A molecular probe based on bcma antigen and preparation method and application thereof

Abstract

The application relates to a molecular probe based on a BCMA antigen and a preparation method and application thereof, and the molecular probe comprises a BCMA antigen derived polypeptide or protein fragment, a labeling group connected with the polypeptide or protein fragment, and the polypeptide or protein fragment can be specifically combined with a chimeric antigen receptor expressing an anti-BCMA single-chain antibody variable region, so as to be used for PET / SPECT imaging to trace chimeric antigen receptor positive cells. The molecular probe of the application can be used for specifically imaging CAR cells in vivo, has high sensitivity, and supports repeated administration and longitudinal observation.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to a molecular probe based on BCMA antigen, its preparation method and application. Background Technology

[0002] The distribution, expansion, and persistence of CAR (Chimeric Antigen Receptor) cells in vivo are key determinants of therapeutic efficacy and relapse rates. Traditional peripheral blood testing cannot provide information on their spatial distribution throughout the body. Publicly available literature indicates that CAR cell PET (Positron Emission Tomography) tracing primarily includes:

[0003] (a) Direct labeling with exogenous radioactivity: The advantage is that the process is relatively simple, but the signal is diluted with cell division and the radioactivity is redistributed after cell death, which makes interpretation difficult and makes it difficult to achieve longitudinal tracking at the weekly level.

[0004] (b) Reporter gene imaging: allows for longitudinal tracking, but requires additional genetic engineering of therapeutic cells, leading to process complexity, potential immunogenicity, and regulatory burden;

[0005] (c) Metabolic imaging (e.g.) 18 F-FDG: It has high uptake of both inflammation and tumors, and it is usually difficult to distinguish between tumor progression / inflammatory response and CAR-related malignant clones or CAR cell residues, resulting in insufficient specificity.

[0006] In recent years, antigen probe-based CAR-PET imaging strategies have emerged, with the core idea of ​​"antigen as probe and CAR as receptor" (for example, using the extracellular domain of CD19 as an imaging probe). Related studies have confirmed that this method can achieve specific imaging of CAR without modifying CAR-T cells. However, in practical applications, this technology still faces challenges such as probe molecular weight, in vivo clearance rate, specificity, sensitivity, frequency of repeated administration, and inhibition of CAR-T effector function. Summary of the Invention

[0007] The technical problem to be solved by this invention is to address the aforementioned problems faced by existing CAR cell imaging molecular probes in practical applications, thereby providing a molecular probe based on BCMA antigen, its preparation method, and its application.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] A molecular probe based on BCMA (B Cell Maturation Antigen) antigen, comprising:

[0010] BCMA antigen-derived polypeptides or protein fragments;

[0011] The labeling group attached to the polypeptide or protein fragment;

[0012] The polypeptide or protein fragment can specifically bind to the chimeric antigen receptor expressing the variable region of the anti-BCMA single-chain antibody for use in PET / SPECT imaging to detect the chimeric antigen receptor-positive cells.

[0013] Preferably, in the BCMA antigen-based molecular probe of the present invention, the amino acid sequence of the polypeptide or protein fragment is SEQ ID NO: 1 or has at least 85% sequence identity with SEQ ID NO: 1, preferably has at least 90% sequence identity with SEQ ID NO: 1, more preferably has at least 95% sequence identity with SEQ ID NO: 1, and retains Cys residues.

[0014] Preferably, in the BCMA antigen-based molecular probe of the present invention, the labeling group includes a chelating agent selected from one or more of NOTA, DOTA, NODAGA, DFO, or their derivatives.

[0015] Preferably, in the BCMA antigen-based molecular probe of the present invention, the labeling group comprises a radionuclide that binds to the chelating agent, wherein the radionuclide is preferably... 68 Ga、 64 Cu、 89 Zr、 111 In、 99m Tc or 18 F(Al 18 One or more of the following (F forms).

[0016] The advantages of using labeled groups are: using chelating agents such as NOA allows for stable coordination of metal radionuclides under mild conditions, improving radiochemical purity and in vivo stability; 68 Short-half-life nuclides such as Ga, combined with small molecular weight probes, can reduce the cumulative radiation burden and support multiple longitudinal imaging sessions.

[0017] Preferably, the BCMA antigen-based molecular probe of the present invention further includes a linker arm for linking the polypeptide or protein fragment to the labeling group; the linker arm is preferably a PEG linker arm, an alkyl chain linker arm, or an amino acid oligopeptide linker arm; the length of the linker arm is preferably 1 to 200 atoms; the technical effect of the linker arm is to adjust steric hindrance and hydrophilicity, reduce potential obstruction to other CAR binding arms, and improve in vivo pharmacokinetics.

[0018] A method for preparing a molecular probe based on BCMA antigen includes the following steps:

[0019] The core fragment of the extracellular domain of human BCMA was selected, and an expression vector with an N-terminal tag was constructed. The expression vector was transformed into host cells to induce the expression of the fusion protein. After purification, tag removal by enzyme digestion, and purification, BCMA antigen-derived peptides or protein fragments were obtained.

[0020] By linking the polypeptide or protein fragment with a labeling group, a molecular probe based on the BCMA antigen is obtained.

[0021] Preferably, the amino acid sequence of the core fragment is SEQ ID NO: 1 or has at least 85% sequence identity with SEQ ID NO: 1.

[0022] Preferably, the labeling group includes a chelating agent and a radionuclide bound to the chelating agent. The method for linking the polypeptide or protein fragment to the labeling group is as follows: the chelating agent and the polypeptide or protein fragment are covalently coupled in an alkaline buffer system under elevated temperature conditions, and after purification, a chelating agent coupling intermediate is obtained; then the radionuclide solution is adjusted to a weakly acidic state and incubated with the chelating agent coupling intermediate at elevated temperature to complete the radiolabeling.

[0023] Preferably, the tag is an affinity tag and / or a solubilizing tag, preferably at least one of a polyhistidine tag, a SUMO tag, an MBP tag, and a GST tag, and more preferably a 6×His-SUMO fusion tag.

[0024] Preferably, the host cell is selected from a prokaryotic expression host or a eukaryotic expression host; more preferably...

[0025] Escherichia coli, Pichia pastoris, or mammalian cells.

[0026] Preferably, the purification method is at least one of affinity chromatography, molecular sieve chromatography, ion exchange chromatography, or reversed-phase high-performance liquid chromatography, and the affinity chromatography purification is preferably Ni-NTA affinity chromatography purification.

[0027] Application of a molecular probe in the preparation of a detection reagent for detecting chimeric antigen receptor-positive cells.

[0028] The beneficial effects of this invention are:

[0029] This invention utilizes molecular probes based on the BCMA antigen for the specific detection / imaging of CAR cells (including therapeutic CAR-T cells and CAR-positive malignant clones), supporting longitudinal observation with repeated dosing. Its advantages over existing technologies are as follows:

[0030] With metabolic imaging (e.g.) 18Compared with F-FDG, the accuracy of CAR positive tumor detection is significantly improved;

[0031] Compared with directly radiolabeled CAR cells, it can achieve multiple effective PET / MR scans and distinguish different response kinetic phenotypes, making longitudinal monitoring feasible;

[0032] Compared to "reporter gene PET," which allows for longitudinal tracking but requires additional genetic modification, the molecular probe of this invention does not require additional genetic modification.

[0033] As those skilled in the art will know, arbitrarily truncating BCMA fragments usually results in structural loss or folding failure, preventing binding to CARs. The BCMA antigen-derived polypeptide or protein fragments of this invention are extremely short, retaining key binding sites while avoiding highly unstable N-terminal residues. The labeling group attached to this fragment maintains its conformation and binding function. The molecular probes of this invention possess specific stability and imaging capabilities in vivo; their small molecular weight facilitates rapid background removal and improves the feasibility of repeated imaging, while maintaining nanomolar affinity; the addition of high concentrations of BCMA antigen-derived polypeptide or protein fragments (probe precursors) does not significantly inhibit the tumor-killing function of CAR-T cells, and the radioactive probes […]. 68 When Ga-NOTA-BED is used for in vivo tracking, it does not significantly inhibit the effector function of CAR-T cells.

[0034] This probe can specifically target and identify CAR-T cells containing the BCMA binding domain, enabling non-invasive visualization of CAR-T cells in vivo. In cell implantation models and xenograft tumor models, it can significantly distinguish between CAR-positive and negative groups. In the longitudinal follow-up model of CAR-T therapy, it can perform repeated scans at multiple time points and obtain the kinetic phenotype of CAR amplification / migration.

[0035] Although BCMA-overexpressing tumor cells competitively occupy the CAR on the surface of BC19 CAR-T cells, reducing probe binding, the probe of this invention still retains measurable sensitivity even under severe lesioning, providing experimental evidence for interpreting "signal degradation caused by lesioning" in clinical imaging monitoring.

[0036] Furthermore, the method for preparing the molecular probe of the present invention has the advantages of simplified steps, high labeling efficiency, and ease of on-site preparation of radiopharmaceuticals. Attached Figure Description

[0037] The technical solution of this application will be further described below with reference to the accompanying drawings and embodiments.

[0038] Figure 1 This is a structural configuration of the BC19 CAR and a characterization diagram of the BED in the embodiment of this application; Figure 1A shows a schematic diagram of the structure of CD19 CAR, BCMACAR and bispecific BC19 CAR, and a simulated diagram of the binding interface between the BED probe and anti-BCMA scFv. Figure 1 B is a schematic diagram of the expression, purification, and tag removal process of the BED probe; Figure 1 C represents the flow cytometry results of CAR expression in different engineered T cells; Figure 1 D is the biolayer interference binding kinetics curve of the unmodified BED probe and BC19 scFv; Figure 1 E represents the biolayer interference binding kinetics curve of the Nota-modified BED probe and BC19 scFv.

[0039] Figure 2 This is an embodiment of the present application. 68 Ga]Ga-NOTA-BED can achieve specific and highly sensitive CAR-T cell detection in vitro and can be quantitatively traced in vivo; Figure 2 A is [ 68 Figure showing the time-dependent uptake and competitive blockade results of Ga-NOTA-BED in BC19 CAR-T cells and Mock-T cells; Figure 2 B is [ 68 Image showing the uptake results of Ga-NOTA-BED in cells with different CAR phenotypes; Figure 2 C shows the results of the competition experiment for probe uptake by BC19 CAR-T cells under tumor cell co-culture conditions (R:C represents Raji cells compared to CAR-T cells; U:C represents U266 cells compared to CAR-T cells). Figure 2 D represents the bilateral axillary cell mass model. 68 Representative PET images of Ga-NOTA-BED; Figure 2 E is Figure 2 Quantitative analysis of ROI at various cell seeding sites and muscle background in Figure D; Figure 2 F shows PET / MR fusion imaging images of different numbers of BC19 CAR-T cell seeding models; Figure 2 G is a graph showing the quantitative analysis of probe uptake corresponding to different numbers of seeded cells; Figure 2 H represents the correlation analysis between PET signal intensity and the number of BC19 CAR-T cells injected.

[0040] Figure 3 This is an embodiment of the present application. 68 Evaluation of the effect of Ga]Ga-NOTA-BED probe on CAR-T cell activity; Figure 3A is a representative bioluminescent image of BC19 CAR-T cells or Mock-T cells co-cultured with luciferase-labeled target cells; Figure 3 B shows the quantitative results of specific lysis of Raji-Luc cells by BC19 CAR-T cells; Figure 3 C represents the quantitative results of specific lysis of U266-Luc cells by BC19CAR-T cells; Figure 3 D shows representative bioluminescence images of BC19 CAR-T cells co-cultured with target cells under different concentrations of NOA-BED treatment. Figure 3 E is a graph showing the quantitative results of the lysis of Raji-Luc cells by BC19 CAR-T cells after NOA-BED treatment; Figure 3 F shows the quantitative results of lysis of U266-Luc cells by BC19 CAR-T cells after NOTA-BED treatment; Figure 3 G and Figure 3 H represents the results of TNF-α secretion level detection in the Raji-Luc and U266-Luc co-culture systems; Figure 3 I and Figure 3 J represents the results of IFN-γ secretion level detection in the Raji-Luc and U266-Luc co-culture systems.

[0041] Figure 4 This is a longitudinal PET / MR tracing of CAR-T cell dynamics under non-antigen competition conditions in the Raji lymphoma model of this application; Figure 4 A represents the Raji tumor model [ 68 Schematic diagram of the experimental procedure for Ga-NOTA-BED PET / MR imaging and bioluminescence imaging; Figure 4 B shows representative IVIS, PET / MR, and MIP images of mice in the BC19 CAR-T treatment group and the PBS control group at different time points. Figure 4 C is a graph showing the changes in tumor burden and intratumoral probe uptake over time in rapid-response mice; Figure 4 D is a graph showing the changes in tumor burden and intratumoral probe uptake over time in moderately responsive mice. Figure 4 E is a graph showing the changes in tumor burden and intratumoral probe uptake over time in slow-responding mice. Figure 4 F is a graph showing the changes in tumor burden and intratumoral probe uptake over time in PBS control mice; Figure 4 G is a comparison graph of peak radioactive uptake between the treatment group and the control group; Figure 4 H is a graph comparing the peak tumor / muscle ratio between the treatment group and the control group; Figure 4I is a longitudinal curve showing the changes in probe uptake within the tumor of each experimental mouse; Figure 4 J is a longitudinal quantitative analysis diagram of the bioluminescence signal of subcutaneous tumors; Figure 4 K is a graph showing the change in mouse body weight during treatment; Figure 4 L represents the survival curves of mice in the treatment and control groups; Figure 4 M represents the immunohistochemical staining pattern of CD19 and CD3 in the endpoint tumor or residual tissue.

[0042] Figure 5 This is a longitudinal PET / MR tracing of CAR-T cell dynamics under antigen competition conditions in the U266 multiple myeloma model of this application. Figure 5 A represents the U266 tumor model. 68 Schematic diagram of the experimental procedure for Ga-NOTA-BED PET / MR imaging and bioluminescence imaging; Figure 5 B shows representative IVIS and PET / MR images of mice in the BC19 CAR-T treatment group, Mock-T group, and PBS group at different time points; Figure 5 C is a graph showing the changes in tumor burden and intratumoral probe uptake over time in the cured subgroup of mice; Figure 5 D is a graph showing the changes in tumor burden and intratumoral probe uptake over time in the progressive subgroup of mice. Figure 5 E is a graph showing the changes in tumor burden and intratumoral probe uptake over time in Mock-T group mice; Figure 5 F is a graph showing the changes in tumor burden and intratumoral probe uptake over time in mice in the PBS group. Figure 5 G is a comparison graph of peak tumor uptake in each experimental group; Figure 5 H is a comparison chart of the peak tumor / muscle ratio of each experimental group; Figure 5 I is a longitudinal curve showing the changes in probe uptake within the tumor of each experimental mouse; Figure 5 J is a longitudinal quantitative analysis graph of the bioluminescence signal of subcutaneous tumors in each experimental group; Figure 5 K represents the curve of weight change in mice in each experimental group; Figure 5 L represents the survival curves of mice in each experimental group; Figure 5 M represents the H&E, BCMA, and CD3 staining pattern of the endpoint tumor or residual tissue. Detailed Implementation

[0043] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0044] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below in conjunction with the accompanying drawings and 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 experimental conditions. Unless otherwise specified, all reagents and raw materials used in this invention are commercially available.

[0045] In the following description, numerous specific details are set forth in order to provide a more thorough understanding of this application. However, it will be apparent to those skilled in the art that this application can be practiced without one or more of these details. In other instances, to avoid confusion with this application, some technical features well-known in the art have not been described; that is, not all features of actual embodiments are described herein, nor are well-known functions and steps described in detail.

[0046] Unless otherwise specified, the techniques or conditions described in the following embodiments are generally performed in accordance with conventional techniques or conditions described in the literature in this field, or in accordance with the product manual and the manufacturer's recommendations. All numerical ranges in the following embodiments include endpoint values.

[0047] Example 1: Expression and purification of the BCMA minimal fragment BED

[0048] The core fragment of the extracellular domain of human BCMA (residues 5–54) was used to construct an N-terminal 6×His-SUMO (small ubiquitin-like modified protein) fusion expression vector, which was transformed into E. coli BL21(DE3) and induced for expression at 16°C with 0.1 mM IPTG for 16 hours. After purification of the fusion protein by Ni-NTA (Nickel-Nitrilotriacetic Acid) affinity chromatography, the SUMO tag was removed by specific cleavage with Ulp1 protease, followed by a second Ni-NTA removal of the tag and enzyme. The flow-through was collected and further purified using Superdex 75 molecular sieves to obtain BED lyophilized powder. Purity >98%, yield approximately 5 mg / L; molecular weight and integrity were verified using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), SEC-HPLC (size exclusion-high performance liquid chromatography), and TOF-MS (time-of-flight mass spectrometry).

[0049] Results: A small molecular weight, soluble, and easily coupled and radiolabeled BCMA antigen-derived polypeptide or protein fragment BED was obtained, with the amino acid sequence SEQ ID NO:1: AGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA.

[0050] Example 2: Preparation of NOTA-BED

[0051] Lyophilized BED was dissolved in anhydrous DMSO (dimethyl sulfoxide), and a Nota-NHS ester solution (50 mg / mL) was added dropwise. The reaction was carried out at a lyophilized BED:NOTA-NHS ester molar ratio of 1:8. DIPEA (N,N-diisopropylethylamine, also known as Hünig's base) was added to adjust the pH to approximately 9.0, and the mixture was continuously stirred at 40°C for 8 hours. The reaction solution was purified by preparative C18 column RP-HPLC (flow rate 5.0 mL / min; mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA acetonitrile solution). The target peak was collected, and the molecular weight was confirmed by TOF-MS to be approximately 5672.52 Da (corresponding to the introduction of single Nota), yielding Nota-BED. Figure 1 As shown, BLI demonstrates that even after Nota coupling (i.e., Nota-BED), it still maintains nanomolar affinity (K). D (Approximately 1.11 nM). It should be noted that the coupling site can be achieved through site engineering (such as introducing Cys).

[0052] Results: Stable coordination was obtained without significantly disrupting the epitopes. 68 Ga's precursor, Nota-BED.

[0053] Example 3: [ 68 Preparation and Quality Control of Ga-NOTA-BED

[0054] Take freshly washed 68 GaCl3 (500 μL, 0.05 M HCl) was adjusted to pH 3.0–4.5, preferably 4.0–4.5, with 1.25 M sodium acetate buffer. 50 μg of the Nota-BED precursor was added, and the mixture was incubated at 60°C for 15 minutes. The reaction solution was purified using a C18 solid-phase extraction column: activated sequentially with ethanol and water, and the free molecules were eluted with water after loading. 68 Ga was eluted with 300 μL of ethanol containing 10 mM HCl to obtain the product - a molecular probe based on BCMA antigen ([ 68[Ga]Ga-NOTA-BED). Radiochemical purity (RCP) > 95% and radiochemical yield (RCY) > 50% were determined by radioactive HPLC, and specific activity ≥ 5 GBq / μmol was calculated.

[0055] Example 4: CAR Expression Validation and Cell Platform Construction (Flow Cytometry)

[0056] Take CAR-T cells or CAR-Jurkat cells (human acute T-lymphoblastic leukemia cell line) in logarithmic growth phase, at 5×10 5 Add FACS buffer (PBS + 1% BSA) to each well, block Fc receptors at 4°C for 10 minutes, then stain with anti-FMC63 antibody (APC, 1:50) and labeled BCMA protein (FITC, 3 μg / mL) at 4°C in the dark for 60 minutes. After removing dead cells by adding 7-AAD, collect ≥10,000 live cells for analysis.

[0057] The results are as follows Figure 1 As shown: CD19 CAR-T was approximately 93.6% positive, BCMA CAR-T was approximately 77.1% positive, BC19 dual-target CAR-T was approximately 68.5% double positive; CAR-Jurkat cells were approximately 93.8% positive.

[0058] Example 5: [ 68 In vitro uptake and competitive blocking of Ga-NOTA-BED probe

[0059] Take 5 x 10⁵ BC19 CAR-T and Mock-T. 5 / tube, add the [prepared in Example 3] 68 Ga-NOTA-BED (approximately 37 kBq, 1 μCi) was incubated at 37°C for 60 or 120 minutes. For the blocking group: 1000 times molar excess of unlabeled NOA-BED (50 μg) was added and pre-incubated at 37°C for 1 hour, followed by the addition of the radioactive probe prepared in Example 3. 68 Ga]Ga-NOTA-BED. After incubation, wash three times with ice-cold PBS + 1% BSA, and determine the gamma count using %AD / 5×10. 5 Cellular representation. Results as follows: Figure 2 As shown: BC19 CAR-T uptake was approximately 5.45±0.46% (1 h) and 5.06±0.51% (2 h); after blockade, it decreased to 0.24±0.08% and 0.17±0.09%, respectively, with Mock-T <0.1%.

[0060] Example 6: Specificity verification of different CAR subtypes

[0061] Under the same conditions, BCMA CAR-T, BC19 CAR-T, CD19 CAR-T, and Mock-T were compared, and the results showed (see...). Figure 2 Intake was strictly dependent on the presence of anti-BCMA scFv: BCMA CAR-T was taken up at approximately 13.54±3.14% in 1 h, BC19 CAR-T at approximately 6.53±1.54%, and CD19 CAR-T and Mock-T were close to the background.

[0062] Example 7: Binding retention under high antigen loading competition conditions

[0063] Establishing co-culture of tumor cells and effector cells: When BCMA-negative Raji cells were co-cultured with BC19 CAR-T cells, even with an E:T ratio of 5:1, the radioactive probe prepared in Example 3 [ 68 The Ga]Ga-NOTA-BED uptake remained at approximately 82.63±2.61% relative to the control group (BC19 CAR-T cells cultured alone, without any tumor cells). However, when BC19 CAR-T cells were co-cultured with BCMA-overexpressing U266 cells, the relative uptake percentages at E:T=1:1 and 5:1 decreased to approximately 66.53±0.94% and 57.01±2.21%, respectively, although the absolute uptake was still measurable.

[0064] Results: Although BCMA-overexpressing tumor cells competitively occupy the CAR on the surface of BC19 CAR-T cells, reducing probe binding, the probe still retains measurable sensitivity under severe lesion conditions, providing experimental evidence for interpreting "signal decrease caused by lesion" in clinical imaging monitoring.

[0065] Example 8: In vivo specificity (bilateral implantation model of cell clusters)

[0066] Different cell clusters (5 × 10⁶ cells per side) were implanted subcutaneously on both sides of BALB / c mice. 6 Cells / 100 μL), intravenous injection after implantation [ 68 Ga]Ga-NOTA-BED 3.7–5.5 MBq (100–150 μCi), followed by micro-PET or PET / MR static scanning after 1 hour. Figure 2 As shown, ROI quantification revealed that BCMA CAR-T aggregates were approximately 1.05±0.18 %ID / g, BC19 CAR-T aggregates were approximately 0.90±0.04 %ID / g, while CD19 CAR-T and Mock-T were approximately 0.15±0.04 and 0.11±0.03 %ID / g, respectively. Therefore, [ 68Ga]Ga-NOTA-BED can specifically target and recognize CAR-T cells containing the BCMA binding domain, enabling in vivo PET visualization and tracing of BCMA CAR-T and BC19 CAR-T.

[0067] Example 9: Sensitivity and Linearity (LOD)

[0068] like Figure 2 As shown, CAR-T cells were divided into groups of 5 × 10⁻⁶. 3 Up to 5×10 6 Gradient implantation was performed using a Bruker 9.4T small animal PET / MR scan with muscle as the background area of ​​interest (ROI). Results were observed at approximately 2 × 10⁻⁶. 4 Cells still showed aggregation signals higher than the background (0.18±0.02 %ID / g vs muscle 0.04±0.01 %ID / g), and the PET signal was linearly correlated with cell number (R0). 2 =0.856).

[0069] Example 10: Non-interference verification (killing) of probe binding to CAR effect function

[0070] like Figure 3 As shown, a luciferase killing system was constructed using Raji-Luc-GFP (CD19+) and U266-Luc-GFP (BCMA+) as target cells, with E:T ratios of 4:1, 2:1, 1:1, and 1:2 (n=3). Here, E represents effector cells (BC19 CAR-T), and T represents target cells (Raji-Luc-GFP or U266-Luc-GFP), co-cultured for 48 hours. The interference group was treated with a fixed E:T ratio of 2:1, with the addition of NOTA-BED to 100 nM. Results showed that BC19 CAR-T cells exhibited high lysis rates against both target cell types (maximum lysis: Raji approximately 96.92±0.67%, U266 approximately 99.36±0.39%). The addition of NOTA-BED (≤100 nM) did not reduce lysis (Raji approximately 89.87±5.85%, U266 approximately 98.89±1.08%, with no statistically significant difference).

[0071] Example 11: Non-interference validation of probe binding to CAR activation (cytokines)

[0072] A mixture of cells with an E:T ratio of 2:1 (where E represents effector cells (BC19 CAR-T) and T represents target cells (Raji-Luc-GFP or U266-Luc-GFP)) was added, along with different concentrations of NOTA-BED (0.1, 1, 10, and 100 nM). The cells were co-cultured for 48 hours, and the supernatant was collected and centrifuged at 350 g for 5 minutes. The mixture was then diluted 1:10 and analyzed using an IFN-γ and TNF-α ELISA kit. Readings were taken at 450 nm, and a four-parameter standard curve was fitted. Results showed that compared to the control group (0 nM) without NOTA-BED, there was no statistically significant difference in the amount of TNF-α and IFN-γ secreted by BC19 CAR-T cells after adding 0.1, 1, 10, and 100 nM NOTA-BED.

[0073] Example 12: Longitudinal PET / MR tracking of the Raji model (therapeutic kinetic phenotype)

[0074] like Figure 4 As shown, a subcutaneous + intravenous Raji model was established: Day-10, 1×10⁻⁶ mmol / L was injected subcutaneously. 6 Raji-Luc, Day 2, tail vein injection 2×10 5 Raji-Luc; Day 0: Students were screened and grouped according to bioluminescence loading and injected with BC19 CAR-T (5×10⁻⁶). 6 (Administration route: iv). Longitudinal imaging: The Raji model was injected with the probe prepared in Example 3 (approximately 3.7 MBq) on Days 1, 8, 12, 17, and 22, respectively. One hour later, a 10-minute static PET / MR was performed, followed by bioluminescence imaging (IVIS). Example results showed that different response phenotypes (rapid response / intermediate response / slow response) could be distinguished, and the peak probe uptake (example average peak 1.29 ± 0.48 %ID / g) and high T / M ratio (the ratio of radioactivity of tumor (T) to background muscle (M), example 15.29 ± 2.29) showed a kinetic relationship with changes in tumor burden.

[0075] Example 13: Longitudinal PET / MR tracking of the U266 model (high antigen load conditions)

[0076] like Figure 5 As shown, the U266 composite model was established: Day-7, 3.5 × 10⁻⁶ mg / L was injected subcutaneously. 6 U266-Luc, Day 2, intravenous injection 8×10 5 U266-Luc; Day 0: Screening and grouping followed by injection of BC19 CAR-T (5×10⁻⁶ T cells). 6(iv). Longitudinal imaging: The U266 model was injected with the probe prepared in Example 3 (approximately 3.7 MBq) on Days 4, 11, 14, 18, and 21, respectively. One hour later, a 10-minute static PET / MR scan was performed, followed by bioluminescence imaging (IVIS). The results showed that the Cure and Progress subgroups could be distinguished, with a peak probe uptake of 1.19 ± 0.28 %ID / g and a high T / M ratio (the ratio of radioactivity of tumor (T) to background muscle (M)) of 11.33 ± 2.94.

[0077] Example 14: Dosimetry and Acute Toxicity (Supported by Repeat Imaging)

[0078] Based on animal distribution and OLIDA / EXM estimation, the kidney is the dose-limiting organ (8.71 × 10⁻⁶). -1 mSv / MBq (meaning that after injecting a 1 MBq probe, the kidneys receive approximately 0.871 mSv of radiation), and the effective systemic dose is 0.0128 mSv / MBq, which is lower than... 18 F-FDG 0.019 mSv / MBq; No abnormal vital signs were observed after a single intravenous injection of 10 times the "high dose", and no structural damage was observed in the major organs by HE.

[0079] Example 15: Specific detection of CAR-positive T-cell lymphoma (CAR-Jurkat and xenograft)

[0080] After preparing a BED (approximately 5.4 kDa) and coupling it with Nota, it was used to... 68 Ga is marked as [ 68 Ga]Ga-NOTA-BED, RCP>95%, RCY>50%, specific activity ≥5 GBq / μmol; in vitro uptake in CAR-Jurkat cells was approximately 6.14±0.38%AD / 5×10⁻⁶ at 60 min. 5 Cells showed parental Jurkat uptake of approximately 0.15±0.04, which could be blocked by a 100-fold cold probe (2 μg). In an in vivo cell embolization model (BALB / c, n=3 / group), CAR-Jurkat uptake at 1 h was approximately 0.78±0.05 %ID / g, parental uptake was approximately 0.24±0.03, and blockade resulted in approximately 0.41±0.03. In a xenograft tumor model (NCG), tumor uptake at 1 h was approximately 2.56±0.61 %ID / g for positive CAR and approximately 0.21±0.05 for negative CAR, decreasing to approximately 0.65±0.11 after blockade; and a T / M ratio of approximately 16.0±3.8, showing better correlation with tumor burden than [previous model]. 18 F-FDG (R) 2 =0.93 vs 0.76).

[0081] Alternative implementation: The application scenario can be expanded from "CAR-positive tumors" to "screening of suspected CAR-positive lesions after treatment" to guide biopsy and CAR transgenic detection.

[0082] Based on the preferred embodiments described above, and through the foregoing explanation, those skilled in the art can make various changes and modifications without departing from the technical spirit of this application. The technical scope of this application is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A molecular probe based on BCMA antigen, characterized in that, include: BCMA antigen-derived polypeptides or protein fragments; The labeling group attached to the polypeptide or protein fragment; The polypeptide or protein fragment can specifically bind to the chimeric antigen receptor expressing the variable region of the anti-BCMA single-chain antibody for use in PET / SPECT imaging to detect the chimeric antigen receptor-positive cells.

2. The molecular probe based on BCMA antigen according to claim 1, characterized in that, The amino acid sequence of the polypeptide or protein fragment is SEQ ID NO: 1 or has at least 85% sequence identity with SEQ ID NO: 1, preferably at least 90% sequence identity with SEQ ID NO: 1, more preferably at least 95% sequence identity with SEQ ID NO: 1, and retains Cys residues.

3. The molecular probe based on BCMA antigen according to claim 1 or 2, characterized in that, The labeling group includes a chelating agent and a radionuclide bound to the chelating agent. The chelating agent is preferably one or more of NOTA, DOTA, NODAGA, DFO, or their derivatives. The radionuclide is preferably... 68 Ga、 64 Cu、 89 Zr、 111 In、 99m Tc or 18 F(Al 18 One or more of the following (F forms).

4. The molecular probe based on BCMA antigen according to claim 1 or 2, characterized in that, It also includes a linker arm for connecting the polypeptide or protein fragment to the labeling group; the linker arm is preferably a PEG linker arm, an alkyl chain linker arm, or an amino acid oligopeptide linker arm; the length of the linker arm is preferably 1 to 200 atoms.

5. A method for preparing a molecular probe based on BCMA antigen according to any one of claims 1-4, characterized in that, Includes the following steps: The core fragment of the extracellular domain of human BCMA was selected, and an expression vector with an N-terminal tag was constructed. The expression vector was transformed into host cells to induce the expression of the fusion protein. After purification, tag removal by enzyme digestion, and purification, BCMA antigen-derived peptides or protein fragments were obtained. By linking the polypeptide or protein fragment with a labeling group, a molecular probe based on the BCMA antigen is obtained.

6. The method for preparing the molecular probe based on BCMA antigen according to claim 5, characterized in that, The amino acid sequence of the core fragment is SEQ ID NO: 1 or has at least 85% sequence identity with SEQ ID NO:

1.

7. The method for preparing the BCMA antigen-based molecular probe according to claim 5 or 6, characterized in that, The labeling group includes a chelating agent and a radionuclide bound to the chelating agent. The method for linking the polypeptide or protein fragment to the labeling group is as follows: the chelating agent and the polypeptide or protein fragment are covalently coupled in an alkaline buffer system under elevated temperature conditions, and after purification, a chelating agent coupling intermediate is obtained. The radionuclide solution is then adjusted to a weakly acidic state, coupled with a chelating agent, and incubated at a higher temperature to complete the radiolabeling.

8. The method for preparing the BCMA antigen-based molecular probe according to claim 5 or 6, characterized in that, The tag is an affinity tag and / or a solubilizing tag, preferably at least one of a polyhistidine tag, a SUMO tag, an MBP tag, and a GST tag, and more preferably a 6×His-SUMO fusion tag; the host cell is preferably a prokaryotic expression host or a eukaryotic expression host, and more preferably Escherichia coli, Pichia pastoris, or mammalian cells.

9. The method for preparing the BCMA antigen-based molecular probe according to claim 5 or 6, characterized in that, The purification method is at least one of affinity chromatography, molecular sieve chromatography, ion exchange chromatography, or reversed-phase high-performance liquid chromatography, preferably Ni-NTA affinity chromatography purification.

10. The use of the molecular probe based on BCMA antigen according to any one of claims 1-4 in the preparation of a detection reagent for detecting chimeric antigen receptor positive cells.

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