B7h3 affibody and method for preparing diagnostic and therapeutic nuclide marker thereof, and use thereof

Abstract

The present invention belongs to the technical fields of organic chemistry, radiochemistry, tumor diagnostics and clinical nuclear medicine. Provided are a B7H3 affibody and a method for preparing a diagnostic and therapeutic nuclide marker thereof, and the use thereof. The B7H3 affibody has the sequence of: Ac-PEG4-AEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQAPK[(Acp)-(Acp)]. The provided B7H3 affibody has good in-vivo stability, and metabolic and pharmacokinetic properties, and a nuclear medicine molecular probe synthesized from same and a diagnostic and therapeutic radionuclide marker has both good affinity and functional activity for B7H3 targets, and is expected to become a radioactive drug for B7H3-targeted imaging and tumor treatment with good application prospects.

Description

Preparation method and application of B7H3 affibody and its diagnostic and therapeutic radionuclide marker Technical Field

[0001] The present invention belongs to the technical fields of organic chemistry, radiochemistry, tumor diagnosis and clinical nuclear medicine, and specifically relates to a preparation method and application of a B7H3 affibody and a diagnostic and therapeutic radionuclide marker thereof. Background Art

[0002] CD276, also known as B7-H3, is a member of the B7 family of transmembrane glycoproteins and a T cell regulator that is highly specifically overexpressed in different subtypes of human malignant tumors compared to normal tissues and benign lesions. Furthermore, B7-H3, as a cell surface receptor protein, is highly correlated with tumor drug resistance, metastasis, and immune regulation. Furthermore, B7-H3 is overexpressed on endothelial cells in the tumor vasculature but not on angiogenic vessels in normal tissue. These properties make B7-H3 an ideal candidate for the development of targeted therapeutics that would simultaneously ablate tumor cells and the tumor vasculature with high specificity. According to a recent study, a B7-H3-targeting antibody-drug conjugate shows great potential for treating a variety of tumor types.

[0003] Research on anti-B7-H3 antibody-drug conjugates for the diagnosis and treatment of malignant tumors has made progress in recent years. However, antibody-based ligands may have many problems for clinical translation due to inefficient and random binding, high cost, and potential immune responses, especially in the case of repeated administration. Current studies have shown that smaller protein fragments can achieve efficient and site-specific binding, gradually replacing antibodies, especially in tumor diagnosis research. Recently, affibodies have been shown to be promising binding ligands for the design of molecular imaging. ABY is a 58-amino acid protein (approximately 7 kDa). Compared with antibodies, ABY exhibits faster liver and kidney clearance, higher biocompatibility and stronger in vitro and in vivo stability, is more suitable for large-scale production, and can achieve site-specific binding.

[0004] With the rapid development of nuclear medicine molecular imaging, nuclear medicine has gradually moved from non-specific diagnosis to disease-specific diagnosis and treatment based on small molecule drugs, peptides, monoclonal antibodies and other prodrugs. Among them, positron emission tomography (PET) is the most widely used and has shown its advantages. These new probes provide a basis for early diagnosis, clinical staging, and efficacy evaluation of tumors, and can also be used for prognosis evaluation. They can also be used in targeted tumor treatment. After being modified with bifunctional coupling agents such as DOTA and H3RESCA-TFP, B7H3 affimers can be used for diagnostic radionuclides. 68Ga, 18 F. 99m Tc and other labels, as well as therapeutic radionuclides 90 Y. 177 Lu, 225 Ac and 213 Bi and other markers, which can be used for tumor-specific radionuclide diagnosis and treatment. These radionuclides each have their own advantages and unique application scenarios, among which 68 The cost of obtaining Ga is relatively low, the labeling method is simple and efficient, the half-life is relatively short, and it can reduce radiation to a certain extent. 18 F has a long half-life (T1 / 2 = 109 min) and is suitable for some probes with slow metabolism in the body. 18 F can be obtained by medical cyclotrons with high yield and is currently the most widely used PET imaging nuclide. 99m Tc is the most widely used radionuclide in SPECT imaging. It has an ideal energy peak for single-photon imaging, and has advantages in half-life and acquisition methods. SPECT imaging has a greater cost control advantage than PET imaging. 177 Lu, alpha therapeutic nuclide 225 Ac has broad application prospects. After the B7H3 probe is labeled with therapeutic radionuclides, it can be enriched in large quantities at the target site, has a strong ionizing radiation effect, and emits rays to destroy cell DNA, achieving a targeted therapeutic effect.

[0005] Therefore, by constructing B7H3-targeted affinity body diagnostic and therapeutic radioactive molecular probes for B7H3 target detection, drug feasibility analysis and specific radionuclide therapy, a non-invasive and effective molecular imaging method is provided for the screening of patients with high-expressing B7H3-positive tumors and the monitoring of B7H3 expression; B7H3-targeted affinity body radionuclide therapy probes provide a new treatment method for patients with B7H3-positive malignant tumors who become resistant to targeted therapy. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method and application of a B7H3 affibody and its diagnostic and therapeutic radionuclide label.

[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides a B7H3 affibody, wherein the sequence of the B7H3 affibody is (N-terminus-C-terminus): Ac-PEG4-AEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQAPK[(Acp)-(Acp)];

[0008] Among them, PEG4 is aminotetraethylene glycol carboxyethyl (1-amino-3, 6, 9, 12-tetraoxapentadecan-15-oicacid), which is connected to the N-terminus; Acp is 6-aminohexanoic acid, which is connected to the C-terminus of K.

[0009] The structure of the B7H3 Affibody is shown in Formula I:

[0010] The structure of the B7H3 (CD276) affibody of the present invention is designed based on the structure of a simulated anti-B7H3 monoclonal antibody fragment. The N-terminus of the structure is directly modified with a bifunctional coupling agent, or a PEG4 structure is added before modification with a bifunctional coupling agent, or two Acp structures are added to the C-terminus, thereby improving the biocompatibility and stability of the affibody in the human body, increasing the in vivo circulation time, and optimizing the metabolic rate and pathway.

[0011] The B7H3 (CD276) affibody and modified B7H3 (CD276) affibody of the present invention can be prepared by various polypeptide synthesis / modification methods in the art. For example, the amino acid sequence is first synthesized and then reacted with a bifunctional coupling agent (DOTA, H3RESCA-TFP). The method for modifying the B7H3 (CD276) affibody with a bifunctional coupling agent can adopt various conventional methods in the art. These methods are well known to those skilled in the art and are not particularly limited in the present invention. For example, in a 0.1M NaHCO3 buffer system with a pH of 8.5-9.0, B7H3 (CD276) and DOTA are mixed at a molar ratio of (5-10): 1 and reacted at room temperature for 30 minutes to 2 hours. The synthesis steps are shown in Figure 1.

[0012] In a second aspect, the present invention provides a modified B7H3 affibody, which is the B7H3 affibody modified with a bifunctional coupling agent, wherein the bifunctional coupling agent includes but is not limited to DOTA, H3RESCA-TFP, NOTA, HBED-CC, DTPA, and 3pC-NETA-NCS.

[0013] The structure of the H3RESCA-TFP modified B7H3 affibody is shown in Formula II:

[0014] Preferably, the bifunctional coupling agent is modified at the N-terminus of the B7H3 (CD276) affinity body.

[0015] In a third aspect, the present invention provides a radioactive diagnostic and therapeutic nuclide label, which is the B7H3 affibody or the modified B7H3 affibody labeled with a radionuclide.

[0016] According to one embodiment of the present invention, the radionuclide may be a diagnostic radionuclide, and the diagnostic radionuclide is preferably 68 Ga, 18 F. 99m Tc.

[0017] When the radionuclide is 68 When Ga is used, the preparation method of the radionuclide-labeled B7H3 (CD276) affibody comprises the following steps:

[0018] 1) Prepared by using a columnar germanium gallium generator 68 Ga nuclide;

[0019] 2) Use 1-4 mL of 0.04-0.06 M HCl solution to elute the germanium gallium generator and mix with 0.8-1.2 M NaAc to 68 Ga in the eluent;

[0020] 3) Add 20-100 μg of the modified B7H3 affibody to the 68 Add 1-4 mL of Ga solution, mix well, and heat to 37-40°C or 85-100°C for 10-20 minutes;

[0021] 4) Purify the product obtained in step 3) and elute the product with anhydrous ethanol to obtain 68 Ga-labeled B7H3 (CD276) Affibody.

[0022] Specifically, taking the modification of the bifunctional chelator DOTA as an example, 68 The following methods can be used to label the modified B7H3 Affibody with Ga:

[0023] After the B7H3 affibody sequence was modified with the bifunctional coupling agent DOTA, the sequence (DOTA)-PEG4-AEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQAPK[(Acp)-(Acp)](DOTA-B7H3 affibody) was obtained; the prepared DOTA-B7H3 affibody was subjected to 68 Ga(T 1 / 2 =68min;β + : 89%; E = 511keV) nuclide labeling, 68 Ga use 68 Ge- 68 Prepare the Ga generator. Take 3mL of 0.05M HCl solution to elute 68Ga to 195 μL 1M NaAc; 0.1 mL (60 μg) of DOTA-B7H3 affinity body precursor was added to the above system, mixed, reacted at 95 ° C for 10 min, and radioactive impurities were eluted with 3 mL of normal saline, and then the target compound was eluted with 0.8 mL of 80% ethanol. 68 The labeling rate and radiochemical purity of Ga-B7H3 affinity were determined by radio-HPLC or radio-TLC. 68 The radiochemical purity of the Ga-B7H3 affimer is greater than 95%. If the labeling efficiency is less than 90%, separate and purify using a Sep-pak C18 column. The Sep-pak column should be activated with 5 mL of anhydrous ethanol and 5 mL of high-purity water before use. An appropriate amount of the sterile-filtered product preparation should undergo quality control testing. Further studies should proceed only after all parameters pass.

[0024] When the radionuclide is 18 When F, the preparation method of the radionuclide-labeled B7H3 affibody comprises the following steps:

[0025] 1) Prepared using a medical cyclotron 18 F solution;

[0026] 2) The cyclotron produced 18 F's H2 18 O was adsorbed by a QMA ion exchange column; 0.45-0.55 mL of normal saline was used to rinse the QMA column and elute 18 F to saline to obtain 18 Solution of F;

[0027] 3) 50-150 μL of the 18F-containing solution obtained in step 2) was mixed with 10-15 μL of KHP (potassium hydrogen phthalate, 0.5 M) and 5-10 μL of AlCl 3 solution (2 mM), shaken and allowed to stand at room temperature for 4-10 minutes, and then 20-100 μg of the modified B7H3 affibody was added. The reaction was carried out at 37-40°C or 100-120°C for 10-20 minutes. After the reaction solution was cooled, the product was loaded onto a C18 separation column, washed with saline, and eluted with ethanol to obtain the product. 18 F-labeled B7H3 (CD276) Affibody.

[0028] Specifically, taking the modification of the bifunctional chelator H3RESCA as an example, 18 The following methods can be used to label the modified B7H3 affinity body:

[0029] The sequence of the B7H3 affibody modified with the bifunctional coupling agent H3RESCA is (H3RESCA)-PEG4-AEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQAPK[(Acp)-(Acp)]; the prepared H3RESCA-B7H3 affibody was subjected to 18 F(T 1 / 2 =109.8min;β + : 96.7%; E = 511keV) nuclide labeling, 18 F is prepared using a cyclotron. 18 F - H2 18 O passes through a QMA ion exchange column, 18 F - was adsorbed onto the QMA column, and the QMA column was washed with 0.5 mL of normal saline; 0.1 mL of 18 F - Place the saline in a reaction tube containing 11 μL of 10-fold KHP and 6 μL of 2mM AlCl3 solution, mix well and place at room temperature for 5 minutes; add 10 μL of 10 mg / mL labeled precursor H3RESCA-B7H3 affinity body and react at 37°C for 15 minutes; after the reaction solution is cooled to room temperature, the product is purified using a C18 separation column, washed with 5 mL of saline, and then eluted with 0.6 mL of 80% ethanol and passed through a 0.22 μm sterile filter membrane; blow dry the solvent with N2 and dilute with saline to obtain the product preparation; use radio-HPLC or radio-TLC to determine the labeling rate and radiochemical purity. 18 After isolation and purification, the radiochemical purity of the F-B7H3 affibody was greater than 95%. If the labeling efficiency was less than 90%, the affibody was isolated and purified using a Sep-pak C18 column. The Sep-pak column was activated with 5 mL of anhydrous ethanol and 5 mL of high-purity water before use. An appropriate amount of the sterile-filtered product preparation was subjected to quality control testing. Further studies were performed only after all parameters passed the test.

[0030] When the radionuclide is 99m When Tc is used, the preparation method of the radionuclide-labeled B7H3 affibody comprises the following steps:

[0031] 1) Prepared by Molybdenum-Technetium Generator 99m TcO4 solution;

[0032] 2) Add 20-100 μg of the modified B7H3 affibody to 0.5-1 mL of 0.2 M Na2HPO4 solution and mix thoroughly;

[0033] 3) Take 0.01-0.02 mL of 1 mg / mL stannous chloride and add it to the solution obtained in step 2), then add 99m Mix the TcO4 solution and heat to 90-100℃ for 10-20 minutes;

[0034] 4) Purify the product obtained in step 3) and elute the product with 80% ethanol to obtain 99m Tc-labeled B7H3 affibody.

[0035] According to the present invention, the radionuclide can also be a therapeutic radionuclide, in order to achieve the purpose of B7H3 high expression tumor targeted molecular imaging therapy. The therapeutic radionuclide is preferably 90 Y. 177 Lu, 225 Ac and 213 At least one of Bi.

[0036] B7H3 affimers modified with bifunctional coupling agent DOTA, bifunctional coupling agent DTPA, bimodal bifunctional ligand 3pC-NETA-NCS, and bimodal bifunctional ligand H3RESCA-TFP can be used for therapeutic radionuclides 90 Y, 177 Lu, 225 Ac or 213 Bi mark, get 90 Y-B7H3 affibody, 177 Lu-B7H3 affibody, 225 Ac-B7H3 affibody, 213 Bi-B7H3 affibody molecular probe for therapeutics.

[0037] The labeling of therapeutic radionuclides can be carried out by various conventional methods in the art. According to a preferred embodiment of the present invention, 90 Y, 177 Lu, 225 Ac or 213 Bi-labeled B7H3 Affibody can be prepared using the following method:

[0038] When the radionuclide is 177 When Lu, the preparation method comprises the following steps:

[0039] 1) Purchase 177 Lu nuclide bottle, containing about 50-150mCi nuclide;

[0040] 2) Add 0.39 mL of 1.0 M sodium acetate to 6 mL of 0.05 M HCl. Measure the pH to approximately 4.0 using pH paper. This serves as the labeling buffer.

[0041] 3) Prepare 0.1 M NaHCO3 as a neutralization buffer to adjust the pH of the labeled total product sample to 7.4;

[0042] 4) Add 0.5 mL of labeling buffer to the reaction flask and add 20-100 μg of the modified B7H3 Affibody;

[0043] 5) To 177 Add 0.5 mL of labeling buffer to the Lu nuclide bottle to obtain 177 Lu buffer solution, then take 0.6mL 177 Add the Lu buffer solution to the reaction flask in step 4), mix well, and heat at 85-100°C for 10-20 minutes;

[0044] 6) Purify the product obtained in step 5) and elute the product with anhydrous ethanol to obtain 177 Lu-labeled B7H3 Affibody.

[0045] Further, 177 Taking Lu as an example, the B7H3 affibody modified with bifunctional chelating agents DOTA, DTPA, 3pC-NETA-NCS, and H3RESCA-TFP was used to obtain the corresponding labeled precursors. The labeling buffer was prepared according to 3mL 0.05M HCl corresponding to 195μL 1M NaAc and set aside; 100μL (60μg) of labeled precursors (DOTA-B7H3 affibody, DTPA-B7H3 affibody, 3pC-NETA-B7H3 affibody, H3RESCA-B7H3 affibody) was added with 100-150μL of labeling buffer and then added 177 Lu; adjust the pH to 5.5, react at 95°C for 10-15 minutes, and when the labeling rate is less than 90%, separate and purify with Sep-pak C18 column to obtain 177 Lu-B7H3 affinity body. The labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC. 177 The radiochemical purity of Lu-B7H3 affibody after separation and purification was greater than 95%.

[0046] In a fourth aspect, the present invention provides the use of a B7H3 affibody labeled with a diagnostic radionuclide in the preparation of a B7H3-targeted tumor PET or SPECT imaging agent.

[0047] In a fifth aspect, the present invention provides the use of a B7H3 affibody labeled with a therapeutic radionuclide in the preparation of a B7H3-targeted tumor radionuclide therapeutic drug.

[0048] In a sixth aspect, the present invention provides a modified affinity body structure targeting B7H3, and performs labeling and evaluation of diagnostic and therapeutic radionuclides.

[0049] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0050] (i) The B7H3 affinity body structure has good in vivo stability, pharmacokinetic properties, in vivo metabolism, affinity and specificity.

[0051] (2) Diagnostic radioactive molecular probes can be used 68 Ga / 18 F / 99m PET or SPECT imaging of Tc-B7H3 affimers; 68 Ga / 18 F / 99m Tc-B7H3 affibody can be used for screening, treatment prediction and efficacy monitoring of patients with B7H3-positive hepatocellular carcinoma, breast cancer, gastric cancer and other tumors.

[0052] (three) 68 Ga / 18 F / 99m Tc-B7H3 affimer PET imaging can determine the patient's response to B7H3 targeted therapy and whether the patient is suitable for therapeutic radionuclide therapy. 90 Y / 177 Lu / 225 Ac / 213 Bi replacement 68 Ga / 18 F / 99m Tc for radionuclide targeted therapy.

[0053] (Four) 90 Y / 177 Lu / 225 Ac / 213 Radionuclide targeted therapy such as Bi-B7H3 affibodies can provide new treatment options for patients with drug-resistant B7H3 tumors.

[0054] (5) of the present invention 68 Ga / 18 F / 99m The preparation method of Tc-B7H3 affibody has high labeling rate. 68 The Ga labeling rate can reach more than 80%, 18 The F labeling rate can reach more than 70%, 99m The Tc labeling rate can reach over 85%. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1a and Figure 1b are respectively 68Ga-RESCA-B7H3 affibody and control experiments 68 Structural formula of Ga-labeled proaffmbody fragment.

[0056] FIG2a and FIG2b are respectively 68 Ga-RESCA-B7H3 affibody and control experiments 68 Mass spectrometry and HPLC characterization of Ga-labeled proaffmbody fragments.

[0057] FIG3a and FIG3b are respectively 68 Ga-RESCA-B7H3 affibody and control experiments 68 In vitro stability analysis of Ga-labeled proaffmabody fragments.

[0058] FIG4a and FIG4b are respectively 68 Ga-RESCA-B7H3 affibody and control experiments 68 Affinity detection of Ga-labeled pro-affiliate fragments.

[0059] FIG5a and FIG5b are respectively 68 Ga-RESCA-B7H3 affibody and control experiments 68 Cellular uptake analysis of Ga-labeled proaffmbody fragments in tumor cells A549, A549 (CD276 transfected), H1975, and H1975 (CD276 transfected).

[0060] FIG6a and FIG6b are respectively 68 Ga-RESCA-B7H3 affibody and control experiments 68 In vivo pharmacokinetic analysis of Ga-labeled proaffmabody fragments.

[0061] FIG. 7a and FIG. 7b are respectively 68 Ga-RESCA-B7H3 affibody and control experiments 68 Distribution of Ga-labeled proaffmbody fragments in normal Kunming mice.

[0062] FIG8a and FIG8b are respectively 68 Ga-RESCA-B7H3 affibody and control experiments 68 Dynamic micro-PET / CT images of Ga-labeled proaffmbody fragments in H1975(CD276) mice.

[0063] FIG9 is a preferred embodiment of the present invention 68Micro-PET / CT images of Ga-RESCA-B7H3 affibody in various tumor models (SW780, LS174T, U87, H3122, H1975). DETAILED DESCRIPTION

[0064] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0065] Example 1

[0066] This example provides the synthesis and characterization of B7H3 (CD276) affibody sequences.

[0067] The sequence of the B7H3 (CD276) Affibody is:

[0068] Ac-PEG4-AEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQAPK[(Acp)-(Acp)], the structure of which is shown in Formula I. After the synthesis of the B7H3 (CD276) affibody, it was verified that the amino acid sequence was correct.

[0069] The molecular structure of the B7H3 (CD276) affibody sequence was characterized by HPLC and mass spectrometry. HPLC analysis conditions: Kromasil 100-5C18 gel filtration / size exclusion chromatography column, flow rate 1 mL / min; mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid (TFA); mobile phase B: water containing 0.1% trifluoroacetic acid (TFA). Mobile phase gradient settings: 30% A and 70% B at 0.0 min; 60% A and 40% B at 20 min; 100% A and 0% B at 20.1 min.

[0070] Example 2

[0071] This example provides the preparation and characterization of a modified B7H3 (CD276) Affibody and a control experiment of a DOTA-modified original Affibody fragment, wherein the sequence of the original Affibody fragment is: AEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQAPK.

[0072] The B7H3 affibody sequence was modified with DOTA or H3RESCA-TFP. The B7H3 affibody was mixed with DOTA (10 mg / mL) or H3RESCA-TFP (5-10 mg / mL) at a molar ratio of 1:10 in a 0.1 M NaHCO3 buffer system at pH 8.8. The reaction was allowed to proceed at room temperature for 1 hour to obtain the modified DOTA-B7H3 affibody or RESCA-B7H3 affibody, the structure of which is shown in Figure 1a. The DOTA-modified original affibody fragment for the control experiment was prepared using the same method as the DOTA-B7H3 affibody preparation, the structure of which is shown in Figure 1b. The molecular structures of the modified RESCA-B7H3 affibody and the DOTA-modified original affibody fragment were characterized by HPLC and mass spectrometry, respectively. The HPLC analysis conditions were the same as in Example 1, and the results are shown in Figures 2a and 2b, respectively.

[0073] It should be noted that the present invention is intended for application in tumor-specific nuclear medicine molecular imaging, and therefore the target molecule (the original affibody fragment) must be modified using a bifunctional coupling agent. This modification will affect the affinity, stability, and in vivo biodistribution of the modified affibody. Furthermore, in studies of the original affibody fragment, the probe's in vivo biological half-life was too short, making it difficult to achieve sufficient intratumoral accumulation. Therefore, it is necessary to further modify the probe to improve the affinity, stability, and in vivo biological half-life of the radiolabeled affibody, and to achieve an in vivo distribution more suitable for nuclear medicine diagnosis and treatment. The results showed that the modified affibody-labeled radionuclide probe had significantly increased affinity, similar or slightly increased in vitro stability, improved in vivo pharmacokinetic parameters (significantly increased biological half-life), reduced renal uptake and accelerated excretion during in vivo distribution, and significantly higher uptake at the tumor site than the unmodified probe in tumor PET imaging.

[0074] Example 3

[0075] This embodiment provides 68 Ga-labeled RESCA-B7H3 affibody and 68 Preparation method of Ga-labeled proaffmbody fragment.

[0076] The prepared RESCA-B7H3 affibody was 68 Ga(T 1 / 2 =68min;β + : 89%; E = 511keV) nuclide labeling, 68 Ga use 68 Ge- 68 Prepare the Ga generator. Take 1mL 0.05M HCl solution to elute 68Ga was added to 65 μL 1M NaAc; 0.1 mL (100 μg) of RESCA-B7H3 affinity body precursor was added to the above system, mixed, reacted at 37 ° C for 15 min, and the reaction solution was cooled to room temperature. The product was loaded onto a C18 separation column, and radioactive impurities were eluted with 4 mL of normal saline. The target compound was washed with 0.5 mL of anhydrous ethanol. 68 The Ga-B7H3 affinity body was eluted and passed through a 0.22 μm sterile filter membrane; the solvent was blown dry with N2 and then the product preparation was prepared with normal saline. The labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC. 68 The labeling rate of Ga-B7H3 affinity body is about 60%, and the radiochemical purity is greater than 95%. 68 The method for Ga labeling the original affibody fragment is basically the same as above, except that the reaction temperature is adjusted to 95°C. The labeling rate is about 50% and the radiochemical purity is about 95%.

[0077] The above experimental results show that 68 The Ga-B7H3 affinity structure has a high labeling efficiency and radiochemical purity.

[0078] Example 4

[0079] This example provides the purified 68 Ga-B7H3 affimer and 68 Comparison of in vitro stability analysis of Ga-labeled affibody fragments.

[0080] Take 10 μL of purified product containing 1.11 MBq (30 μCi) 68 Ga-B7H3 affinity was added to 200 μL of physiological saline (or 5% HSA solution) and incubated at 4°C; 37-74 kBq (1-2 μCi) samples were taken at 0 h, 2 h, 12 h, 24 h, 36 h and 60 h of incubation for radio-TLC analysis; analysis method: 2 μL of the avidin containing 37-74 kBq (1-2 μCi) of radioactivity was taken and the avidin was analyzed. 68 Ga-B7H3 affinity body physiological saline solution or 68 Add 5% HSA solution of Ga-B7H3 affinity to 20μL saturated EDTA and mix well. Perform radio-TLC analysis. Take 2μL sample and drop it 1cm from the bottom of Xinhua No. 1 filter paper. Place it in physiological saline development system. After complete development, remove the filter paper and dry it. Perform radio-TLC detection. 68 Ga and 68 The Rf values ​​of Ga-B7H3 affinity were 9-10cm and 0-1cm respectively; the results showed that in physiological saline solution or 5% HSA solution, 68 Ga-B7H3 affimer and 68The Ga-labeled affibody fragments had good in vitro stability within 4 hours. 68 The in vitro radiochemical purity of Ga-B7H3 affinity body is slightly higher than 68 Ga-labeled affibody fragments, and the specific results are shown in Figures 3a and 3b.

[0081] The above experimental results show that 68 The Ga-B7H3 affinity structure has good stability.

[0082] Example 5

[0083] This example provides the purified 68 Ga-B7H3 affimer and 68 Comparison of targeted affinity analysis of Ga-labeled affibody fragments.

[0084] Dilute B7H3 protein to a working concentration of 1-2 μg / mL in carbonate coating buffer and coat a 96-well microtiter plate with 100 μL per well. Incubate at 4°C overnight (16-24 hours). Remove the plate the next day, discard the liquid, and wash five times with 200-300 μL of PBST (0.01 mol / L, pH 7.4) per well. Discard the wash buffer and pat dry on absorbent paper. Add 200 μL of blocking buffer per well and incubate at 37°C for 1.5-2 hours. Discard the liquid and pat dry on absorbent paper. Wash five times with 200 μL of PBST per well before use. Remove the coated plate and add 100 μL of radiolabeled probe per well at various concentrations (0.001-200 μCi / mL, n=4). Incubate at room temperature for 1 hour, wash 5 times with PBST (200 μL / well), separate each well, place in a counting tube, and count and analyze using a gamma counter. After statistical analysis, the equilibrium dissociation constant (Kd) was obtained. The results are shown in Figures 4a and 4b. 68 The equilibrium dissociation constant Kd of Ga-B7H3 affinity is 4.5nM, which is significantly lower than 68 Ga labeled affibody fragment (Kd = 8.3 nM).

[0085] The above experimental results show that 68 After modification with a bifunctional coupling agent and radionuclide labeling, the Ga-B7H3 affibody structure retains its high affinity for the B7H3 target. Furthermore, modifications at both ends of the affibody yield even higher affinity than the original affibody fragment.

[0086] Example 6

[0087] This example provides the purified 68 Ga-B7H3 affimer and 68 The Ga-labeled affibody fragment was expressed in human tumor cells A549, H1975, and A549 cells transfected with B7H3 protein.CD276 、H1975 CD276 Uptake and uptake inhibition experiments in cells.

[0088] Human lung cancer cells A549, H1975, and A549 grown to the logarithmic phase CD276 and H1975 CD276 2×10 5 Each well was evenly plated in a 24-well plate, 500 μL of PRIM 1640 medium without fetal bovine serum was added to each well, and the cells were incubated in an incubator for 24 hours. 68 Ga-B7H3 affimer or 68 Ga-labeled affinity fragments were evenly added to the well plate (20 μCi per well), incubated in an incubator for a period of time, and the well plate was taken out at 5 min, 30 min, 60 min, and 120 min, and cells (n=5) were lysed with 1M NaOH and collected and placed in a γ-Counter for measurement. Cell competition inhibition experiment: The experimental steps are roughly the same as the cell uptake experiment, except that 68 20 μg / well B7H3 affinity was added to some wells (n=4) 30 min before Ga-B7H3 affinity, and then probe solution (20 μCi per well) was added to each well. The lysed cell solution was collected after 60 min and placed in a γ-Counter for measurement.

[0089] The statistical results of uptake are shown in Figure 5a ( 68 Ga-B7H3 affibody), Figure 5b ( 68 Ga (denoted as the original affibody fragment), 68 The Ga-B7H3 affinity probe showed a high uptake in cells after B7H3 transfection, which was significantly higher than the uptake value of tumor cells before transfection. 68 Ga-B7H3 affinity probe in A549 CD276 、H1975 CD276 The uptake of cells can be inhibited by B7H3 affinity body, while the inhibitory effect on non-transfected cells is not obvious, which proves the specific targeting of the probe to B7H3.

[0090] The above experimental results also show that 68 Ga-B7H3 affinity probe 68 Ga-labeled affibody fragments achieved higher uptake, with 60 min, A549 CD276 Taking the cell uptake value as an example, 68 The uptake value of Ga-B7H3 affinity probe was 3.7±0.2%ID / g, which was significantly higher than 68 The uptake value of Ga-labeled affibody fragment was 2.4±0.2% ID / g.

[0091] Example 7

[0092] This example provides the purified 68 Ga-B7H3 affimer and 68 Pharmacokinetic analysis of Ga-labeled affibody fragments in normal KM mice.

[0093] Prepare 5 KM mice (female, 5-6 weeks old, 18-20g) and 68 Ga-B7H3 affimer or 68 The Ga-labeled affibody fragment was diluted with saline to 18.5 MBq / mL (0.5 mCi / mL). Each mouse was injected with 3.7 MBq (0.1 mCi, 200 μL) of the labeled product via the tail vein. Blood was collected from the periocular venous plexus of the mice using capillary tubes at the corresponding time points after injection of the labeled product (1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 45 min, and 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h, and 36 h). 1% of the injected dose, or 0.037 MBq (1 μCi, 2 μL), was used as the reference activity for the assay. The reference activity was measured along with the collected blood samples using a gamma counter. After attenuation correction, the data were analyzed and the percentage injected dose per gram of blood sample was calculated. The results are expressed as %ID / g ± SD. Specific results are shown in Figures 6a and 6b. 68 The clearance phase half-life of Ga-B7H3 affinity body is 28.34min, and the distribution phase half-life is 1.34min, which is significantly higher than 68 The clearance phase half-life (10.31 min) and the partition phase half-life (0.81 min) of the Ga-labeled affibody fragment were significant.

[0094] The above experimental results show that 68 After modification, the Ga-B7H3 affinity body exhibits better pharmacokinetic properties than the original affinity body fragment and increases the drug circulation time in the body, which will to a certain extent prevent the probe from being quickly cleared and increase the target uptake value.

[0095] Example 8

[0096] This embodiment provides 68 Ga-B7H3 affimer and 68 Comparative analysis of the in vivo distribution of Ga-labeled affibody fragments in normal Kunming mice.

[0097] Thirty normal Kunming mice (female, 5-6 weeks old, 18-20 g) were randomly divided into two experimental groups, and each group was randomly divided into five time groups (n=3). 7.4 MBq (0.2 mCi, 200 μL) of the drug was injected into the tail vein of each group. 68 Ga-B7H3 affimer or68 After Ga-labeling the original affinity body fragments, the mice were anesthetized and killed at 5 min, 30 min, 60 min, 120 min, and 240 min, respectively. The blood, heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, and brain were weighed, and the radioactivity counts in each organ were detected using a γ-counter. After attenuation correction, the in vivo biodistribution of each organ at different time points was statistically analyzed.

[0098] The results are shown in Figure 7a and Figure 7b. 68 Ga-B7H3 affimer and 68 The Ga-labeled affibody fragments are mainly metabolized by the kidneys in mice, and the non-specific uptake of other tissues and organs is extremely low, reflecting excellent drug distribution characteristics in vivo, which matches the distribution of the B7H3 target in vivo. 68 The maximum uptake of Ga-B7H3 affimer in kidney was significantly lower than 68 Ga-labeled affibody fragments (161±13%ID / g vs 721±22%ID / g), while 68 The renal uptake of Ga-B7H3 affinity body decreased rapidly. At 240 min, the renal uptake value dropped to 27±8.4%ID / g, which was significantly lower than that of 68 The renal uptake value of Ga-labeled affibody fragment was (442±110% ID / g).

[0099] The above experimental results show that 68 The modified Ga-B7H3 affibody has better in vivo biodistribution than the original affibody fragment, greatly accelerating the excretion rate of the probe after nonspecific uptake in the kidney. The accelerated renal clearance rate can reduce the radiation dose received by the patient and increase the target-to-species ratio to a certain extent, which will achieve better tumor imaging effects in PET imaging.

[0100] Example 9

[0101] This embodiment provides 68 Ga-B7H3 affimer and 68 Ga-labeled affibody fragments in B7H3-transfected human lung cancer cells H1975 CD276 Dynamic micro-PET / CT imaging in the model.

[0102] Prepare 2 mice for transplantation of human lung cancer cells H1975 CD276 Nude mice (female, 5-6 weeks old, 18-20 g, with tumors 0.8-1.0 cm in diameter) were anesthetized with 3 L / min of isoflurane gas and fixed in the center of the Micro-PET scanner bed in a prone position. 7.4 MBq (0.2 mCi, 200 μL) of cytotoxicity was injected into the tail vein. 68Ga-B7H3 affimer and 68 After Ga-labeling of the affibody fragments, the mice were scanned immediately using dynamic scanning mode with an energy window of 350-700 keV. The dynamic scanning time was 1 hour, and static scanning was extended for 2 hours and 4 hours. Image reconstruction was performed using ordered subsets expectation maximization (OSEM) software.

[0103] The specific results are shown in Figure 8a ( 68 Ga-B7H3 affibody), Figure 8b ( 68 Ga marker (affine fragment): 68 Ga-B7H3 affibody in B7H3-transfected H1975 cells CD276 In the tumor model, 1-hour dynamic imaging showed good in vivo stability and uptake distribution. The molecular probe was mainly metabolized by the kidneys, and the nonspecific uptake of other tissues in the whole body was extremely low. In the 10-minute dynamic imaging, 68 The Ga-B7H3 affinity body showed specific radioactive accumulation in tumor tissue, which gradually increased and persisted over time (more than 2 hours). 68 Ga-labeled affinity body PET imaging distribution and 68 Ga-B7H3 affibody is similar, but its tumor uptake is significantly lower 68 Ga labeled affibody fragment.

[0104] The above experimental results show that 68 The tumor-specific imaging effect of Ga-B7H3 affimer PET imaging is significantly better than that of the original affimer fragment, which is due to 68 The Ga-B7H3 affibody has higher affinity, more suitable pharmacokinetic parameters and better in vivo biodistribution, and can be used as a qualified PET imaging probe for clinical translational research.

[0105] Example 10

[0106] This embodiment provides 68 Ga-B7H3 affibody was used for micro-PET / CT imaging in various tumor models (human bladder cancer model SW780, human colon cancer model LS174T, human brain glioma model U87, human lung cancer models H3122 and H1975).

[0107] Three mice (female, 5-6 weeks old, 18-20 g, with tumor diameter of 0.8-1 cm) were prepared for each tumor model. Each mouse was injected with 7.4 MBq (0.2 mCi, 200 μL) via the tail vein. 68Ga-B7H3 affibody; micro-PET imaging was performed on each animal model in each group 1 and 2 hours after injection. Mice were anesthetized with 3 L / min of isoflurane gas and fixed in the prone position at the center of the micro-PET scanner bed. Static scanning mode was used with an energy window of 350-700 keV and a scan time of 10 minutes. The scan time was appropriately extended as the probe was metabolized and decayed. During the scan, the mice were maintained anesthetized with 1 L / min of isoflurane gas. Image reconstruction was performed using ordered subsets expectation maximization (OSEM) software, and image analysis and processing were performed using MMWKS software after attenuation correction. The specific results are shown in Figure 7. 68 Ga-B7H3 affibody showed different degrees of radioactive uptake in various tumors at 1h and 2h after tail vein injection, which is consistent with the characteristics of broad-spectrum expression of B7H3 in tumor cells. 68 Ga-B7H3 affibodies have potential for multiple tumor-specific diagnostics.

[0108] The above experiments demonstrate that the B7H3 (CD276) affibody sequence designed and synthesized by the present invention has high affinity, good in vivo stability and pharmacokinetic properties. The molecular probes synthesized by the affibody and labeled with diagnostic or therapeutic radionuclides have high specificity and functional activity for the B7H3 (CD276) molecule, and are expected to become radiopharmaceuticals with good application prospects for targeted B7H3 (CD276) imaging and tumor treatment.

[0109] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A B7H3 affibody, characterized in that The sequence of the B7H3 affibody is: Ac-PEG4-AEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQAPK[(Acp)-(Acp)]; Among them, PEG4 is aminotetraethylene glycol carboxyethyl, and Acp is 6-aminohexanoic acid; The structure of the B7H3 Affibody is shown in Formula I:

2. A modified B7H3 affibody, characterized in that It is the B7H3 affibody according to claim 1 modified with a bifunctional coupling agent, wherein the bifunctional coupling agent is DOTA, H3RESCA-TFP, NOTA, HBED-CC, DTPA or 3pC-NETA-NCS; The structure of the H3RESCA-TFP modified B7H3 affibody is shown in Formula II:

3. The modified B7H3 affibody according to claim 2, characterized in that The bifunctional coupling agent is modified at the N-terminus of the B7H3 affibody.

4. A radioactive diagnostic and therapeutic nuclide marker, characterized in that: It is the B7H3 affibody according to claim 1 or the modified B7H3 affibody according to claim 2 or 3 labeled with a radionuclide.

5. The diagnostic and therapeutic radionuclide marker according to claim 4, characterized in that: The radionuclide is a diagnostic radionuclide selected from 68 Ga, 18 F and 99m At least one of Tc.

6. The diagnostic and therapeutic radionuclide marker according to claim 4, characterized in that: The radionuclide is a therapeutic radionuclide selected from 90 Y. 177 Lu, 225 Ac and 213 At least one of Bi.

7. Use of the diagnostic and therapeutic radionuclide marker according to claim 5 in the preparation of a B7H3-targeted tumor PET or SPECT imaging agent.

8. Use of the diagnostic and therapeutic radionuclide marker according to claim 6 in the preparation of a B7H3-targeted tumor radionuclide therapeutic drug.

9. The method for preparing the diagnostic and therapeutic radionuclide marker according to claim 5, characterized in that: When the radionuclide is 68 When Ga, the preparation method comprises the following steps: 1) Prepared by columnar germanium gallium generator 68 Ga nuclide; 2) Use 1-4 mL of 0.04-0.06 M HCl solution to elute the germanium gallium generator and mix with 0.8-1.2 M NaAc to 68 Ga in the eluent; 3) Add 20-100 μg of the modified B7H3 affibody to the 68 Add 1-4 mL of Ga solution, mix well, and heat to 37-40°C or 85-100°C for 10-20 minutes; 4) Purify the product obtained in step 3) and elute the product with anhydrous ethanol to obtain 68 Ga-labeled B7H3 affibody; When the radionuclide is 18 F, the preparation method comprises the following steps: 1) Prepared using a medical cyclotron 18 F solution; 2) The cyclotron produced 18 F's H2 18 O was adsorbed by a QMA ion exchange column; the QMA ion exchange column was rinsed with 0.45-0.55 mL of saline and eluted. 18 F to saline to obtain 18 Solution of F; 3) Take the product obtained in step 2) 18 50-150 μL of F solution was mixed with 10-15 μL of 0.5 M KHP and 5-10 μL of 2 mM AlCl3 solution, shaken and placed at room temperature for 4-10 min, then added with 20-100 μg of the modified B7H3 affibody, reacted at 37-40°C or 100-120°C for 10-20 min, and after the reaction solution was cooled, the product was loaded onto a C18 separation column, washed with physiological saline and eluted with ethanol to obtain 18 F-labeled B7H3 affibody; When the radionuclide is 99m When Tc, the preparation method comprises the following steps: 1) Prepared by Molybdenum-Technetium Generator 99m TcO4 solution; 2) Add 20-100 μg of the modified B7H3 affibody to 0.5-1 mL of 0.2 M Na2HPO4 solution and mix thoroughly; 3) Take 0.01-0.02 mL of 1 mg / mL stannous chloride and add it to the solution obtained in step 2), then add 99m Mix the TcO4 solution and heat to 90-100℃ for 10-20 minutes; 4) Purify the product obtained in step 3) and elute the product with 80% ethanol to obtain 99m Tc-labeled B7H3 affibody.

10. The method for preparing the diagnostic and therapeutic radionuclide marker according to claim 6, characterized in that: When the radionuclide is 177 When Lu, the preparation method comprises the following steps: 1) Purchase 177 Lu nuclide bottle, containing 50-150mCi nuclide; 2) Take 6 mL of 0.05 M HCl and add 0.39 mL of 1.0 M sodium acetate to it as labeling buffer; 3) Prepare 0.1 M NaHCO3 as a neutralization buffer to adjust the pH of the labeled total product sample to 7.4; 4) Add 0.5 mL of labeling buffer to the reaction flask and add 20-100 μg of the modified B7H3 Affibody; 5) To 177 Add 0.5 mL of labeling buffer to the Lu nuclide bottle to obtain 177 Lu buffer solution, then take 0.6mL 177 Add the Lu buffer solution to the reaction flask in step 4), mix well, and heat at 85-100°C for 10-20 minutes; 6) Purify the product obtained in step 5) and elute the product with anhydrous ethanol to obtain 177 Lu-labeled B7H3 Affibody.

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