CN116893215A - Clinical marker for monitoring CRS (cancer therapy) process after CAR-T treatment and application thereof - Google Patents

Clinical marker for monitoring CRS (cancer therapy) process after CAR-T treatment and application thereof Download PDF

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CN116893215A
CN116893215A CN202310257282.2A CN202310257282A CN116893215A CN 116893215 A CN116893215 A CN 116893215A CN 202310257282 A CN202310257282 A CN 202310257282A CN 116893215 A CN116893215 A CN 116893215A
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treatment
monitoring
car
crs
expression
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王兴兵
郭天南
王有名
孙瑞
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Anhui Provincial Hospital First Affiliated Hospital of USTC
Westlake University
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Anhui Provincial Hospital First Affiliated Hospital of USTC
Westlake University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation

Abstract

The invention relates to the biomedical field, in particular to a clinical marker for monitoring CRS process after CAR-T treatment and application thereof, and the expression of apolipoprotein A-1 is used for judging Cytokine Release Syndrome (CRS) process, so that effective treatment can be carried out and a novel treatment method can be developed. The invention discloses that APOA1 is used for monitoring the Cytokine Release Syndrome (CRS) process after CAT-T treatment for the first time, and mass spectrum and biochemical detection show that the APOA1 has the lowest expression when the inflammation is the heaviest in a patient with the Cytokine Release Syndrome (CRS) and gradually returns to the treatment before the inflammation is recovered, and has good consistency with the Cytokine Release Syndrome (CRS) process of the patient. These results suggest that APOA1 is an important clinical indicator in monitoring Cytokine Release Syndrome (CRS).

Description

Clinical marker for monitoring CRS (cancer therapy) process after CAR-T treatment and application thereof
Technical Field
The invention relates to the biomedical field, in particular to a clinical marker for monitoring CRS (cancer cell therapy) progress after CAR-T treatment and application thereof.
Background
The antigen-combining receptor T cell therapy (Chimeric Antigen Receptor T-Cell Immunotherapy, CAR-T) is an accurate targeting therapy for treating tumors, T cells are activated by genetic engineering technology, a positioning navigation device CAR (tumor chimeric antigen receptor) is arranged, tumor cells in a human body are specially identified, a large number of various effector factors are released by immunization, and the tumor cells can be effectively killed, so that the purpose of treating malignant tumors is achieved.
Recognition of antigen-positive target cells after CAR-T cell reinfusion into the body results in massive activation and proliferation of T cells, which can lead to immune cells producing massive inflammatory factors, leading to the appearance of cytokine release syndrome (cytokine release syndrome, cytokine Release Syndrome (CRS)). The clinical characteristics of the traditional Chinese medicine composition are fever, hypotension, multiple organ dysfunction and the like, and life is endangered when the traditional Chinese medicine composition is serious. The existing clinical observations found that IFN-gamma (interferon-gamma), fracktalkine (fractal chemokine), GM-CSF (granulocyte-macrophage growth factor), IL-5 (interleukin-5), IL-6 (interleukin-6), flt-3L (human FMS-like tyrosine kinase 3 ligand) and IL-10 (interleukin-10) increase in serum had a clear correlation with the patient's Cytokine Release Syndrome (CRS).
However, rapid real-time detection of serum Cytokine Release Syndrome (CRS) -associated cytokines per day is not practical due to technical limitations. Although studies have found that increasing or decreasing levels of C-reactive protein (CRP) in serum are significantly correlated with serum IL-6 levels in patients with Cytokine Release Syndrome (CRS) and therapeutic efficacy of drug therapy, the present invention still requires more clues to understand and monitor the progression of Cytokine Release Syndrome (CRS) for more effective treatment.
Disclosure of Invention
In view of the above, the present invention aims to provide a clinical marker for monitoring the progress of cytokine release syndrome after chimeric antigen receptor T cell (CAR-T) treatment, and its use, by judging the progress of Cytokine Release Syndrome (CRS) by the expression of Apolipoprotein a-1 (APOA 1, abbreviated as a-1), so that effective treatment can be performed and new therapeutic methods can be developed.
In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:
in a first aspect, in one embodiment of the invention, there is provided a clinical marker for monitoring CRS progression following CAR-T treatment, the clinical marker being apolipoprotein a-1, for use in determining cytokine release syndrome progression by expression of apolipoprotein a-1.
In a second aspect, in one embodiment provided by the present invention, there is also provided the use of a clinical marker for monitoring CRS progression after CAR-T treatment, comprising the steps of:
collecting serum samples of patients before and after CAR-T treatment, and collecting data in a data dependent collection mode in a mass spectrometer after the serum samples are subjected to pancreatin digestion to peptide fragments;
matching information detected in a data dependent acquisition mode in a database, analyzing protein expression, and determining APOA1 expression;
selecting a patient serum sample to be monitored, processing the patient serum sample to be monitored into polypeptide, then carrying out parallel reaction monitoring, carrying out data analysis on the parallel reaction monitoring detection result, selecting a peptide fragment of a target protein to represent the expression of the protein, and using LSPLGEEMR to quantify the APOA1 expression;
the correlation of APAO1 expression and cytokine release syndrome progression was confirmed by serum IL6 expression and CAR-T cell expansion numbers.
As a further scheme of the invention, in the data dependent acquisition mode, peptide ions with relatively strong signals are preferentially acquired in each detection cycle to carry out fragmentation, a secondary spectrogram is obtained, the secondary spectrogram is analyzed to carry out peptide qualitative analysis, and proteins are identified.
As a further aspect of the invention, data is collected in a data dependent collection mode in a mass spectrometer, and raw mass spectrometry data is subjected to library searching by Proteome Discoverer software to analyze protein expression to determine APOA1 expression.
As a further scheme of the invention, parallel reaction monitoring is to selectively detect target proteins and target peptide fragments based on ion monitoring technology of high-resolution and high-precision mass spectrum and quantify the target proteins/peptide fragments.
As a further scheme of the invention, the serum sample of the patient to be monitored is processed into polypeptide, then parallel reaction monitoring is carried out, and liquid chromatography-tandem mass spectrometry is adopted for analysis when the polypeptide is analyzed; in the liquid chromatography tandem mass spectrometry, the liquid chromatography is responsible for separating an object to be detected and an interfering object, and the mass spectrometry is responsible for detection;
the liquid chromatography-tandem mass spectrometry analysis of the polypeptide comprises the following steps:
after sample injection, the sample enters a chromatographic column under the carrying of a mobile phase, and enters a mass spectrum for detection after being separated by the chromatographic column;
and performing parallel reaction monitoring after treating serum samples of patients to be monitored into polypeptides, wherein the parallel reaction monitoring comprises:
selectively detecting parent ion information of the target peptide fragment in the primary mass spectrum by utilizing the selective detection capability of the quaternary rod mass analyzer;
fragmentation of parent ions in a collision cell, detection of information of all fragments within a selected parent ion window in a secondary mass spectrum, and specific analysis of target proteins/peptides;
and obtaining parallel reaction monitoring detection results at the detector for data analysis.
As a further scheme of the invention, the parallel reaction monitoring detection result is imported into Skyline open source software for data analysis.
The technical scheme provided by the invention has the following beneficial effects:
the invention discloses that APOA1 is used for monitoring the Cytokine Release Syndrome (CRS) process after CAT-T treatment for the first time, and mass spectrum and biochemical detection show that the APOA1 has the lowest expression when the inflammation is the heaviest in a patient with the Cytokine Release Syndrome (CRS) and gradually returns to the treatment before the inflammation is recovered, and has good consistency with the Cytokine Release Syndrome (CRS) process of the patient. These results suggest that APOA1 is an important clinical indicator in monitoring Cytokine Release Syndrome (CRS).
These and other aspects of the invention will be more readily apparent from the following description of the embodiments. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following description will briefly introduce the drawings that are needed in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are only some embodiments of the present invention. In the drawings:
figure 1 is a schematic representation of serum IL6 expression at 6 time points before and after CAR-T treatment in patients.
Figure 2 is a graphical representation of the number of CAR-T cell expansion at 6 time points before and after CAR-T treatment of patients.
FIG. 3 is a schematic representation of mass spectrometry detection of APOA1 expression in serum following CAR-T treatment of patients.
FIG. 4 is a schematic diagram showing the biochemical detection of APOA1 expression in patient serum.
FIG. 5 is a schematic representation of PRM verification of APOA1 expression in patient serum.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art. The following examples are given by way of illustration of the invention and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Because the target cells which recognize antigen positivity after the CAR-T cells are returned into the body lead to massive activation and proliferation of the T cells, immune cells can generate massive inflammatory factors, and thus, cytokine release syndrome appears, and the clinical characteristics of the cytokine release syndrome are shown as fever, hypotension, multiple organ dysfunction and the like, and the life is endangered in severe cases. To enable more clues to understand and monitor the progression of the Cytokine Release Syndrome (CRS) for more effective treatment. The present invention provides a clinical marker for monitoring the progress of Cytokine Release Syndrome (CRS) after chimeric antigen receptor T cell (CAR-T) treatment, and uses thereof, judging the progress of Cytokine Release Syndrome (CRS) by the expression of Apolipoprotein a-1 (apoprotein A1, APOA1, abbreviated as a-1), so that effective treatment can be performed and new therapeutic methods can be developed.
One embodiment of the invention provides a clinical marker for monitoring CRS progression following CAR-T treatment, which is apolipoprotein a-1, for use in judging cytokine release syndrome progression by expression of apolipoprotein a-1.
An embodiment of the invention also provides for the use of a clinical marker for monitoring CRS progression after CAR-T treatment, comprising the steps of:
collecting serum samples of patients before and after CAR-T treatment, and collecting data in a data dependent collection mode in a mass spectrometer after the serum samples are subjected to pancreatin digestion to peptide fragments;
matching information detected in a data dependent acquisition mode in a database, analyzing protein expression, and determining APOA1 expression;
selecting a patient serum sample to be monitored, processing the patient serum sample to be monitored into polypeptide, then carrying out parallel reaction monitoring, carrying out data analysis on the parallel reaction monitoring detection result, selecting a peptide fragment of a target protein to represent the expression of the protein, and using LSPLGEEMR to quantify the APOA1 expression;
the correlation of APAO1 expression and cytokine release syndrome progression was confirmed by serum IL6 expression and CAR-T cell expansion numbers.
In this example, serum samples of patients before and after CAR-T treatment were collected, and after pancreatin was digested to peptide fragments, the serum samples were subjected to a mass spectrometer (Q exact was used TM HF-X combined quadrupole rod Orbitrap TM Mass spectrometer or Orbitrap Exploris, 480 mass spectrometer) data is acquired in data-dependent acquisition (data-dependent acquisition, DDA) mode.
The data dependent acquisition mode (DDA) is the simplest and most original data acquisition mode in tandem mass spectrometry, and is mainly used for non-targeted quantitative proteomics research.
The data dependent acquisition mode (DDA) is based on the principle of the shotgun method, in each detection cycle, peptide ions with relatively strong signals are preferentially acquired for fragmentation, a secondary spectrogram is obtained, then the peptide ions are fragmented and analyzed in the second stage of tandem mass spectrometry, and thousands of proteins can be generally identified by the DDA data dependent scanning mode. Mass spectrometry raw data were searched by Proteome Discoverer software (Version 2.4.0.305, thermo Fisher Scientific/sammer, feier germany) to analyze protein expression and thereby determine APOA1 expression.
New patient serum samples were taken and treated as polypeptides for parallel response monitoring (Parallel Reaction Monitoring, PRM). Parallel Reaction Monitoring (PRM) is an ion monitoring technology based on high-resolution and high-precision mass spectrum, and can selectively detect target proteins and target peptide fragments, so that the quantification of the target proteins/peptide fragments is realized.
Parallel response monitoring after treatment of a patient serum sample to be monitored for polypeptides, comprising:
the parent ion information of the target peptide fragment is selectively detected in a primary mass spectrum by utilizing the selective detection capability of a quaternary rod mass analyzer. Then, the parent ions are fragmented in a collision cell; and finally, detecting information of all fragments in the selected parent ion window in the secondary mass spectrum, so that the target protein/peptide fragment in the complex sample can be accurately and specifically analyzed.
In this example, the parallel reaction monitoring is performed after the patient serum sample to be monitored is processed into polypeptide, and liquid chromatography-tandem mass spectrometry (Liquid Chromatograph Mass Spectrometer, LC-MS/MS) is used for analyzing the polypeptide; in the liquid chromatography tandem mass spectrometry, the liquid chromatography is responsible for separating an object to be detected and an interfering object, and the mass spectrometry is responsible for detection.
Therein, nanooflow DIONEX UltiMate can be used TM 3000 RSLCnano system and Qexact TM HF-X combined quadrupole rod Orbitrap TM The mass spectrometer is used for carrying out liquid chromatography-tandem mass spectrometry analysis on the polypeptide, and the specific operation is as follows:
after sample injection, the sample firstly enters a chromatographic column under the carrying of a mobile phase, and enters a mass spectrum for detection after being separated by the chromatographic column. In a triple quaternary rod, a primary mass spectrum scans ions of a specific range or allows the ions to enter a collision chamber, in the collision chamber, molecular ions collide and split to form sub-ions which enter a secondary mass spectrum, the secondary mass spectrum scans the ions of the specific range or allows the ions to enter a detector, parallel Reaction Monitoring (PRM) detection results are imported into Skyline open source software for data analysis, peptide fragments of target proteins are selected in the software to represent the expression of the proteins, and LSPLGEEMR is used for quantifying the APOA1 expression.
The Skyline open source software is open source software used for quantitative data processing and proteomics analysis.
Finally, the correlation of APAO1 expression and Cytokine Release Syndrome (CRS) progression was confirmed by serum IL6 expression and CAR-T cell expansion numbers.
The invention discloses that APOA1 is used for monitoring the Cytokine Release Syndrome (CRS) process after CAT-T treatment for the first time, and mass spectrum and biochemical detection show that the APOA1 has the lowest expression when the inflammation is the heaviest in a patient with the Cytokine Release Syndrome (CRS) and gradually returns to the treatment before the inflammation is recovered, and has good consistency with the Cytokine Release Syndrome (CRS) process of the patient. These results suggest that APOA1 is an important clinical indicator in monitoring Cytokine Release Syndrome (CRS).
In particular, embodiments of the present invention are further described below with reference to the accompanying drawings.
Embodiments of the invention provide for the use of clinical markers for monitoring CRS progression after CAR-T treatment, comprising in particular:
(1) Sample collection
Referring to FIGS. 1 and 2, the present invention was validated by collecting 45 serum samples from 8 patients with CD19 CAR-T treated acute B-lymphoblastic leukemia (B-cell acute lymphoblastic leukemia, B-ALL) at 6 time points (D-1, D4, D7, D14, D28) before and after treatment, and measuring the expression of APOA1 in serum after CAR-T treatment of the patients by mass spectrometry, as shown in FIG. 3, and collecting 32 serum samples from another 6 patients for Parallel Response Monitoring (PRM). It is noted that the study was approved by the medical ethics committee, and all subjects were informed and provided written informed consent prior to group entry.
Samples were drawn in the morning with serum separation tubes. After the blood had coagulated at room temperature for about 30 minutes, it was centrifuged at 1000g for 10 minutes. Serum was extracted and stored at-80 ℃ prior to use. All samples were not thawed more than twice before analysis was performed.
(2) Sample pretreatment
After the serum was returned to room temperature, 4. Mu.L of serum was mixed with 400. Mu.L of PBS (phosphate buffered saline), and 14 kinds of high-abundant proteins in the serum were first depleted using a high-selectivity 14 kinds of high-abundant protein removal resin kit, serum albumin, igG, igA, igM, igD, igE, kappa and lambda light chains, alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-2-macroglobulin, apolipoprotein A1, fibrin, binding globin and transferrin, the presence of high abundance proteins interfering with the detection of low abundance proteins. Wherein, 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。
Serum samples were concentrated to 50 μl using a 3K MWCO filtration device, which is a disposable ultrafiltration centrifuge tube, equipped with Polyethersulfone (PES) membrane for concentration, desalting and buffer replacement of biological samples, the molecular weight cut-off (molecular weight cutoff, MWCO) of the PES membrane being 3K.
200ul of lysis buffer, which may be 10M urea in triethylammonium bicarbonate buffer (Triethylammonium bicarbonateBuffer, TEAB), (Sigma), was then added to a volumetric sample volume of 250 ul. Next, 25ul 100mM tris (2-carboxyethyl) phosphine (tris (2-carboxythio) phosphine, TCEP, efficient disulfide reducing agent, disulfide bond opening) and 12.5ul 800mM Iodoacetamide (Iodoacetamide, IAA, alkylating agent of cysteine and histidine) were used to complete denaturation of the protein sample and maintain the reduced state, respectively, disulfide bond reduction and alkylation reactions were carried out at 31.5℃during which disulfide bonds of the protein were opened, followed by alkylation to modify sulfhydryl groups to prevent free sulfhydryl groups from regenerating disulfide bonds, so that protein molecules were chain-like, increasing protein solubility and exposing as many cleavage sites as possible, followed by two-step pancreatin digestion of the protein as a polypeptide.
Digestion was stopped by adding 1% final concentration of trifluoroacetic acid (Trifluoroacetic acid, TFA, a strong acid, pancreatin deactivated under acidic conditions) (Thermo Fisher). Continuous mass tag (TMTpro) 16plex (Thermo Fisher) reagent incubation for 60min at 1200rpm for labeling polypeptides, TMTpro 16plex labeling reagent is a new generation tandem mass spectrometry tag that can label 17 samples simultaneously: 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.
All tags in a set of tag reagents bear the same mass (e.g., homogenous), and the 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 amino acid N-terminus or lysine, and in primary mass spectrometry, identical peptide fragments of different origin are labeled to give the same mass-to-charge ratio and flow out at the same retention time. In the secondary mass spectrum, the balance group is disconnected, different report groups appear on the same peptide segment marked by different labels, the peak areas of different report groups can reflect the content of a certain peptide segment in a current sample, and the expression difference of a certain peptide segment in different samples can be found by comparing the peak areas of different report groups.
TMT-tagged polypeptides were separated into 96 fractions in a high performance liquid chromatograph (Dinex Ultimate 3000 UHPLC) (Thermo Fisher) at a flow rate of 500. Mu.L/min using a 96min high pH reverse phase liquid chromatography (reverse-phase liquid chromatography, RPLC) gradient. Finally, 96 fractions were equally combined (example: no. 1, no. 25, no. 49, no. 72 into one tube) into 24 fractions, which were dried in vacuo.
(3) Mass spectrometric detection of APOA1 expression
Prior to mass spectrometry, the dry peptide was redissolved in 2% Acetonitrile (ACN)/0.1% formic acid solvent and then analyzed in a Data Dependent Acquisition (DDA) mode using a mass spectrometer (Q exact HF-X hybrid Quadrupole-Orbitrap or Orbitrap Exploris 480) (Thermo Fisher), with a liquid phase (LC) gradient of 60 minutes and a flow rate of 300nL/min, as described above.
Mass spectra raw data were analyzed by Proteome Discoverer (Version 2.4.0.305,Thermo Fisher Scientific) using FASTA files (20377 proteins) downloaded from the UniProt website on day 5 and 7 of 2020. The precursor ion mass tolerance was set to 10ppm and the production mass tolerance was set to 0.02Da. The proteomic data Quality Control (QC) method was that we used the same 30 sample mixture per batch, labeled with TMT pro-126, to calibrate the data from the different batches and evaluate its quantitative accuracy.
(4) Biochemical detection of serum APOA1 expression
After serum sample collection, clinical blood lipid was measured in a Beckmann Coulter AU5800 full-automatic biochemical analyzer, which measures protein expression level by spectrophotometry and potentiometry, and biochemical detection of APOA1 expression in patient serum is shown in FIG. 4.
(5) PRM further verifies the expression of APOA1
The additional 32 serum samples collected were processed as described above into polypeptides and analyzed in Parallel Reaction Monitoring (PRM) mode using the nanofluidic UltiMate 3000 RSLCnano system (Thermo Scientific, san Jose, USA) in combination with Q exact HF-X hybrid Quadrupole-Orbitrap (Thermo Fisher Scientific, san Jose, USA) in combination with LC-MS/MS. PRM results were analyzed by Skyline (MacCoss Lab, university of Washington), an open source software application for quantitative data processing and proteomic data analysis. The common internal retention time is used to predict retention time, and the isolation time window is set to 10 minutes. All integrated peaks were manually checked to ensure correct peak detection and integration, and PRM validated APOA1 expression in patient serum as shown in figure 5.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (9)

1. A clinical marker for monitoring CRS progression following CAR-T treatment, wherein the clinical marker is apolipoprotein a-1 for use in determining cytokine release syndrome progression by expression of apolipoprotein a-1.
2. Use of a clinical marker for monitoring CRS progression after CAR-T treatment, comprising the steps of:
collecting serum samples of patients before and after CAR-T treatment, and collecting data in a data dependent collection mode in a mass spectrometer after the serum samples are subjected to pancreatin digestion to peptide fragments;
matching information detected in a data dependent acquisition mode in a database, analyzing protein expression, and determining APOA1 expression;
selecting a patient serum sample to be monitored, processing the patient serum sample to be monitored into polypeptide, then carrying out parallel reaction monitoring, carrying out data analysis on the parallel reaction monitoring detection result, selecting a peptide fragment of a target protein to represent the expression of the protein, and using LSPLGEEMR to quantify the APOA1 expression;
the correlation of APAO1 expression and cytokine release syndrome progression was confirmed by serum IL6 expression and CAR-T cell expansion numbers.
3. The use of a clinical marker according to claim 2 for monitoring CRS progression after CAR-T treatment, wherein in a data dependent acquisition mode, peptide ions with relatively strong signals are preferentially acquired for fragmentation in each detection cycle, a secondary spectrum is obtained, the secondary spectrum is resolved for peptide qualitative analysis, and protein is identified.
4. The use of a clinical marker according to claim 3 for monitoring CRS progression after CAR-T treatment, wherein data is collected in a data dependent collection mode in a mass spectrometer, and wherein raw mass spectrometry data is searched by a proteome discover software pair to analyze protein expression to determine APOA1 expression.
5. Use of a clinical marker according to claim 2 for monitoring CRS progression after CAR-T treatment, wherein parallel response monitoring is based on ion monitoring technology of high resolution, high precision mass spectrometry for selective detection of target proteins, target peptide fragments and quantification of target proteins/peptide fragments.
6. The use of a clinical marker according to claim 5 for monitoring CRS progression after CAR-T treatment, wherein parallel response monitoring is performed after treatment of the patient serum sample to be monitored for polypeptides, comprising:
selectively detecting parent ion information of the target peptide fragment in the primary mass spectrum by utilizing the selective detection capability of the quaternary rod mass analyzer;
the parent ion is fragmented in a collision cell, information of all fragments within a selected parent ion window is detected in a secondary mass spectrum, and specific analysis is performed on the target protein/peptide fragment.
7. The use of a clinical marker according to claim 6 for monitoring CRS progression after CAR-T treatment, wherein parallel response monitoring is performed after treatment of a patient serum sample to be monitored for polypeptides, and wherein the polypeptides are analyzed by liquid chromatography-tandem mass spectrometry; in the liquid chromatography tandem mass spectrometry, the liquid chromatography is responsible for separating an object to be detected and an interfering object, and the mass spectrometry is responsible for detection.
8. The use of a clinical marker according to claim 7 for monitoring CRS progression after CAR-T treatment, wherein the polypeptide is subjected to liquid chromatography-tandem mass spectrometry analysis, comprising the steps of:
after sample injection, the sample enters a chromatographic column under the carrying of a mobile phase, and enters a mass spectrum for detection after being separated by the chromatographic column;
the mass spectrum is detected according to the mass-to-charge ratio of the measured object, and the measured object is converted into gas-phase ions in an ion source and enters the mass spectrum;
scanning ions in a specific range by a primary mass spectrum in a triple quaternary rod or allowing the ions to enter a collision chamber, and performing collision and fragmentation on molecular ions in the collision chamber to form ion ions which enter a secondary mass spectrum, wherein the secondary mass spectrum scans the ions in the specific range or allows the ions to enter a detector;
and obtaining parallel reaction monitoring detection results at the detector for data analysis.
9. The use of a clinical marker according to claim 7 for monitoring CRS progression after CAR-T treatment, wherein parallel response monitoring assays are imported into Skyline open source software for data analysis.
CN202310257282.2A 2023-03-17 2023-03-17 Clinical marker for monitoring CRS (cancer therapy) process after CAR-T treatment and application thereof Pending CN116893215A (en)

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