Polypeptides, radioprobes targeting thomsen-friedenreich antigens and uses

Abstract

The application provides a polypeptide, a radioactive probe and application targeting Thomsen-Friedenreich antigen. The application provides a polypeptide specifically binding to Thomsen-Friedenreich antigen and a radionuclide probe coupled with Ga and containing the polypeptide 68 The polypeptide targeting Thomsen-Friedenreich antigen can specifically target Thomsen-Friedenreich antigen; the radioactive probe targeting Thomsen-Friedenreich antigen can be effectively accumulated and retained at a tumor site, has a higher tumor-to-background uptake ratio, and has a good clinical application prospect in the field of tumor detection.

Description

Technical Field

[0001] This invention relates to the field of nuclear medicine. Specifically, this invention relates to peptides, radioactive probes, and applications targeting the Thomsen-Friedenreich antigen. Background Technology

[0002] The Thomsen-Friedenreich antigen (T antigen, TF antigen, or CD176) was accidentally discovered by O. Thomsen in Copenhagen in 1927. Thomsen and Friedenreich studied the "polyagglutinin" phenomenon in contaminated blood samples and found that bacteria producing neuraminidase could expose the TF antigen on red blood cells. In 1960, E. Klenk and G. Uhlenbruck revealed the disaccharide chemical nature of the TF antigen as a blood group precursor. In 1975, G. Springer discovered that the TF antigen is also a tumor antigen for epithelial cancer. The sequence of the TF antigen is D-Gal-β1,3-GalNAc-Ser / Thr (GalNAc: N-acetyl-D-galactosamine, Gal: D-galactose), a disaccharide D-Gal-β1,3-GaINAc linked to serine or threonine via an O-glycosidic bond. It is the core component of O-type glycan glycoproteins and is widely present in cell membranes. In normal cells, TF antigens are masked by sialic acid and other glycans, but are exposed when tumor cells undergo abnormal glycosylation. Exposed TF antigens are considered a "pan-cancer antigen," expressed in most tumor types, and their mediated adhesion plays a crucial role in tumor metastasis. Furthermore, TF antigens can also adhere to vascular endothelial cells, and studies have shown that TF antigens expressed on the surface of tumor cells may be involved in the intravascular adhesion process. Therefore, TF antigens are an ideal biomarker for highly sensitive and specific cancer lesion localization and targeted therapy.

[0003] Currently reported nuclear medicine diagnostic reagents targeting TF antigens include radionuclides. 124 I-labeled antibody hJAA-F11 and 99m Tc, 111 In and 90 While monoclonal antibodies can be labeled with Y molecules such as 170H.82, their direct clinical application is limited by drawbacks such as large molecular weight, long metabolic time in vivo, high blood background, and poor cell membrane penetration. Peptide drugs, with molecular weights between small molecules and biological macromolecules, possess advantages such as high selectivity, high activity, low concentration, and low toxicity, giving them a natural advantage in clinical translation. 64Cu-NO2A-TFpep is the first radioactive peptide probe targeting the TF antigen, but it has drawbacks such as long sequence, difficulty in synthesis, and high radioactivity retention in the kidneys even 2 hours after injection.

[0004] Therefore, there is a need in the field to develop a peptide probe that targets TF antigens, is easy to design and synthesize, has less non-specific uptake, and can be rapidly cleared from non-target tissues, for the early diagnosis and treatment of tumors. Summary of the Invention

[0005] The purpose of this invention is to provide a class of targeted TF antigen peptides, molecular probes and their applications.

[0006] In a first aspect of the invention, a polypeptide targeting the Thomsen-Friedenreich (TF) antigen is provided, the amino acid sequence of which is shown in RWYAWSPT (SEQ ID NO:1), or has ≥85% sequence similarity to which it has specific binding activity to the Thomsen-Friedenreich antigen.

[0007] In another preferred embodiment, the polypeptide sequence is as shown in RWYAWSPT (SEQ ID NO:1).

[0008] In a second aspect of the invention, a method for preparing a polypeptide targeting the Thomsen-Friedenreich antigen as described in the first aspect of the invention is provided, wherein the polypeptide is prepared by solid-phase synthesis.

[0009] In another preferred embodiment, the preparation method includes the following steps:

[0010] (1) Provide 2-Chlorotrityl Chloride resin as a peptide synthesis carrier;

[0011] (2) Starting from the C-terminus of the polypeptide sequence, the following amino acid synthesis raw materials are sequentially added to the resin: (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-butoxy)butyric acid (Fmoc-Thr(tBu)-OH), N-fluorenmethoxycarbonyl-L-proline (Fmoc-Pro-OH), N-fluorenmethoxycarbonyl-O-tert-butyl-L-serine (Fmoc-L-Ser(tBu)-OH), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(tert-butoxycarbonyl)-1H-indole-3-yl)propionic acid (Fmoc-L-T The following compounds were used to obtain crude peptide resin: rp(Boc)-OH), N-fluorenmethoxycarbonyl-L-alanine (Fmoc-L-Ala-OH), N-fluorenmethoxycarbonyl-O-tert-butyl-L-tyrosine (Fmoc-L-Tyr(tBu)-OH), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(tert-butyloxycarbonyl)-1H-indol-3-yl)propionic acid (Fmoc-L-Trp(Boc)-OH), and N-fluorenmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-L-Arg(Pbf)-OH).

[0012] In this process, for each amino acid synthesis raw material, in the presence of a condensation reagent, the synthesis raw material is reacted with the resin to attach it to the resin, and then Fmoc is removed.

[0013] (3) In the presence of a cleavage reagent, the crude peptide resin obtained in step (2) is deresined to obtain the polypeptide.

[0014] In another preferred embodiment, the molar ratio of each of the synthetic raw materials to the resin is (1-10):1; preferably (4-6):1.

[0015] In another preferred embodiment, the cleavage reagent includes trifluoroacetic acid (TFA) and triisopropylsilane (TIS).

[0016] In another preferred embodiment, step (3) includes:

[0017] The obtained crude peptide resin was replaced with dichloromethane, and trifluoroacetic acid (TFA), triisopropylsilane (TIS) and double-distilled water (H2O) were used as cleavage reagents to obtain crude peptides. The crude peptides were then freeze-dried and purified by HPLC to obtain the polypeptide.

[0018] In another preferred embodiment, in step (2), the Fmoc protecting group is removed using piperidine N,N-dimethylformamide (DMF).

[0019] In another preferred embodiment, in step (2), the condensing agent is N,N-diisopropylethylamine (DIEA) and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU).

[0020] In a third aspect of the invention, a polypeptide conjugate targeting the Thomsen-Friedenreich antigen is provided, said polypeptide conjugate comprising:

[0021] (a) a polypeptide portion, said polypeptide portion being the polypeptide described in the first aspect of the present invention; and

[0022] (b) A coupling portion, wherein the coupling portion is a detectable marker or drug.

[0023] In another preferred embodiment, the coupling portion is selected from: radionuclides, fluorescent labeling units, and nuclear magnetic resonance imaging labeling units.

[0024] In another preferred embodiment, the coupling portion is a radionuclide.

[0025] In another preferred embodiment, the structure of the polypeptide conjugate is shown in formula (I) or (II) below:

[0026] Nu-X-JC04(I)

[0027] Nu-XL-JC04(II);

[0028] in,

[0029] Nu is a radioactive nuclide;

[0030] X represents a radionuclide chelating group;

[0031] L is a linking group;

[0032] JC04 is a polypeptide targeting the TF antigen as described in the first aspect of this invention.

[0033] In another preferred embodiment, the radionuclide chelating group or linking group is covalently linked to the N-terminus, C-terminus, or reactive side chain of the polypeptide targeting the TF antigen.

[0034] In another preferred embodiment, the reactive side chain refers to the reactive group on the amino acid side chain of the targeted TF antigen polypeptide.

[0035] In another preferred embodiment, the reactive group is selected from the group consisting of amino, carboxyl, mercapto, or combinations thereof.

[0036] In another preferred embodiment, the linking group is a sequence composed of lysine, glutamic acid, serine, glycine, and / or its L-type non-natural amino acids.

[0037] In another preferred embodiment, the linking group is a monosaccharide, disaccharide, or trisaccharide group.

[0038] In another preferred embodiment, the linking group is (PEG)m or (CH2)n, where m is an integer from 1 to 10 and n is an integer from 1 to 18.

[0039] In another preferred embodiment, the radionuclide chelating group X is a group formed by a bifunctional chelating agent, wherein the bifunctional chelating agent is DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A / NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3, TCMC, DOTAGA, Macropa, or isonitrile.

[0040] In another preferred embodiment, the radionuclide is a diagnostic radionuclide or a therapeutic radionuclide.

[0041] In another preferred embodiment, the diagnostic radionuclide is selected from the group consisting of: 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I, 124 I, or a combination thereof.

[0042] In another preferred embodiment, the therapeutic radionuclide is selected from the group consisting of: 177 Lu、 125 I, 131 I, 211 At、 111 In、 1 53 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 Pb, 161 Tb, 227Th 223 Ra, or combinations thereof.

[0043] In another preferred embodiment, the structure of the polypeptide conjugate is as shown in formula (I), and Nu is... 68 Ga,X is a nuclide chelate group derived from DOTA.

[0044] In a fourth aspect of the invention, a method for preparing the conjugate as described in the third aspect of the invention is provided, the method comprising the steps of:

[0045] (1) Provide a conjugate precursor comprising a polypeptide and a radionuclide chelating group as described in the first aspect of the present invention;

[0046] (2) Under suitable conditions, the coupling precursor and the coupling part are coupled together to obtain the coupling.

[0047] In another preferred embodiment, in step (1), the coupling precursor is prepared by the following steps:

[0048] (1-1) In the presence of N,N-diisopropylethylamine (DIEA), DOTA-NHS and the polypeptide as described in the first aspect of the present invention are reacted to prepare the conjugate precursor.

[0049] In another preferred embodiment, step (1) further includes purifying the conjugate precursor obtained in step (1-1), for example, by HPLC purification.

[0050] In another preferred embodiment, the coupling portion in step (2) is a radionuclide.

[0051] In another preferred embodiment, the coupling portion in step (2) is a radionuclide. 68 Ga.

[0052] In another preferred embodiment, step (2) includes the following steps:

[0053] (2-3) Under suitable conditions, the coupling precursor is combined with... 68 The product is obtained by reacting GaCl3 to yield a product containing the polypeptide conjugate, and

[0054] (2-2) Optionally, the product obtained from the reaction is purified.

[0055] In another preferred embodiment, the method has a radionuclide labeling efficiency of ≥95%, more preferably ≥99%.

[0056] In a fifth aspect of the invention, a TF protein detection reagent is provided, the detection reagent comprising a conjugate as described in the third aspect of the invention and a detection-acceptable carrier.

[0057] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0058] In another preferred embodiment, the detection reagent is a contrast agent, and the contrast agent further comprises other preparations for contrast imaging.

[0059] In another preferred embodiment, the contrast agent is a contrast agent used for MRI (magnetic resonance imaging) or CT (computed tomography).

[0060] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0061] In another preferred embodiment, the test reagent is in liquid form.

[0062] In a sixth aspect of the invention, a pharmaceutical composition is provided comprising a conjugate as described in the third aspect of the invention and a pharmaceutically acceptable carrier.

[0063] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating tumors, such as cytotoxic drugs.

[0064] In another preferred embodiment, the pharmaceutical composition is used to treat or prevent tumors that express TF protein (i.e., TF protein positive).

[0065] In another preferred embodiment, the tumor is selected from the group consisting of breast cancer, lung cancer, and colon cancer.

[0066] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0067] In a seventh aspect of the invention, a kit for detecting TF antigen is provided, the kit containing a conjugate as described in the third aspect of the invention or a TF protein detection reagent as described in the fifth aspect of the invention.

[0068] In another preferred embodiment, the kit further includes instructions for use in the non-invasive detection of TF protein expression in a test subject.

[0069] In another preferred embodiment, the kit is used for the detection of tumors expressing TF protein (i.e., TF protein positive).

[0070] In an eighth aspect of the invention, the use of the polypeptide as described in the first aspect of the invention, or the conjugate as described in the third aspect of the invention, is provided for the preparation of (a) a detection reagent, detection kit, or detection plate for detecting TF protein; and (b) a pharmaceutical composition for treating or preventing tumors expressing TF protein (TF protein positive).

[0071] In another preferred embodiment, the conjugate is used to prepare a contrast agent for in vivo detection of TF antigen.

[0072] In a ninth aspect of the invention, the use of a polypeptide targeting the Thomsen-Friedenreich antigen as described in the first aspect of the invention is provided in the preparation of a radionuclide reagent.

[0073] In a tenth aspect of the invention, a method for treating a disease is provided, the method comprising administering to a desired subject a conjugate as described in a third aspect of the invention or a pharmaceutical composition as described in a sixth aspect of the invention.

[0074] In another preferred embodiment, the object includes mammals, such as humans.

[0075] In another preferred embodiment, the disease is cancer.

[0076] In another preferred embodiment, the disease is selected from: breast cancer, lung cancer, and colon cancer.

[0077] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0078] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0079] Figure 1 The structure (A), synthetic route (B), and mass spectrometry analysis (C) of the peptide targeting the TF antigen are shown.

[0080] Figure 2 The structure (A) and mass spectrometry analysis (B) of DOTA-JC04, a radioactive probe precursor targeting TF antigen, are shown.

[0081] Figure 3 A radioactive probe targeting the TF antigen is shown. 68 The results of radiochemical purity (A) and stability of Ga-DOTA-JC04 after incubation in PBS (B) and human serum (C) for 30 min, 60 min and 120 min are shown in the figure.

[0082] Figure 4 A radioactive probe targeting the TF antigen is shown. 68 Figure showing the cellular uptake of Ga-DOTA-JC04 in mouse breast cancer cells 4T1.

[0083] Figure 5 A radioactive probe targeting the TF antigen is shown. 68 PET / CT images of Ga-DOTA-JC04 applied to the experimental group and the blocking group. From left to right, the images are the images of the experimental group 30 min, 60 min, and 120 min after probe injection, and the blocking group 120 min after probe injection.

[0084] Figure 6 A radioactive probe targeting the TF antigen is shown. 68 Biodistribution of Ga-DOTA-JC04 in breast cancer 4T1 tumor-bearing mice (highly expressing TF antigen). Detailed Implementation

[0085] Through extensive and in-depth research, the inventors have developed for the first time a polypeptide, a radioactive probe, and applications targeting the Thomsen-Friedenreich antigen. This invention provides a polypeptide targeting the Thomsen-Friedenreich antigen and a polypeptide-radionoid conjugate constructed based on this polypeptide. The polypeptide-radionoid conjugate of this invention exhibits significant uptake at tumor sites, enabling tumor imaging. The conjugate of this invention provides a new method for the detection and treatment of tumors such as breast cancer, lung cancer, and colorectal cancer. Based on this, this invention was completed.

[0086] the term

[0087] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0088] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0089] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0090] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0091] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0092] Peptides targeting TF antigens

[0093] As used herein, the terms "polypeptide of the present invention", "polypeptide targeting Thomsen-Friedenreich (TF) antigen" and "polypeptide JC04" are used interchangeably and all refer to the polypeptide that specifically binds to the TF antigen as described in the first aspect of the present invention.

[0094] The polypeptide of the present invention has the following amino acid sequence from the N-terminus to the C-terminus: NH2-Arg-Trp-Tyr-Ala-Trp-Ser-Pro-Thr-COOH (RWYAWSPT, SEQ ID NO:1);

[0095] This invention provides a method for preparing the targeted TF antigen polypeptide JC04, which includes the following steps:

[0096] (1-1) 2-Chlorotrityl Chloride Resin was selected as the polypeptide synthesis vector;

[0097] (1-2) Starting from the C-terminus of the polypeptide sequence, (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-butoxy)butyric acid (Fmoc-Thr(tBu)-OH), N-fluorenmethoxycarbonyl-L-proline (Fmoc-Pro-OH), and N-fluorenmethoxycarbonyl-O-tert-butyl-L-serine (Fmoc-L-) are sequentially added to the resin. Ser(tBu)-OH), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)propionic acid (Fmoc-L-Trp(Boc)-OH), N-fluorenmethoxycarbonyl-L-alanine (Fmoc-L-Ala-OH), N-fluorenmethoxycarbonyl-O-tert-butyl-L-tyrosine (Fmoc -L-Tyr(tBu)-OH), (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)propionic acid (Fmoc-L-Trp(Boc)-OH), N-fluorenemethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-L-Arg(Pbf)-OH), with each amino acid used in an amount four times the amount of resin material; in a specific embodiment, an N,N-dimethylformamide (DMF) solution containing 20% ​​(v / v) piperidine is used as the 9-fluorenemethoxycarbonyl group removal reagent; and N,N-diisopropylethylamine (DIEA) and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) are used as condensation reagents for polypeptide synthesis.

[0098] (1-3) The crude peptide resin obtained was replaced with dichloromethane, and trifluoroacetic acid (TFA), triisopropylsilane (TIS) and double-distilled water (H2O) were used as cleavage reagents to obtain crude peptides. The peptides were then freeze-dried and purified by HPLC to obtain white powder JCO4.

[0099] peptide conjugates

[0100] As used herein, the terms "peptide conjugate," "peptide conjugate of the present invention," "peptide diagnostic drug," and "peptide therapeutic drug" are used interchangeably and all refer to the peptide conjugate described in the third aspect of the present invention.

[0101] The polypeptide conjugate of the present invention contains:

[0102] (a) a polypeptide portion, said polypeptide portion being the polypeptide described in the first aspect of the present invention; and

[0103] (b) A coupling portion, wherein the coupling portion is a detectable marker or drug.

[0104] In a preferred embodiment, the polypeptide portion of the conjugate has an amino acid sequence as shown in SEQ ID NO:1.

[0105] In one embodiment, the structure of the polypeptide conjugate is shown in formula (I) or (II) below:

[0106] Nu-X-JC04(I)

[0107] Nu-XL-JC04(II);

[0108] in,

[0109] Nu indicates a radionuclide used for diagnosis or treatment.

[0110] X represents a nuclide chelating group.

[0111] L represents a linker group that increases the distance between the targeting peptide and the radionuclide chelating group and regulates the pharmacokinetic properties in vivo.

[0112] JC04 indicates a polypeptide targeting the TF antigen.

[0113] Specifically, X or XL can be covalently linked to the N-terminus, C-terminus, or reactive side chain of the TF-targeting antigen polypeptide JC04. The reactive side chain refers to a reactive group, such as an amino or carboxyl group, on the side chain of the TF-targeting antigen polypeptide.

[0114] In some embodiments, the radionuclide chelating group is selected from DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A / NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3, or isonitriles.

[0115] In some embodiments, the diagnostic radionuclide is 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I and 124 At least one of I. In some embodiments, the therapeutic radionuclide is 177 Lu、 125 I, 131 I,211 At、 111 In、 1 53 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

[0116] Preparation method of peptide conjugates

[0117] This invention also provides a method for preparing the peptide conjugate targeting TF of this invention.

[0118] In some embodiments, the radioactive probe targeting the TF antigen is represented by the general formula Nu-X-JC04, and Nu is derived from... 68 When Ga,X is derived from DOTA, the method includes the following steps:

[0119] (2-1) Dissolve DOTA-NHS in DMSO, then add JCO4 and N,N-diisopropylethylamine (DIEA), and react in the dark;

[0120] (2-2) After the reaction is complete, distilled water is added to dilute the solution, and the solution is loaded into a C18 preparative column. Gradient elution is performed by HPLC. The eluent of the target probe is collected, and the eluent is concentrated and lyophilized. The resulting white powder is DOTA-JC04.

[0121] (2-3) Add 1.5M sodium acetate buffer and 37-74 MBq to the precursor DOTA-JC04. 68 The reaction was carried out with GaCl3 as the eluent at 95°C for 10 min. The product was optionally purified using a Sep-pak C18 column to obtain... 68 Ga-DOTA-JC04; Optimally obtained 68 Ga-DOTA-JC04 has a radiochemical purity greater than 95%.

[0122] Pharmaceutical Compositions and Therapeutic Uses

[0123] In this invention, a pharmaceutical composition containing the polypeptide conjugate of this invention is also provided.

[0124] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the polypeptide conjugate of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the polypeptide conjugate of the present invention can also be used with other therapeutic agents. The aforementioned peptide conjugates can be combined with pharmaceutically acceptable excipients to form drug formulations, thereby achieving more stable therapeutic effects. These formulations ensure the structural integrity of the amino acid core sequence of the TF-targeting peptide conjugates of this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation). The formulations can be in various forms. Generally, liquid formulations can be stably stored for at least one year at 2°C-8°C, and lyophilized formulations remain stable for at least six months at 30°C. The formulations can be commonly used in the pharmaceutical industry, such as suspensions, injections, or lyophilized formulations, with injections or lyophilized formulations being preferred.

[0125] For the pharmaceutical compositions of the present invention (such as aqueous injections or lyophilized formulations), pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonic modifiers, and buffers. Surfactants include nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleic, or octadecyl sarcosine; Pluronics; MONAQUAT™, etc., added in an amount that minimizes the tendency of protein granulation. Solution stabilizers can be sugars, including reducing and non-reducing sugars; amino acids, including monosodium glutamate or histidine; alcohols, including one or a combination of triols, higher sugar alcohols, propylene glycol, and polyethylene glycol. The amount of solution stabilizer added should be such that the final formulation is considered by a person skilled in the art to remain stable for a stable period of time. Isotonic modifiers can be one of sodium chloride and mannitol. Buffers can be one of TRIS, histidine buffer, and phosphate buffer.

[0126] When using the pharmaceutical composition, a safe and effective amount of the polypeptide conjugate of the present invention is administered to mammals, wherein the safe and effective amount is generally at least about 50 micrograms per kilogram of body weight, and in most cases does not exceed about 100 milligrams per kilogram of body weight, preferably between about 100 micrograms per kilogram of body weight and about 50 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise. Typically, the total dosage should not exceed a certain range, for example, an intravenous dose of 10 to 3000 mg / day / 50 kg, preferably 100 to 1000 mg / day / 50 kg.

[0127] The TF-targeting polypeptide conjugates and pharmaceutical preparations containing them of the present invention can be used as anti-tumor drugs for tumor treatment. The anti-tumor drugs referred to in the present invention are drugs that inhibit and / or treat tumors, and may include delaying the development of symptoms associated with tumor growth and / or reducing the severity of these symptoms. It further includes alleviating existing symptoms associated with tumor growth and preventing the occurrence of other symptoms, as well as reducing or preventing metastasis.

[0128] The aforementioned TF-targeting peptide conjugates and their pharmaceutical formulations can also be administered in combination with other antitumor drugs for the treatment of tumors. These antitumor drugs used for combination administration include, but are not limited to: 1. Cytotoxic drugs (1) Drugs that act on the chemical structure of DNA: alkylating agents such as nitrogen mustard, nitrosamines, and methanesulfonates; platinum compounds such as cisplatin, carboplatin, and oxalate platinum; mitomycin C (MMC); (2) Drugs that affect nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta; thymidine synthase inhibitors such as fluorouracil (5F) U, FT-207, capecitabine, etc.; purine nucleoside synthase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG, etc.; nucleotide reductase inhibitors such as hydroxyurea (HU), etc.; DNA polymerase inhibitors such as cytarabine (Ara-C) and Gemz, etc.; (3) drugs that act on nucleic acid transcription: drugs that selectively act on DNA templates and inhibit DNA-dependent RNA polymerase, thereby inhibiting RNA synthesis, such as: actinomycin D, daunorubicin, doxorubicin, epirubicin, aclarubicin, scintillans, etc.; (4) drugs that mainly act on tubulin Synthetic drugs: paclitaxel, texotericin, vinorelbine, podophyllodes, homoharringtonine; (5) Other cytotoxic drugs: asparaginase mainly inhibits protein synthesis; 2. Hormonal anti-estrogens: tamoxifen, droloxifene, exemestane, etc.; aromatase inhibitors: ammoniaglutide, lantron, letrozole, renin, etc.; anti-androgens: flutamethasone RH-LH agonists / antagonists: phenadol, phenadol, etc.; 3. Biological response modifiers: mainly inhibit tumor interferon through the body's immune function; interleukin-2; thymopeptides; 4. Monoclonal antibodies: meropenem MabThera; Cetuximab (C225); Herceptin (Trastuzumab); Bevacizumab (Avastin); Yervoy (Ipilimumab); Nivolumab (OPDIVO); Pembrolizumab (Keytruda); Atezolizumab (Tecentriq); 5. Others, including some drugs with unclear mechanisms and requiring further research; cell differentiation inducers such as retinoids; apoptosis inducers.

[0129] The present invention also provides the use of TF-targeting peptide conjugates in the preparation of diagnostic reagents and therapeutic drugs.

[0130] In specific embodiments, the polypeptide conjugates of the present invention (e.g., fluorescence imaging reagents or radionuclide imaging reagents) can be prepared as tumor diagnostic agents or therapeutic agents, wherein the tumor is a tumor that highly expresses TF, including but not limited to breast cancer, lung cancer, colorectal cancer, etc.

[0131] The main advantages of this invention include:

[0132] (1) The present invention provides a polypeptide that targets TF antigen, which has small molecular weight, low immunogenicity, good tissue penetration ability and high affinity and selectivity for TF antigen, and has good application prospects.

[0133] (2) The radionuclide probe targeting TF antigen provided by this invention has high affinity and specific targeting for TF antigen, high radiochemical purity, high stability, and rapid in vivo metabolic properties. The molecular probe targeting TF antigen provided by this invention shows significant cellular uptake in mouse breast cancer cells 4T1 that highly express TF antigen, and high uptake at the tumor site in 4T1 breast cancer tumor-bearing mice, exhibiting a high target-to-non-target ratio, and has the potential to conduct radionuclide imaging and therapy.

[0134] (3) This invention utilizes radioactive nuclides 68 Ga is easier to obtain and has milder and simpler labeling conditions, which will... 68 Ga-labeled peptides have shown good performance in radiographic imaging.

[0135] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0136] Example 1: Preparation of JCO4

[0137] (1) Resin swelling

[0138] Add a certain amount of 2-ChlorotritylChloride Resin to the reaction flask, then add an appropriate amount of dichloromethane (DCM), and gently purge with nitrogen gas for 30 minutes to allow the resin to swell fully. Drain the dichloromethane solution, then wash three times with N,N-dimethylformamide (DMF) and drain again.

[0139] (2) Removal of Fmoc

[0140] Add a DMF solution containing 20% ​​(v / v) piperidine to the reaction flask, and remove the protective agent every 5 min and every 8 min. After the reaction is complete, wash the resin three times each with DMF, DCM, and DMF respectively.

[0141] (3) Coupling

[0142] Weigh out 4 times the molar amount of Fmoc-Thr(tBu)-OH and O-benzotriazole-tetramethylurea hexafluorophosphonate (HBTU) and dissolve them completely in DMF. After adding N,N-diisopropylethylamine (DIEA) to activate the carboxyl group, add the solution to the reaction flask and react. After reacting for 30 min, wash the solution three times each with DMF, DCM and DMF respectively. Repeat the above steps, sequentially coupling other amino acids (N-fluorenylmethoxycarbonyl-L-proline (Fmoc-Pro-OH), N-fluorenylmethoxycarbonyl-O-tert-butyl-L-serine (Fmoc-L-Ser(tBu)-OH), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(tert-butyloxycarbonyl)-1H-indol-3-yl)propionic acid (Fmoc-L-Trp(Boc)-OH), N-fluorenylmethoxycarbonyl-L-alanine (Fmoc-L-Ala-OH), N-fluorenylmethoxycarbonyl-O- -tert-butyl-L-tyrosine (Fmoc-L-Tyr(tBu)-OH), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(tert-butyloxycarbonyl)-1H-indol-3-yl)propionic acid (Fmoc-L-Trp(Boc)-OH), N-fluorenmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-L-Arg(Pbf)-OH)), until the last amino acid is coupled, then replaced with DCM, and thoroughly dried in a vacuum drying oven.

[0143] (4) Pyrolysis

[0144] Add 10 mL of lysis buffer (95% TFA + 2.5% TIS + 2.5% H2O) to the resin, shake at room temperature for 2 h, then separate the lysis buffer from the resin using a sintered glass funnel, retaining the filtrate. Add ice-cold anhydrous diethyl ether to the filtrate, centrifuge to obtain a solid. Wash three times with ice-cold diethyl ether, freeze-dry the resulting precipitate to obtain a crude powder.

[0145] (5) Purification

[0146] Using acetonitrile as a buffer, the sample was loaded into a C18 preparative column and eluted using HPLC with a gradient. The target eluent was collected. The sample was purified by cyclic injection, concentrated to remove acetonitrile, and finally lyophilized. The resulting white powder was the target peptide. Mass spectrometry analysis confirmed the expected product JC04.

[0147] Example 2: Preparation of the radioactive probe precursor DOTA-JCO4

[0148] (1) Dissolve 0.2 nmol DOTA-NHS in DMSO (200 μL), add 0.6 nmol JCO4 and 0.2 nmol N,N-diisopropylethylamine (DIEA), and react at 37 °C in the dark for 2 h.

[0149] (2) After the reaction was complete, distilled water was added for dilution, and the product was separated and purified by HPLC. The preparative HPLC conditions were as follows: using an Agilent series HPLC system, the sample was loaded onto a C18 preparative column for gradient elution for 30 min at a flow rate of 3 mL / min. The mobile phase A was ultrapure water (containing 0.1% TFA), and the mobile phase B was acetonitrile (containing 0.1% TFA). The elution gradient was set as follows: 95% A + 5% B for 3 min; 5% A + 95% B for 23 min; 5% A + 95% B for 26 min; and 95% A + 5% B for 30 min. The final product was analyzed by HPLC and mass spectrometry, confirming the expected product DOTA-JC04.

[0150] Example 3 68 Preparation of Ga-DOTA-JC04

[0151] Add 132 μL of 1.5 M sodium acetate solution to a glass reaction flask, then add 20 μg of DOTA-JCO4, and then add 1 mL of Ci to the mixture. 68 The GaCl3 eluent (1 mL) was heated in a 95°C water bath for 10 min. After the reaction was complete, the mixture was cooled to room temperature to obtain the radioactive probe. 68 Ga-DOTA-JC04.

[0152] Test Example 1: 68 Ga-DOTA-JC04 Test

[0153] 68 PET / CT imaging and biodistribution of Ga-DOTA-JC04 in breast cancer 4T1 tumor-bearing mice (high expression of TF antigen) in the experimental group and thyroid cancer K1 tumor-bearing mice (low expression of TF antigen) in the blocking group.

[0154] Mice in the experimental and blocking groups were injected via the tail vein with 150 μCi / 200 μL. 68 Ga-DOTA-JC04 was used for PET / CT imaging at 30 min, 60 min, and 120 min post-injection. The imaging results are as follows: Figure 4 As shown, the probe showed significant aggregation in the tumor from 30 minutes onwards, with a clear tumor margin, and remained lodged in the tumor until 120 minutes. There was a significant difference in radiation signal at the tumor site between the experimental group and the blocking group at 120 minutes. This indicates that the probe... 68Ga-DOTA-JC04 binds specifically to tumor sites in vivo.

[0155] The tail vein of mice in the experimental and blocking groups was 100 μCi / 100 μL. 68 After injection of Ga-DOTA-JC04, animals were sacrificed at 30, 60, and 120 minutes, and organs of interest were harvested to measure the radiation intensity of each organ and tissue. The results for the experimental group are as follows: Figure 5 As shown, the probe mainly accumulates in tumors and metabolic organs such as the liver and kidneys, and is rapidly cleared from non-target organs.

[0156] Biodistribution results of the experimental group and the blocking group 120 min after injection are as follows: Figure 6 As shown, there were significant differences in radioactive signals at the tumor sites (P<0.01). Biodistribution experiments further illustrated the probe's effectiveness. 68 Ga-DOTA-JC04 has the potential for in vivo tumor imaging.

[0157] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A polypeptide targeting the Thomsen-Friedenreich (TF) antigen, characterized in that, The amino acid sequence of the polypeptide is shown in RWYAWSPT (SEQ ID NO:1), or has ≥85% sequence similarity to it and has specific binding activity to the Thomsen-Friedenreich antigen.

2. A method for preparing the polypeptide targeting the Thomsen-Friedenreich antigen as described in claim 1, characterized in that, The polypeptide was prepared by solid-phase synthesis.

3. A polypeptide conjugate targeting the Thomsen-Friedenreich antigen, characterized in that, The polypeptide conjugate contains: (a) a polypeptide portion, said polypeptide portion being the polypeptide of claim 1; and (b) A coupling portion, wherein the coupling portion is a detectable marker or drug.

4. The polypeptide conjugate as described in claim 3, characterized in that, The coupling component is selected from: radionuclides, fluorescent labeling units, and nuclear magnetic resonance imaging labeling units.

5. The polypeptide conjugate as described in claim 3, characterized in that, The structure of the polypeptide conjugate is shown in formula (I) or (II) below: Nu-X-JC04(I) Nu-XL-JC04(II); in, Nu is a radioactive nuclide; X represents a radionuclide chelating group; L is a linking group; JC04 is the polypeptide targeting the TF antigen as described in claim 1.

6. The polypeptide conjugate as described in claim 5, characterized in that, The structure of the polypeptide conjugate is shown in formula (I), and Nu is... 68 Ga,X is a nuclide chelate group derived from DOTA.

7. A method for preparing the conjugate as described in claim 3, the method comprising the steps of: (1) Provide a conjugate precursor, said conjugate precursor comprising the polypeptide and nuclide chelating group as described in claim 1; (2) Under suitable conditions, the coupling precursor and the coupling part are coupled together to obtain the coupling.

8. A TF protein detection reagent, said detection reagent comprising the conjugate as described in claim 3 and a detection-acceptable carrier.

9. A pharmaceutical composition comprising the conjugate as described in claim 3 and a pharmaceutically acceptable carrier.

10. A kit for detecting TF antigen, said kit containing the conjugate as described in claim 3 or the TF protein detection reagent as described in claim 8.

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