B7-h3-based dual-modality imaging probes, methods of making and uses thereof

Abstract

The application discloses a B7-H3-based bimodal imaging probe, a preparation method and application. The bimodal imaging probe comprises a B7-H3 affibody, a near-infrared fluorescent dye IR-Dye800CW is connected to the C terminal of the B7-H3 affibody, a Gd-DOTA chelate is connected to the N terminal of the B7-H3 affibody, and the Gd-DOTA chelate chelates gadolinium ions Gd 3+ The near-infrared fluorescent dye IR-Dye800CW is used for near-infrared two-region fluorescence imaging for magnetic resonance imaging. The bimodal imaging probe of the application combines the advantages of magnetic resonance imaging and near-infrared two-region fluorescence imaging, and can be used for both magnetic resonance imaging and near-infrared two-region fluorescence imaging, overcoming the limitations of single detection means.

Description

TECHNICAL FIELD

[0001] The present application generally relates to the technical field of biomedicine. More particularly, the present application relates to a B7-H3-based dual-modality imaging probe, a preparation method and use thereof. BACKGROUND

[0002] Prostate cancer is one of the most common malignant tumors in men worldwide. At present, its diagnosis mainly relies on invasive prostate biopsy, and non-invasive and highly sensitive and accurate detection methods are one of the current research hotspots. Existing non-invasive detection methods such as prostate MRI and PET-CT have certain accuracy, but have limitations. The diagnostic accuracy of prostate MRI is about 80%, and the diagnostic accuracy of PSMA-PET is close to 90%. However, the expression of PSMA molecules decreases during the development of prostate cancer, affecting its diagnostic sensitivity.

[0003] In terms of treatment, radical prostatectomy is the main treatment for locally advanced prostate cancer, but there are problems such as positive surgical margins and complications after surgery, so finding a new method to assist accurate resection during surgery is a key clinical problem. In the past, indocyanine green (ICG) was used for real-time fluorescence imaging to identify the boundaries of prostate cancer lesions and positive lymph nodes during surgery, but ICG lacks tumor specificity, making it difficult to accurately delineate the boundaries and unable to meet actual needs. Near-infrared two-zone fluorescence molecular imaging can visualize tumors in the operating room through targeted fluorescent probes.

[0004] At present, a single imaging method cannot meet the clinical needs. Magnetic resonance imaging has high resolution and deep tissue penetration capability, but the cost is high and the display of fine structures is limited; near-infrared two-zone fluorescence imaging has high sensitivity and high temporal resolution, but the tissue penetration depth is limited. Therefore, developing a technology that combines the advantages of multiple imaging to overcome the limitations of a single detection method is a problem that needs to be solved urgently. In addition, existing fluorescent probes also have deficiencies in specificity, stability and imaging effect, which cannot meet the needs of clinical precise diagnosis and treatment of prostate cancer. SUMMARY

[0005] To solve at least one or more of the above-mentioned technical problems, the present application provides, in various aspects, a B7-H3-based dual-modality imaging probe, a preparation method and use thereof.

[0006] In a first aspect, the present application provides a B7-H3-based dual-modality imaging probe, which comprises a B7-H3 affibody, wherein the C-terminus of the B7-H3 affibody is connected with a near-infrared fluorescent dye IR-Dye800CW, and the N-terminus of the B7-H3 affibody is connected with a Gd-DOTA chelate, and the Gd-DOTA chelate chelates gadolinium ions Gd 3+For magnetic resonance imaging, the near-infrared fluorescent dye IR-Dye800CW is used for near-infrared two-region fluorescence imaging.

[0007] In some embodiments, the structure of the bimodal imaging probe is as follows:

[0008]

[0009] In some embodiments, the molecular weight of the B7-H3 affibody is 8 kDa.

[0010] In some embodiments, the excitation wavelength of the bimodal imaging probe is 782 nm, and the emission wavelength is 807 nm.

[0011] In some embodiments, the T1 relaxation time of the bimodal imaging probe in an aqueous solution gradually shortens as the concentration of the bimodal imaging probe increases.

[0012] In a second aspect, the present application provides a preparation method for preparing the aforementioned bimodal imaging probe, comprising the following steps: dissolving DOTA-NHS and B7-H3 affibody in an organic solvent, adding triethylamine, stirring the reaction under an inert gas atmosphere, to obtain a first intermediate with DOTA connected to the N-terminus of the B7-H3 affibody; dissolving the first intermediate and IRDye800CW-maleimide in an aqueous solution containing an organic solvent, stirring the reaction under an inert gas atmosphere, to obtain a second intermediate with IR-Dye800CW connected to the C-terminus of the B7-H3 affibody; dissolving the second intermediate and GdCl3 in deionized water, and reacting under stirring conditions to obtain a B7-H3-based bimodal imaging probe.

[0013] In some embodiments, the organic solvent is N,N-dimethylformamide; and / or the inert gas comprises nitrogen; and / or the second intermediate and GdCl3 are dissolved in deionized water with a pH of 6.0.

[0014] In some embodiments, the preparation method further comprises: after obtaining the first intermediate, purifying the first intermediate; and / or after obtaining the second intermediate, purifying the second intermediate; and / or purifying the bimodal imaging probe.

[0015] In a third aspect, the present application provides use of the aforementioned bimodal imaging probe in the preparation of a prostate cancer diagnostic reagent.

[0016] In some embodiments, the diagnostic reagent is used for at least one of: preoperative tumor localization by magnetic resonance imaging; and / or intraoperative real-time navigation of prostate cancer tumor boundaries; and / or screening of patients suitable for B7-H3 targeted treatment of prostate cancer.

[0017] By the B7-H3 based bimodal imaging probe provided above, the IR-Dye800CW and Gd-DOTA chelate are connected at the C terminal and N terminal of the B7-H3 affibody respectively, so as to fuse the advantages of magnetic resonance imaging and near-infrared two-region fluorescence imaging, which can be used for both magnetic resonance imaging and near-infrared two-region fluorescence imaging, overcoming the limitations of single detection means, and thus can be applied to preoperative accurate positioning of prostate cancer tumors, improving the diagnostic accuracy of prostate cancer, and intraoperative real-time guidance of surgical resection, assisting in accurate resection of tumors and reducing complications, and can also be applied to screening of patients suitable for B7-H3 targeted treatment. The bimodal imaging probe of the embodiments of the present application has the characteristics of high specificity and stability, as well as good biocompatibility and operability, and has significant clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0018] The above and other objects, features and advantages of the example embodiments of the present application will be readily understood through reading the detailed description of the example embodiments of the present application below, with reference to the accompanying drawings. In the drawings, several embodiments of the present application are shown by way of example and not limitation, and the same or corresponding reference numbers indicate the same or corresponding parts, in which:

[0019] Figure 1 A structural diagram of the bimodal imaging probe of the embodiments of the present application is shown;

[0020] Figure 2 An excitation spectrum diagram of the bimodal imaging probe of the embodiments of the present application is shown;

[0021] Figure 3 An emission spectrum diagram of the bimodal imaging probe of the embodiments of the present application is shown;

[0022] Figure 4 A T1 relaxation time trend diagram of the bimodal imaging probe of the embodiments of the present application is shown;

[0023] Figure 5 A magnetic resonance imaging diagram of a mouse injected with the bimodal imaging probe is shown;

[0024] Figure 6 A near-infrared two-region fluorescence imaging diagram of a mouse injected with the bimodal imaging probe is shown. DETAILED DESCRIPTION

[0025] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0026] It should be understood that the terms "comprising" and "including" used in the specification and claims of the present application indicate the presence of the described features, integers, steps, operations, elements, and / or components, but do not exclude one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0027] It should also be understood that the terms used in the specification of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. As used in the specification and claims of the present application, the singular forms "a", "an" and "the" are intended to include the plural forms, unless the context clearly indicates otherwise. It should be further understood that the term "and / or" used in the specification and claims of the present application means any combination of one or more of the associated listed items and all possible combinations thereof.

[0028] As used in the specification and claims of the present application, the term "if" can be interpreted as "when" or "upon" or "in response to a determination" or "in response to detecting" depending on the context. Similarly, the phrases "if it is determined" or "if [a described condition or event] is detected" can be interpreted to mean "upon determining" or "in response to determining" or "upon detecting [the described condition or event]" or "in response to detecting [the described condition or event]" depending on the context.

[0029] The present applicant finds that B7-H3, as an immunomodulatory glycoprotein specifically highly expressed in prostate cancer, has important clinical value, and antibody conjugate drugs against B7-H3 also show good effects in early clinical trials, so enhanced MR examination carrying B7-H3 molecular markers is expected to significantly improve the detection rate of prostate cancer. At the same time, near-infrared two-region fluorescent molecular imaging can be used for tumor visualization during surgery, and if combined with the special value of B7-H3 in prostate cancer, it will bring opportunities for precise surgery of prostate cancer. Based on this, the present application aims to provide a dual-modality imaging probe that can effectively integrate enhanced MR imaging and near-infrared two-region fluorescent imaging technology with high sensitivity and high temporal resolution, to overcome the limitations of single detection means and meet the clinical needs for precise diagnosis and treatment of prostate cancer. The specific embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0030] In a first aspect, the present application provides a B7-H3-based dual-modality imaging probe, which comprises a B7-H3 affibody, a near-infrared fluorescent dye IR-Dye800CW connected to the C-terminus of the B7-H3 affibody, and a Gd-DOTA chelate connected to the N-terminus of the B7-H3 affibody, wherein the Gd-DOTA chelate chelates a gadolinium ion Gd 3+ For magnetic resonance imaging, the near-infrared fluorescent dye IR-Dye800CW is used for near-infrared two-zone fluorescence imaging.

[0031] B7-H3 is an immunomodulatory glycoprotein highly expressed in prostate cancer, which is closely related to processes such as cancer immune escape and accelerated tumor growth, and is a key target for the diagnosis and treatment of prostate cancer. The B7-H3 affibody is a molecule that can bind to the B7-H3 protein specifically expressed on the surface of prostate cancer cells.

[0032] In some embodiments, the sequence of the B7-H3 affibody is:

[0033] KAEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQGG GC (SEQ ID NO: 1).

[0034] The dual-modality imaging probe prepared from the B7-H3 affibody can be represented as:

[0035] (Gd-DOTA)2-KAEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQGGGC-(IRDye 800CW Mal) (SEQ ID NO: 2).

[0036] The N-terminus and the C-terminus of the B7-H3 affibody are two important parts in its molecular structure. The N-terminus generally refers to the amino-terminal end of a protein or polypeptide chain, while the C-terminus is the carboxyl-terminal end, i.e., the N-terminus has an amino group (-NH2), while the C-terminus has a carboxyl group (-COOH). In the present application, the C-terminus of the B7-H3 affibody is connected to the near-infrared fluorescent dye IR-Dye800CW, while the N-terminus is connected to the Gd-DOTA chelate. This connection is usually achieved by covalently binding IR-Dye800CW and Gd-DOTA chelate to the corresponding terminal points of the B7-H3 affibody, respectively.

[0037] The N-terminus and C-terminus of the B7-H3 affibody have different chemical properties. In comparison, the C-terminus can be involved in the folding, stability and formation of the binding site of the molecule in some cases. If a larger molecule such as a Gd-DOTA chelate is connected at the C-terminus, it can change the three-dimensional structure of the B7-H3 affibody, thereby affecting the binding ability of the B7-H3 affibody to the B7-H3 protein. Therefore, connecting the Gd-DOTA chelate to the N-terminus of the B7-H3 affibody and connecting the C-terminus with a stable fluorescent group IRDye800CW can avoid affecting the binding activity of the B7-H3 affibody to the B7-H3 protein, reduce interference with the structure and function of the B7-H3 affibody, and ensure the effectiveness of fluorescence imaging while ensuring that the bimodal imaging probe can effectively bind to the target protein.

[0038] The Gd-DOTA chelate is a magnetic resonance imaging (MRI) contrast agent. DOTA is a chelating agent that can stably bind to gadolinium ions (Gd 3+ ), which are commonly used paramagnetic contrast agents in clinical MRI. In this application, the Gd-DOTA chelate is connected to the N-terminus of the B7-H3 affibody, and by chelating gadolinium ions Gd 3+ , the bimodal imaging probe has the function of magnetic resonance imaging. In some embodiments, the N-terminus of the B7-H3 affibody can be connected to multiple Gd-DOTA chelates, which is beneficial to improve the effect of magnetic resonance imaging. The spatial structure of multiple Gd-DOTA chelates is large, and in this application it is connected to the N-terminus of the B7-H3 affibody instead of the C-terminus, so as not to affect the binding activity of the B7-H3 affibody to the B7-H3 protein. The two ends of the B7-H3 affibody are connected to different groups respectively, and the groups do not affect each other to affect the imaging function.

[0039] In some embodiments, the structure of the bimodal imaging probe is as follows:

[0040]

[0041] In other embodiments, the structure of the bimodal imaging probe is as follows:

[0042]

[0043] In other embodiments, the molecular weight of the B7-H3 affibody is 8 kDa (kilodaltons). This relatively small molecular weight makes the B7-H3 affibody have good tissue penetration and stability in the body, can quickly distribute and penetrate tissues, is beneficial for imaging in time during surgery, and is also easy to operate during synthesis and purification, which is beneficial for large-scale production and clinical application.

[0044] Figure 1A structural diagram of the bimodal imaging probe of the embodiment of the application is shown. As shown in Figure 1 , the bimodal imaging probe comprises a B7-H3 affimer 101, a near-infrared fluorescent dye IR-Dye800CW 102, and a Gd-DOTA chelate 103. The specific structure of the near-infrared fluorescent dye IR-Dye800CW 102 and the Gd-DOTA chelate 103 can be seen from the partial enlarged structural diagram shown in the dashed box of Figure 1 . Figure 1 As can be seen from , the Gd-DOTA chelate 103 can be bonded and connected to the N-terminus of the B7-H3 affimer 101 through an amide bond, and the near-infrared fluorescent dye IR-Dye800CW 102 can be bonded and connected to the C-terminus of the B7-H3 affimer 101 through a sulfide bond.

[0045] Excitation and emission spectrum test:

[0046] In this test example, the FLS980 spectrometer is used, the bimodal imaging probe solution is placed in the sample cell, then the appropriate excitation wavelength is selected through the monochromator, and the bimodal imaging probe is excited by using a xenon lamp or a laser. The fluorescence emitted by the bimodal imaging probe is collected, and the fluorescence intensity is detected by a photomultiplier tube through another monochromator, and the emission spectrum at different wavelengths is recorded. At the same time, the software automatically draws the excitation spectrum to show the excitation and emission characteristics of the bimodal imaging probe. The test results are shown in Figure 2 and Figure 3 . Figure 2 A excitation spectrum diagram of the bimodal imaging probe of the embodiment of the application is shown. Figure 3 A emission spectrum diagram of the bimodal imaging probe of the embodiment of the application is shown.

[0047] The excitation wavelength refers to the wavelength of the light used to excite the fluorescent dye to emit fluorescence. As shown in Figure 2 , the excitation wavelength of the bimodal imaging probe of the embodiment of the application is 782 nm, which indicates that when the bimodal imaging probe is irradiated with light of 782 nm, the IR-Dye800CW molecule in the bimodal imaging probe will absorb light energy of this wavelength, thereby entering an excited state. The excitation light of 782 nm can effectively excite IR-Dye800CW to emit bright fluorescence, providing a good signal basis for subsequent fluorescence imaging.

[0048] The emission wavelength refers to the wavelength of the fluorescence emitted by the fluorescent dye after being excited. As shown in Figure 3As shown in the middle, the emission wavelength of the bimodal imaging probe of the embodiments of the present application is 807 nm, which indicates that the IR-Dye800CW in the bimodal imaging probe will emit fluorescence with a wavelength of 807 nm under excitation by 782 nm light. The bimodal imaging probe also has a high fluorescence signal in the near-infrared second band (900-1200 nm), and the fluorescence in this band has a large penetration depth in biological tissues, which can achieve high-resolution imaging of deep tissues. In addition, the fluorescence signal at 807 nm has low scattering and absorption in tissues, which can provide clearer imaging and help accurately identify tumor boundaries and positive lymph nodes in real-time intraoperative navigation, providing clear visual guidance for doctors and improving the accuracy and success rate of surgery.

[0049] In the application scenario of surgery, by using 782 nm excitation light irradiation, the fluorescence signal in the near-infrared second band 900-1200 nm can be detected in real time, providing real-time information on the location of tumor boundaries and positive lymph nodes for doctors, helping doctors to more accurately resect tumor tissue while preserving normal tissue as much as possible, reducing surgical complications, and improving the postoperative quality of life of patients.

[0050] T1 relaxation time test:

[0051] In magnetic resonance imaging, T1 relaxation time is the time required for longitudinal magnetization to recover to the equilibrium state. In this test example, a probe solution with a gradient concentration in the range of 0-0.4 mM (mmol / L) is prepared. The specific concentrations can include 0 mM (blank control), 0.1 mM, 0.2 mM, 0.3 mM, and 0.4 mM. Deionized water or physiological saline is usually used as the solvent in the probe solution to ensure that the pH value and ionic strength of the probe solution are consistent with the actual application conditions. A standard NMR sample tube is used to ensure that the inner diameter and length of the sample tube are suitable for the probe of the MRI instrument. The prepared probe solutions with different concentrations are respectively loaded into the sample tubes, and the amount of solution in each sample tube is ensured to be the same and free of air bubbles.

[0052] Use a 7.0T or higher field strength MRI instrument to ensure adequate resolution and sensitivity. Select a T1-weighted imaging sequence such as spoiled gradient recalled echo (SPGR) or fast low angle shot (FLASH). Set the following imaging parameters: (1) repetition time (TR): set to a longer time such as 500-1000 ms to ensure adequate T1 relaxation process; (2) echo time (TE): set to a shorter time such as 2-5 ms to reduce the influence of T2 relaxation; (3) flip angle (FA): choose an appropriate flip angle such as 90° or 30° to optimize signal intensity; (4) matrix size: set to 256x256 or higher to ensure image resolution; (5) field of view (FOV): set an appropriate FOV according to the size of the sample tube such as 20-30 mm.

[0053] Place the sample tubes containing different concentrations of probe solutions in the probe of the MRI instrument one by one, ensuring consistent positioning of the sample tubes to reduce the impact of positional differences on measurement results. Start the MRI instrument and acquire T1-weighted images of each sample. Ensure consistent imaging conditions for each sample to ensure data comparability. Calculate the T1 relaxation time using the following formula:

[0054] S = S0(1 - e -TR / T1 ) (Formula One);

[0055] In the formula, S is the measured signal intensity, S0 is the maximum signal intensity (usually measured when TR is much larger than T1), TR is the repetition time, and T1 is the T1 relaxation time.

[0056] Substitute the signal intensity data at different TR values into the above formula and calculate the T1 relaxation time by nonlinear fitting method (such as least squares method). The test results are shown in Table 1, where the T1 relaxation time of the bimodal imaging probe in aqueous solution gradually shortens with increasing concentration of the bimodal imaging probe, indicating that the Gd-DOTA chelate of the bimodal imaging probe can effectively shorten the T1 relaxation time of the aqueous solution and enhance the MRI signal. Figure 4

[0057] Figure 4 In Table 1, r1 = 10.43 s -1 mM -1 represents the T1 relaxation rate, which is an indicator of the relaxation ability of the probe contrast agent. The T1 relaxation rate is related to the concentration and relaxation time of the probe contrast agent, and its calculation formula is:

[0058]

[0059] where T1 is the relaxation time and C is the concentration of the probe contrast agent. In Figure 4 ​In this case, r1=10.43 s -1 mM -1 This means that when the concentration of the probe contrast agent increases, the T1 relaxation time will be shortened, and this relationship is linear. Specifically, for every 1 mM increase in the concentration of the probe contrast agent, the T1 relaxation time will decrease by 1 / 10.43 ms. This indicates that the probe contrast agent can cause a significant change in the T1 relaxation time at a unit concentration, thus reflecting the contrast effect of the probe. In magnetic resonance imaging, a higher r1 value generally means better contrast effect, because they can achieve significant signal changes at lower contrast agent concentrations, helping to improve the contrast and clarity of the image.

[0060] In a second aspect, the present application provides a preparation method for preparing the aforementioned dual-modality imaging probe, comprising the following steps: dissolving DOTA-NHS and B7-H3 affibody in an organic solvent, adding triethylamine, stirring the reaction under an inert gas atmosphere, to obtain a first intermediate with DOTA connected to the N-terminus of the B7-H3 affibody; dissolving the first intermediate and IRDye800CW-maleimide in an aqueous solution containing an organic solvent, stirring the reaction under an inert gas atmosphere, to obtain a second intermediate with IR-Dye800CW connected to the C-terminus of the B7-H3 affibody; dissolving the second intermediate and GdCl3 in deionized water, and reacting under stirring conditions to obtain the B7-H3-based dual-modality imaging probe.

[0061] In some embodiments, DOTA-NHS and B7-H3 affibody can be dissolved in an organic solvent at a mass ratio of 2:1. In other embodiments, the organic solvent is N,N-dimethylformamide. In yet other embodiments, the inert gas includes nitrogen. In some embodiments, the second intermediate and GdCl3 are dissolved in deionized water with a pH of 6.0.

[0062] In yet other embodiments, the aforementioned preparation method further comprises: after obtaining the first intermediate, purifying the first intermediate; and / or after obtaining the second intermediate, purifying the second intermediate; and / or purifying the dual-modality imaging probe. In some embodiments, the purification process can be carried out by preparative high-performance liquid chromatography (HPLC). Preparative high-performance liquid chromatography (HPLC) purification refers to the process of separating and purifying target compounds in a mixture using HPLC technology. HPLC is a solution-phase-based chromatography technique, which is based on the principle of dissolving the mixture to be separated in a mobile phase solution, and achieving separation of different components through partitioning and redistribution on the stationary phase.

[0063] Example 1:

[0064] Step 1), DOTA-NHS (10 mg, Xi'an Ruixi Biotechnology Co., Ltd.) and B7-H3 affibody (5 mg, Qiangyao Biotechnology Co., Ltd.) were dissolved in 5 mL of deionized water containing 10% v / v N,N-dimethylformamide (DMF), then triethylamine (Et3N, 2.6 mg) was added to the solution, and the mixed solution was continuously stirred at 20°C under a nitrogen atmosphere for 12-16 hours; the solvent in the mixed solution was removed under reduced pressure, and the product was purified by preparative high performance liquid chromatography (HPLC) to obtain a first intermediate with DOTA connected to the N-terminus of the B7-H3 affibody.

[0065] Step 2), 4.2 mg of the first intermediate and 7.5 mg of IRDye800CW-maleimide (from Xi'an Ruixi Biotechnology Co., Ltd.) were dissolved in 5 mL of deionized water containing 10% v / v DMF, stirred at 20°C under a nitrogen atmosphere for 8-12 hours, and after evaporation of the solvent under reduced pressure, the product was purified by HPLC to obtain a second intermediate with IR-Dye800CW connected to the C-terminus of the B7-H3 affibody.

[0066] Step 3), 4.9 mg of the second intermediate and 6.3 mg of GdCl3·6H2O were dissolved in deionized water with a pH of 6.0, the resulting mixture was stirred at 20°C for 72 hours, and then the solvent was removed under reduced pressure to obtain a B7-H3-based bimodal imaging probe. The bimodal imaging probe can be purified by preparative HPLC to obtain the final product. In addition, the presence of free Gd3+ions in the bimodal imaging probe can be confirmed by using o-cresol orange as an indicator. 3+

[0067] In order to facilitate understanding of the above steps, the corresponding reaction formula of each step is listed as follows:

[0068] Step 1):

[0069]

[0070] Step 2):

[0071]

[0072] Step 3):

[0073]

[0074] ​In a third aspect, the present application also provides a use of the aforementioned dual-modality imaging probe in the preparation of a diagnostic reagent for prostate cancer. In some embodiments, the diagnostic reagent is used for at least one of the following: preoperative tumor localization by magnetic resonance imaging; and / or intraoperative real-time navigation of the boundary of a prostate cancer tumor; and / or screening of patients suitable for B7-H3-targeted therapy of prostate cancer.

[0075] Specifically, the dual-modality imaging probe of the present application can provide high-resolution tissue structure information through the magnetic resonance imaging (MRI) modality, helping doctors to accurately locate prostate cancer tumors preoperatively, and to determine the size, location and boundary of the tumor, thereby providing an important basis for the formulation of surgical plans.

[0076] In prostate cancer surgery, real-time navigation is crucial to ensure complete removal of the tumor and to reduce damage to normal tissue. Near-infrared-II fluorescence imaging (NIR-II) has high sensitivity and high temporal resolution, and can provide real-time visualization information of the tumor boundary and positive lymph nodes. The dual-modality imaging probe of the present application can guide surgical resection in real time through the near-infrared-II fluorescence imaging modality, helping doctors to more accurately identify and resect tumor tissue while preserving normal tissue as much as possible, reducing surgical complications and improving the postoperative quality of life of patients.

[0077] B7-H3 is an immunomodulatory glycoprotein that is highly specifically expressed in prostate cancer and is a key target for the diagnosis and treatment of prostate cancer. By detecting the expression of B7-H3 in patients, patients suitable for B7-H3-targeted therapy can be screened. The dual-modality imaging probe of the present application can effectively bind to B7-H3 protein, and through the imaging results, it can be determined whether the patient is suitable for receiving B7-H3-targeted therapy, thereby realizing personalized treatment plans, improving treatment effectiveness, and avoiding unnecessary treatment side effects and economic burden.

[0078] In summary, the dual-modality imaging probe of the present application, by combining the advantages of magnetic resonance imaging and near-infrared-II fluorescence imaging, not only improves the accuracy of prostate cancer diagnosis, but also provides an effective means for intraoperative navigation and screening of patients suitable for B7-H3-targeted therapy, and has significant clinical application value.

[0079] Effect experiment example:

[0080] 1. Cell culture

[0081] Human prostate cancer cells 22RV1 were cultured in RPMI-1640 medium containing 10 wt% fetal bovine serum at 37°C, 5% (volume percentage) CO2 and saturated humidity. Subculture was performed every 2 to 3 days to ensure cell viability and proliferation, providing sufficient cell sources for subsequent tumor model establishment.

[0082] 2. Tumor model establishment and probe injection

[0083] 5×10 6 The cultured 22RV1 cells were subcutaneously seeded in the right groin area of ​​mice. At week 2-3 of tumor growth, the bimodal imaging probe prepared according to this application was injected via the tail vein into the mice at a dose of 0.002 mol / kg. This step aims to deliver the bimodal imaging probe into the mice, allowing it to accumulate in the tumor tissue in preparation for subsequent imaging observation.

[0084] 3. Imaging observation

[0085] Mice injected with the dual-modal imaging probe were observed in a 7.0T magnetic resonance imaging (MRI) system and a near-infrared II in vivo imaging system, respectively. MRI can acquire high-resolution images of tumor tissue to accurately determine the location, size, and boundaries of the tumor; while near-infrared II fluorescence imaging can provide real-time visualization of tumor boundaries and positive lymph nodes, which is helpful for intraoperative navigation and precise tumor resection.

[0086] Figure 5 The image shows a magnetic resonance imaging (MRI) image of a mouse injected with a dual-modal imaging probe. Figure 5 As shown in the image, pre-injection T2-weighted imaging (T2WI) reveals the actual location of the tumor. Figure 5 The study also illustrates the dynamic changes in T1-weighted imaging (T1WI) before (Pre) injection of the dual-modal imaging probe and from 0.5 h to 4 h after injection. Before injection, the tumor region exhibited a uniform low signal intensity; 0.5 h after injection, the tumor boundaries began to blur, and the signal intensity slightly increased; after 1 h, scattered high-signal foci appeared within the tumor; at 2 h, the high-signal areas significantly expanded and merged into patches; at 4 h, the overall signal intensity of the tumor reached its peak, and the contrast with the surrounding normal tissue was significantly enhanced.

[0087] from Figure 5 Further analysis revealed that the signal in the tumor region gradually increased over time, while normal tissues such as muscle and fat maintained a low signal. This confirms that the dual-modal imaging probe achieves tumor-specific enrichment by targeting B7-H3 molecules, which aligns with the high spatial resolution advantage of MRI as a preoperative localization method.

[0088] Figure 6 The image shows a near-infrared II fluorescence image of a mouse injected with a dual-modal imaging probe. Figure 6 As shown, it displays a pre-injection white light image and tumor bioluminescence imaging (BLI) revealing the actual location of the tumor, as well as the dynamic process of NIR-II fluorescence imaging from 0.5 h to 4 h post-injection. Figure 6As can be seen, at 0.5h, weak fluorescence signals began to appear in the tumor region, mainly distributed in the tumor edge; at 1h, the fluorescence intensity was significantly enhanced, the tumor boundary was clearly visible, and the central region signal was slightly lower than the periphery; at 2h, the fluorescence signal reached a peak, the tumor was uniformly high in brightness, and was in sharp contrast to the surrounding normal tissue; at 3-4h, the fluorescence intensity of the tumor decreased slightly, but still maintained high contrast, and the liver and kidney regions appeared excretion fluorescence signals.

[0089] It can be seen that the fluorescence signal is always limited to the tumor region, and the intensity change is consistent with the enrichment metabolism process of the probe in the tumor (accumulation first and then slow elimination). The fluorescence signal of the liver and kidney is enhanced at 4h, indicating that the probe is excreted through the liver-biliary system and the kidney, which meets the clinical safety requirements. Normal tissues (such as muscle and bone) do not appear non-specific fluorescence, which verifies the high tumor penetration and low spontaneous fluorescence interference characteristics of IRDye800CW fluorescent dye. At the same time, the fluorescence signal reaches a peak at 1-2h, which is highly consistent with the best time window of surgical resection, providing experimental evidence for real-time visualization of the tumor boundary during surgery.

[0090] Combining Figure 5 and Figure 6 It can be seen that the mice injected with the dual-mode imaging probe still maintain high contrast on MRI at 4h after injection, indicating that the dual-mode imaging probe of the application is suitable for precise positioning of the tumor before surgery; the NIR-II fluorescence reaches a peak at 1-2h, indicating that the dual-mode imaging probe of the application is suitable for real-time navigation of the tumor during surgery, and the time windows of the two modalities are closely linked. The experimental results further verify that the dual-mode imaging probe of the application can simultaneously meet the dual requirements of preoperative diagnosis (MRI) and intraoperative navigation (NIR-II).

[0091] Although several embodiments of the application have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many changes, modifications and substitutions can be made to the embodiments of the application disclosed herein without departing from the spirit and scope of the application. It should be understood that various alternatives to the embodiments of the application described herein can be employed in practicing the application. The appended claims are intended to define the scope of the application and thus cover any equivalents or alternatives within the scope of these claims.

Claims

1. A B7-H3-based dual-modality imaging probe, characterized in that, The bimodal imaging probe comprises a B7-H3 affibody, a near-infrared fluorescent dye IR-Dye800CW is connected to the C-terminal of the B7-H3 affibody, a Gd-DOTA chelate is connected to the N-terminal of the B7-H3 affibody, and the Gd-DOTA chelate chelates gadolinium ions Gd 3+ For magnetic resonance imaging, the near-infrared fluorescent dye IR-Dye800CW is used for near-infrared two-region fluorescence imaging; The sequence of the B7-H3 affibody is: KAEAKYAKEKIAALSEIIWLPNLTHGQIMAFIAALNDDPSQSSELLSEAKKLNDSQGG GC.

2. The bimodal imaging probe of claim 1, wherein, The structure of the dual-modal imaging probe is as follows:

3. The bimodal imaging probe of claim 1 or 2, wherein, The molecular weight of the B7-H3 affibody is 8 kDa.

4. The bimodal imaging probe of claim 1 or 2, wherein, The excitation wavelength of the dual-modal imaging probe is 782 nm, and the emission wavelength is 807 nm.

5. The bimodal imaging probe of claim 1 or 2, wherein, The T1 relaxation time of the dual-modal imaging probe in an aqueous solution gradually shortens with the increase of the concentration of the dual-modal imaging probe.

6. A method for preparing the bimodal imaging probe of any one of claims 1-5, wherein, The method comprises the following steps: DOTA-NHS and B7-H3 affibody are dissolved in an organic solvent, triethylamine is added, and the reaction is stirred under an inert gas atmosphere to obtain a first intermediate with DOTA connected to the N-terminus of the B7-H3 affibody; The first intermediate and IRDye800CW-maleimide are dissolved in an aqueous solution containing an organic solvent, and the reaction is stirred under an inert gas atmosphere to obtain a second intermediate with IR-Dye800CW connected to the C-terminus of the B7-H3 affibody; The second intermediate and GdCl3 are dissolved in deionized water, and the reaction is stirred to obtain a B7-H3-based dual-modal imaging probe.

7. The preparation method of claim 6, wherein, the organic solvent is N,N-dimethylformamide; and / or the inert gas comprises nitrogen; and / or the second intermediate and GdCl3 are dissolved in deionized water with a pH of 6.

0.

8. The production method according to claim 6 or 7, characterized by, The preparation method further comprises: After obtaining the first intermediate, the first intermediate is subjected to a purification treatment; and / or After obtaining the second intermediate, the second intermediate is subjected to a purification treatment; and / or The dual-modal imaging probe is subjected to a purification treatment.

9. Use of the dual-modal imaging probe of any one of claims 1-5 in the preparation of a diagnostic reagent for prostate cancer.

10. Use according to claim 9, characterized in that, The diagnostic reagent is used for at least one of the following: tumor localization by magnetic resonance imaging before surgery; and / or real-time navigation of the boundary of a prostate cancer tumor during surgery; and / or screening of patients suitable for B7-H3-targeted treatment of prostate cancer.

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