Clinical markers for monitoring the progression of CRS after CAR-T treatment and applications thereof

Abstract

The present application relates to the biomedical field, and particularly to a clinical marker for monitoring CRS progress after CAR-T treatment and application thereof, the cytokine release syndrome (CRS) progress is judged by the expression of apolipoprotein A-1, so that effective treatment can be carried out and new treatment methods can be developed. The present application discloses APOA1 for the first time to monitor the cytokine release syndrome (CRS) progress after CAT-T treatment, and it is found through mass spectrometry and biochemical detection that APOA1 is expressed the lowest when the inflammation of the cytokine release syndrome (CRS) patient is the most serious, and with the recovery of the inflammation, its expression is also gradually recovered to the pre-treatment, and the cytokine release syndrome (CRS) progress of the patient has good consistency. These results suggest that APOA1 is an important clinical indicator for monitoring the cytokine release syndrome (CRS).

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a clinical biomarker for monitoring the progression of CRS after CAR-T therapy and its application. Background Technology

[0002] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) is a precision targeted therapy for treating tumors. Through genetic engineering, T cells are activated and equipped with a CAR (chimeric antigen receptor) positioning and navigation device. These cells specifically recognize tumor cells in the body and release a large number of effector factors through immune action. These factors can efficiently kill tumor cells, thereby achieving the goal of treating malignant tumors.

[0003] After CAR-T cells are reinfused into the body, they recognize antigen-positive target cells, leading to massive activation and proliferation of T cells. This results in the production of large amounts of inflammatory factors by immune cells, leading to cytokine release syndrome (CRS). Its clinical features include fever, hypotension, and multiple organ dysfunction, which can be life-threatening in severe cases. Existing clinical observations have found that elevated serum levels of IFN-γ, fracktalkine, GM-CSF, IL-5, IL-6, Flt-3L, and IL-10 are clearly correlated with the development of CRS in patients.

[0004] However, rapid, real-time daily monitoring of serum cytokine release syndrome (CRS)-related cytokines is not practically feasible due to technological limitations. Although studies have found a significant correlation between changes in serum C-reactive protein (CRP) levels and serum IL-6 levels and the efficacy of drug treatment in patients with CRS, this invention still requires more clues to understand and monitor the development of CRS for more effective treatment. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a clinical biomarker for monitoring the progression of cytokine release syndrome (CRS) after chimeric antigen receptor T cell (CAR-T) therapy and its application. The expression of apolipoprotein A1 (APOA1, abbreviated as A-1) is used to determine the progression of CRS, thereby enabling effective treatment and the development of new therapeutic methods.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] In a first aspect, in one embodiment of the present invention, a clinical biomarker for monitoring the progression of CRS after CAR-T therapy is provided, wherein the clinical biomarker is apolipoprotein A-1, and the expression of apolipoprotein A-1 is used to determine the progression of cytokine release syndrome.

[0008] Secondly, in one embodiment of the present invention, the application of a clinical biomarker in monitoring the CRS process after CAR-T therapy is also provided, including the following steps:

[0009] Serum samples were collected from patients before and after CAR-T treatment. The serum samples were digested into peptides by trypsin and then data were collected in data-dependent acquisition mode in a mass spectrometer.

[0010] The information detected in the data-dependent acquisition mode is matched and analyzed in the database to determine the expression of APOA1.

[0011] Serum samples from patients to be monitored were selected, processed into peptides, and then subjected to parallel reaction monitoring. The results of the parallel reaction monitoring were analyzed, and peptides of the target protein were selected to characterize the expression of the protein. LSPLGEEMR was used to quantify APOA1 expression.

[0012] The correlation between APAO1 expression and the progression of cytokine release syndrome was confirmed by serum IL6 expression and CAR-T cell expansion.

[0013] As a further aspect of the present invention, in the data-dependent acquisition mode, in each detection cycle, peptide ions with relatively strong signals are preferentially acquired for fragmentation to obtain secondary spectra. The secondary spectra are then analyzed for peptide qualitative analysis to identify the protein.

[0014] As a further aspect of the present invention, data is acquired in a data-dependent acquisition mode in a mass spectrometer, and the raw mass spectrometry data are analyzed by searching a library using Proteome Discoverer software to determine APOA1 expression.

[0015] As a further aspect of the present invention, parallel reaction monitoring is a selective detection of target proteins and target peptides based on high-resolution, high-precision mass spectrometry ion monitoring technology, and quantification of target proteins / peptides.

[0016] As a further aspect of the present invention, the serum sample of the patient to be monitored is processed into a polypeptide and then subjected to parallel reaction monitoring. When analyzing the polypeptide, liquid chromatography-tandem mass spectrometry is used. In the liquid chromatography-tandem mass spectrometry, liquid chromatography is responsible for separating the analyte from interfering substances, and mass spectrometry is responsible for detection.

[0017] The analysis of the polypeptide by liquid chromatography-tandem mass spectrometry includes the following steps:

[0018] After the sample is injected, it first enters the chromatographic column under the carry of the mobile phase, and after separation by the chromatographic column, it enters the mass spectrometer for detection;

[0019] Serum samples from patients to be monitored were processed into peptides and then subjected to parallel reaction monitoring, which included:

[0020] By utilizing the selective detection capability of a quadrupole mass analyzer, the precursor ion information of the target peptide can be selectively detected in a single-stage mass spectrometer.

[0021] The precursor ion is fragmented in a collision cell, and information on all fragments within the selected precursor ion window is detected in a secondary mass spectrometer to perform specific analysis on the target protein / peptide.

[0022] Data analysis was performed on the parallel reaction monitoring results obtained from the detector.

[0023] As a further aspect of the present invention, the results of parallel reaction monitoring are imported into the Skyline open-source software for data analysis.

[0024] The technical solution provided by this invention has the following beneficial effects:

[0025] This invention discloses for the first time the use of APOA1 to monitor the progression of cytokine release syndrome (CRS) after CAT-T therapy. Mass spectrometry and biochemical assays revealed that APOA1 expression was lowest in CRS patients during periods of peak inflammation, and gradually returned to pre-treatment levels as inflammation subsided, showing good consistency with the progression of CRS. These results suggest that APOA1 is an important clinical indicator for monitoring CRS.

[0026] These or other aspects of the invention will become more apparent from the following description of embodiments. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. In the drawings:

[0028] Figure 1 This is a schematic diagram showing the serum IL6 expression at six time points before and after CAR-T treatment in patients.

[0029] Figure 2 This is a schematic diagram showing the expansion of CAR-T cells at six time points before and after CAR-T treatment in the patient.

[0030] Figure 3 This is a schematic diagram showing the expression of APOA1 in the serum of patients after CAR-T therapy, as detected by mass spectrometry.

[0031] Figure 4 This is a schematic diagram illustrating the expression of APOA1 in patient serum through biochemical testing.

[0032] Figure 5 This is a schematic diagram illustrating the expression of APOA1 in the serum of patients verified by PRM. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. The following embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0035] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0036] Because CAR-T cells, after being reinfused into the body, recognize antigen-positive target cells, leading to massive activation and proliferation of T cells, this results in the production of large amounts of inflammatory factors by immune cells, leading to cytokine release syndrome (CRS). CRS is clinically characterized by fever, hypotension, and multiple organ dysfunction, and can be life-threatening in severe cases. To gain more insights into and monitor the progression of CRS and thus enable more effective treatment, this invention provides a clinical biomarker for monitoring the progression of CRS after chimeric antigen receptor T-cell (CAR-T) therapy and its application. The expression of apolipoprotein A1 (APOA1, abbreviated as A-1) is used to determine the progression of CRS, thereby enabling effective treatment and the development of new therapeutic methods.

[0037] One embodiment of the present invention provides a clinical biomarker for monitoring the progression of CRS after CAR-T therapy, wherein the clinical biomarker is apolipoprotein A-1, and the expression of apolipoprotein A-1 is used to determine the progression of cytokine release syndrome.

[0038] An embodiment of the present invention also provides the application of a clinical biomarker in monitoring the CRS process after CAR-T therapy, comprising the following steps:

[0039] Serum samples were collected from patients before and after CAR-T treatment. The serum samples were digested into peptides by trypsin and then data were collected in data-dependent acquisition mode in a mass spectrometer.

[0040] The information detected in the data-dependent acquisition mode is matched and analyzed in the database to determine the expression of APOA1.

[0041] Serum samples from patients to be monitored were selected, processed into peptides, and then subjected to parallel reaction monitoring. The results of the parallel reaction monitoring were analyzed, and peptides of the target protein were selected to characterize the expression of the protein. LSPLGEEMR was used to quantify APOA1 expression.

[0042] The correlation between APAO1 expression and the progression of cytokine release syndrome was confirmed by serum IL6 expression and CAR-T cell expansion.

[0043] In this embodiment, serum samples were collected from patients before and after CAR-T treatment. The serum samples were digested into peptides by trypsin and then analyzed by mass spectrometry (using Q Exactive). TM HF-X Combined Quadrupole Orbitrap TMData is acquired in data-dependent acquisition (DDA) mode on a mass spectrometer or OrbitrapExploris 480 mass spectrometer.

[0044] Among them, data-dependent acquisition mode (DDA) is the simplest and most primitive data acquisition mode in tandem mass spectrometry, and is mainly used for non-targeted quantitative proteomics research.

[0045] Data-dependent acquisition (DDA) mode is based on the shotgun principle, prioritizing the acquisition of peptide ions with relatively strong signals in each detection cycle for fragmentation to obtain secondary spectra. These fragments are then further fragmented and analyzed in the second stage of tandem mass spectrometry. DDA data-dependent scanning mode can typically identify thousands of proteins. The raw mass spectrometry data was analyzed using Proteome Discoverer software (Version 2.4.0.305, Thermo Fisher Scientific) to search for protein expression and determine APOA1 expression.

[0046] New patient serum samples were selected, processed into peptides, and then subjected to parallel reaction monitoring (PRM). Parallel reaction monitoring (PRM) is an ion monitoring technique based on high-resolution, high-precision mass spectrometry that enables selective detection of target proteins and peptides, thereby achieving quantification of target proteins / peptides.

[0047] Serum samples from patients to be monitored were processed into peptides and then subjected to parallel reaction monitoring, including:

[0048] First, the selective detection capability of a quadrupole mass analyzer is utilized to selectively detect the precursor ion information of the target peptide in the first-stage mass spectrometry. Then, the precursor ion is fragmented in a collision cell; finally, information on all fragments within the selected precursor ion window is detected in the second-stage mass spectrometry. This allows for accurate and specific analysis of target proteins / peptides in complex samples.

[0049] In this embodiment, the serum sample of the patient to be monitored was processed into a peptide and then subjected to parallel reaction monitoring. When analyzing the peptide, liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used. In the liquid chromatography-tandem mass spectrometry, liquid chromatography is responsible for separating the analyte from interfering substances, and mass spectrometry is responsible for detection.

[0050] Among them, nanoflow DIONEX UltiMate can be used. TM3000 RSLCnano system and QExactive TM HF-X Combined Quadrupole Orbitrap TM Mass spectrometry is used to analyze peptides using liquid chromatography-tandem mass spectrometry. The specific operation is as follows:

[0051] After sample injection, it first enters the chromatographic column under the carry-on of the mobile phase. After separation by the column, it enters the mass spectrometer for detection. In a triple quadrupole mass spectrometer, the first-stage mass spectrometer scans a specific range of ions or allows specific ions to enter the collision chamber. In the collision chamber, molecular ions collide and fragment, forming daughter ions that enter the second-stage mass spectrometer. The second-stage mass spectrometer scans a specific range of ions or allows specific ions to enter the detector. The parallel reaction monitoring (PRM) results are imported into the Skyline open-source software for data analysis. In this software, peptides of the target protein are selected to characterize the protein's expression, and LSPLGEEMR is used to quantify APOA1 expression.

[0052] Among them, Skyline is an open-source software for quantitative data processing and proteomics analysis.

[0053] Finally, the correlation between APAO1 expression and the progression of cytokine release syndrome (CRS) was confirmed by serum IL6 expression and CAR-T cell expansion.

[0054] This invention discloses for the first time the use of APOA1 to monitor the progression of cytokine release syndrome (CRS) after CAT-T therapy. Mass spectrometry and biochemical assays revealed that APOA1 expression was lowest in CRS patients during periods of peak inflammation, and gradually returned to pre-treatment levels as inflammation subsided, showing good consistency with the progression of CRS. These results suggest that APOA1 is an important clinical indicator for monitoring CRS.

[0055] Specifically, the embodiments of the present invention will be further described below with reference to the accompanying drawings.

[0056] Embodiments of the present invention provide an application of a clinical biomarker in monitoring the progression of CRS after CAR-T therapy, specifically including:

[0057] (1) Sample collection

[0058] See Figure 1 and Figure 2As shown, this invention collected 45 serum samples from 8 patients with B-cell acute lymphoblastic leukemia (B-ALL) who received CD19 CAR-T therapy at 6 time points (D-1, D1, D4, D7, D14, D28) before and after treatment, and performed mass spectrometry analysis. The expression of APOA1 in the serum of the patients after CAR-T therapy was detected by mass spectrometry as follows: Figure 3 As shown, 32 serum samples were collected from an additional 6 patients for parallel response monitoring (PRM) validation. It should be noted that this study was approved by the medical ethics committee, and all participants were informed and provided written informed consent before enrollment.

[0059] Samples were drawn in the morning using serum separation tubes. After the blood had clotted at room temperature for approximately 30 minutes, it was centrifuged at 1000g for 10 minutes. Serum was extracted and stored at -80°C before use. All samples were not thawed more than twice before analysis.

[0060] (2) Sample pretreatment

[0061] After the serum returned to room temperature, 4 μL of serum was mixed with 400 μL of PBS (phosphate-buffered saline). First, a high-selectivity 14-high-abundance protein removal resin kit was used to deplete the 14 high-abundance proteins in the serum: serum albumin, IgG, IgA, IgM, IgD, IgE, κ and λ light chains, α-1-acid glycoprotein, α-1-antitrypsin, α-2-macroglobulin, apolipoprotein A1, fibrin, haptoglobin, and transferrin. The presence of high-abundance proteins can interfere with the detection of low-abundance proteins. The high-selectivity 14-high-abundance protein removal resin kit can be selected from High Select... TM Top14 abundant protein depletion resin, Thermo Fisher Scientific TM San Jose, USA.

[0062] Serum samples were concentrated to 50 μL using a 3k MWCO filtration device. This device is a disposable ultrafiltration centrifuge tube equipped with a polyethersulfone (PES) membrane for the concentration, desalting, and buffer replacement of biological samples. The molecular weight cutoff (MWCO) of the PES membrane is 3K.

[0063] Then, 200 μL of lysis buffer was added to bring the sample volume to 250 μL. The lysis buffer could be 10 M urea in triethylammonium bicarbonate buffer (TEAB), Sigma. Next, disulfide reduction and alkylation reactions were performed at 31.5 °C using 25 μL of 100 mM tris(2-carboxyethyl)phosphine (TCEP, a highly efficient disulfide bond reducing agent that opens disulfide bonds) and 12.5 μL of 800 mM iodoacetamide (IAA, an alkylating agent for cysteine ​​and histidine that ensures complete denaturation of the protein sample and maintains its reduced state). The reduction process opens the disulfide bonds of the protein, and alkylation modifies the thiol groups, preventing the free thiol groups from regenerating disulfide bonds, making the protein molecules chain-like, increasing protein solubility, and exposing as many restriction enzyme sites as possible. Finally, the protein was digested into peptides using a two-step trypsin digestion process.

[0064] The digestion reaction was terminated by adding 1% trifluoroacetic acid (TFA, a strong acid that inactivates trypsin under acidic conditions) (Thermo Fisher). The TMTpro 16plex (ThermoFisher) continuous mass tagging reagent was used to label peptides by incubation at 1200 rpm at room temperature for 60 min. TMTpro 16plex is a next-generation tandem mass spectrometry tag that can simultaneously label 17 samples: TMTpro 126; TMTpro 127N; TMTpro 127C; TMTpro 128N; TMTpro 128C; TMTpro 129N; TMTpro 129C; TMTpro 130N; TMTpro 130C; TMTpro 131N; TMTpro 131C; TMTpro 132N; TMTpro 132C; TMTpro 133N; TMTpro 133C; TMTpro 134N; TMTpro 134C.

[0065] All tags in a set of labeled reagents have the same mass (e.g., homogeneous) and their chemical structure consists of an amino reactive group, a balancing group, and an isotopic reporter group. After MS / MS fragmentation, the amino reactive group reacts with the N-terminus or lysine of the amino acid. In primary mass spectrometry, the same peptide from different sources, after being labeled, exhibits the same mass-to-charge ratio and elutes at the same retention time. In secondary mass spectrometry, the balancing group breaks down, and the same peptide labeled with different tags exhibits different reporter groups. The peak area of ​​different reporter groups reflects the content of a certain peptide in the current sample. By comparing the peak areas of different reporter groups, the expression differences of a certain peptide in different samples can be found.

[0066] The TMT-labeled peptide was separated into 96 fractions using high-performance liquid chromatography (Dinex Ultimate 3000 UHPLC) (Thermo Fisher) at a flow rate of 500 μL / min and a high-pH reverse-phase liquid chromatography (RPLC) gradient for 96 min. The 96 fractions were then combined at equal intervals (e.g., fractions 1, 25, 49, and 72 were combined into one tube) to form 24 fractions, which were then dried under vacuum.

[0067] (3) Mass spectrometry detection of APOA1 expression

[0068] Prior to mass spectrometry analysis, the dry peptides were redissolved in 2% acetonitrile (ACN) / 0.1% formic acid and then analyzed using a mass spectrometer (Q Exactive HF-X hybrid Quadrupole-Orbitrap or OrbitrapExploris 480) (Thermo Fisher) in data-dependent acquisition (DDA) mode, as described above, with a liquid chromatography (LC) gradient of 60 minutes and a flow rate of 300 nL / min.

[0069] Raw mass spectrometry data were analyzed using Proteome Discoverer (Version 2.4.0.305, Thermo Fisher Scientific) with FASTA files (20,377 proteins) downloaded from the UniProt website on May 7, 2020. Precursor ion mass tolerance was set at 10 ppm, and production mass tolerance was set at 0.02 Da. For proteomics data quality control (QC), we used a mixture of the same 30 samples per batch, labeled with TMT pro-126, to calibrate data from different batches and assess their quantitative accuracy.

[0070] (4) Biochemical detection of serum APOA1 expression

[0071] After serum sample collection, clinical blood lipids were measured using a Beckman Coulter AU5800 fully automated biochemical analyzer. This instrument quantifies protein expression levels using spectrophotometry and potentiometry. The biochemical detection of APOA1 expression in patient serum was also performed. Figure 4 As shown.

[0072] (5) PRM further validated the expression of APOA1.

[0073] The remaining 32 serum samples were processed into peptides as described above. Peptides were analyzed using a DIONEX UltiMate 3000 RSLCnano system (Thermo Scientific, San Jose, USA) and a Q Exactive HF-X hybrid Quadrupole-Orbitrap (Thermo Fisher Scientific, San Jose, USA) combined with LC-MS / MS in parallel reaction monitoring (PRM) mode. PRM results were analyzed using Skyline (MacCoss Lab, University of Washington), an open-source software application for quantitative data processing and proteomics data analysis. Retention time was predicted using a common internal retention time, and the isolation time window was set to 10 minutes. All integrated peaks were manually checked to ensure correct peak detection and integration. PRM validated APOA1 expression in patient serum as follows: Figure 5 As shown.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a clinical biomarker in monitoring the progression of CRS after CAR-T therapy, characterized in that, Includes the following steps: Serum samples were collected from patients before and after CAR-T treatment. The serum samples were digested into peptides by trypsin and then data were collected in data-dependent acquisition mode in a mass spectrometer. The information detected in the data-dependent acquisition mode is matched and analyzed in the database to determine the expression of APOA1. Serum samples from patients to be monitored were selected, processed into peptides, and then subjected to parallel reaction monitoring. The results of the parallel reaction monitoring were analyzed, and peptides of the target protein were selected to characterize the expression of the protein. LSPLGEEMR was used to quantify APOA1 expression. The correlation between APOA1 expression and the progression of cytokine release syndrome was confirmed by serum IL6 expression and CAR-T cell expansion.

2. The application of the clinical biomarker as described in claim 1 in monitoring the CRS progression after CAR-T therapy, characterized in that, In the data-dependent acquisition mode, peptide ions with relatively strong signals are preferentially acquired in each detection cycle for fragmentation to obtain secondary spectra. The secondary spectra are then analyzed to perform qualitative peptide analysis and identify the protein APOA1.

3. The application of the clinical biomarker as described in claim 2 in monitoring the CRS progression after CAR-T therapy, characterized in that, Data was acquired in the data-dependent acquisition mode of the mass spectrometer. The raw mass spectrometry data were analyzed by searching the library using ProteomeDiscoverer software to determine the expression of the protein APOA1.

4. The application of the clinical biomarker as described in claim 1 in monitoring the CRS progression after CAR-T therapy, characterized in that, Parallel reaction monitoring is a technique based on high-resolution, high-precision mass spectrometry to selectively detect and quantify the target protein APOA1 / APOA1 peptides.

5. The application of the clinical biomarker as described in claim 4 in monitoring the CRS progression after CAR-T therapy, characterized in that, Serum samples from patients to be monitored were processed into peptides and then subjected to parallel reaction monitoring, including: By utilizing the selective detection capability of a quadrupole mass analyzer, the precursor ion information of APOA1 peptides can be selectively detected in a single-stage mass spectrometer. The precursor ion is fragmented in a collision cell, and information on all fragments within the selected precursor ion window is detected by secondary mass spectrometry to perform specific analysis on the target protein APOA1 / APOA1 peptide.

6. The application of the clinical biomarker as described in claim 5 in monitoring the CRS progression after CAR-T therapy, characterized in that, Serum samples from patients to be monitored were processed into peptides and then subjected to parallel reaction monitoring. When analyzing the peptides, liquid chromatography-tandem mass spectrometry was used. In the liquid chromatography-tandem mass spectrometry, liquid chromatography is responsible for separating the analyte from interfering substances, and mass spectrometry is responsible for detection.

7. The application of the clinical biomarker as described in claim 6 in monitoring the CRS progression after CAR-T therapy, characterized in that, The results of the parallel reaction monitoring were imported into the Skyline open-source software for data analysis.

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