combination thereof; Preferably, the cleavable linker L is selected from one or more of an acid-cleavable linker, a reduction condition-cleavable linker, an enzyme-cleavable linker, a cathepsin B-cleavable linker, and a GSH-cleavable reduction linker; Preferably, the cleavable linker comprises one or more of a hydrazone bond, a Val-Cit dipeptide, and a disulfide bond; Preferably, the non-cleavable linker comprises a poly-sarcosine, a thioether bond, and / or an amide bond; More preferably, the linker L is selected from a combination of a cleavable linker and a non-cleavable linker; More preferably, the linker L is selected from a 10-unit poly-sarcosine and a valine- citrulline dipeptide.

8. The polypeptide conjugate of claim 3, wherein, The structure is shown as LPDC-1 or LPDC-2:

9. A composition characterized in that, The pharmaceutical composition comprises: (1) the polypeptide according to any one of claims 1-2, or the polypeptide conjugate according to any one of claims 3-8; (2) a pharmaceutically acceptable carrier or excipient. Preferably, the pharmaceutically acceptable excipient is selected from one or more of the following: diluents, binders, lubricants, wetting agents, antioxidants, buffers, bacteriostatic agents, suspending agents, solubilizers, thickening agents, stabilizers, preservatives.

10. The polypeptide of any one of claims 1-2, or the polypeptide conjugate of any one of claims 3-8, or the composition of claim 9 for use in the manufacture of a medicament for the diagnosis and / or treatment of a tumor; Preferably, the tumor is selected from a tumor that positively expresses LYPD3; More preferably, the tumor is selected from bladder cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, cervical squamous cell carcinoma and cervical adenocarcinoma, cutaneous melanoma, breast cancer, prostate cancer, hepatocellular carcinoma, pancreatic cancer, renal cancer, ovarian cancer, endometrial cancer or thyroid cancer; More preferably, the tumor is a refractory tumor; More preferably, the tumor is refractory bladder cancer.

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