WO2022194032A1 - Procédé de criblage de bcr/tcr spécifiques, et système associé - Google Patents

Procédé de criblage de bcr/tcr spécifiques, et système associé Download PDF

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WO2022194032A1
WO2022194032A1 PCT/CN2022/080277 CN2022080277W WO2022194032A1 WO 2022194032 A1 WO2022194032 A1 WO 2022194032A1 CN 2022080277 W CN2022080277 W CN 2022080277W WO 2022194032 A1 WO2022194032 A1 WO 2022194032A1
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bcr
tcr
sequences
sequence
specific
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PCT/CN2022/080277
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张志新
卓越
杨鑫
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成都益安博生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • 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
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • 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
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search
    • 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
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of molecular biology and cellular immunology, and particularly relates to a method and a system for screening specific antibodies (BCR) and T cell receptors (TCR).
  • BCR specific antibodies
  • TCR T cell receptors
  • Monoclonal antibodies are antibodies produced by a single B cell that are highly uniform and recognize specific epitopes. Monoclonal antibodies have broad application prospects in basic medical and biological research, as well as in the diagnosis, prevention and treatment of clinical diseases. Antibody screening is required to obtain monoclonal antibodies against a specific antigen. Existing relatively mature monoclonal antibody screening technologies include:
  • the mouse spleen cells immunized with the antigen are fused with mouse myeloma cells, and the formed hybrid cells can not only produce antibodies, but also proliferate indefinitely.
  • the fused hybrid cells are divided into single cells for inoculation and culture, and the antibodies secreted by the proliferated cells are screened and identified by methods such as enzyme-linked immunosorbent assay (ELISA), and the cell lines that can produce high titer antibodies against the antigen are found, and then cloned from them.
  • ELISA enzyme-linked immunosorbent assay
  • hybridoma technology The main disadvantage of hybridoma technology is that the later identification process requires a large number of repeated cultivation, isolation and identification experiments, which brings high time and material costs.
  • the antibody heavy chain variable region (VH) and light chain variable region (VL) of B lymphocytes are amplified by PCR method, and the amplified fragment is inserted into the phage vector, and the antibody molecule is Fab fragment or single chain antibody. (scFv), it forms a fusion protein with the single-chain phage coat protein and is displayed on the phage surface. Afterwards, the phages are transfected into host cells for proliferation, and released after maturation. Finally, the target antigen-specific phages and corresponding monoclones can be obtained after the steps of affinity adsorption, elution, and amplification by the method of antigen screening. Antibody.
  • yeast surface display technology In order to obtain antibodies that are closer to human antibodies for screening, there are also yeast surface display technology, ribosome display and mRNA display screening technology, etc.
  • the methods are similar to phage display screening.
  • the disadvantages of various display technologies for antibody screening are similar to those of hybridoma technology, the later identification process is labor-intensive, and time and material costs are high.
  • B lymphocytes or T lymphocytes whose surface receptors can recognize specific antigens are screened. Then directly from a single B cell or T cell, clone the B cell receptor (BCR, which is secreted in vitro) or T cell receptor (TCR) gene it carries.
  • BCR B cell receptor
  • TCR T cell receptor
  • the existing monoclonal antibody screening technology has three significant deficiencies: first, it is difficult to screen human antibodies, because it is impossible to repeatedly immunize humans with antigens and adjuvants like immunized animals. If transgenic mice (carrying human immunoglobulin genes) are used, the cost will be higher. This makes the preparation of human antibodies either need to invest more resources in screening and identification, or need to humanize animal antibodies.
  • the second is the difficulty in preparing monoclonal antibodies against complex antigens.
  • complex antigens such as membrane protein complexes, tumor cells, etc.
  • the selection of antigens for immunization itself is difficult and there is a risk of failure, and the function cannot be guaranteed after immunization.
  • Antibodies that meet the requirements such as antibodies obtained by immunizing polypeptide fragments, not necessarily neutralizing antibodies. If multiple antigens are selected for immunization and screening at the same time, the cost of monoclonal antibody preparation will be significantly increased.
  • the present invention provides a method and a system for screening specific BCR/TCR.
  • the present invention compares a huge number of BCR/TCR sequences by comparing the big data of the immune map, and has a higher Specificity and accuracy can greatly improve the efficiency of monoclonal antibody screening and identification.
  • a method for screening specific BCR/TCR comprising the following steps:
  • the BCR/TCR variable region gene is subjected to multiple PCR amplification, followed by high-throughput sequencing, and the nucleic acid and amino acid sequences of the BCR/TCR variable region are determined ;
  • sample to be tested is peripheral blood containing lymphocytes, spleen tissue, tumor tissue or lymphocytes cultured in vitro.
  • sample to be tested is a human-derived sample or an animal-derived sample.
  • step (3) also includes: summarizing and deduplicating all the BCR/TCR sequences of the samples after immunization, then removing all sequences that are repeated with the sequences contained in the sequence set of the sequencing results of the unimmunized samples, and establishing characteristic BCR/TCR sequences. set.
  • the specific acquisition method of the specific immune sequence in the characteristic BCR/TCR sequence set is:
  • the selection of the BCR sequence can also refer to the biochemical properties of the antigen, such as charge and hydrophobicity.
  • the method for screening characteristic antibody heavy chain sequences in peripheral blood after novel coronavirus COVID-19 infection includes the following steps:
  • step (2) After the mRNA obtained in step (1) of reverse transcription processing, multiple PCR amplification of the variable region gene of the antibody heavy chain is performed, and then high-throughput sequencing is performed to determine the nucleic acid and amino acid sequence of the variable region of the antibody heavy chain , try to ensure that the total number of functional antibody heavy chain sequences in each sample is not less than 30,000, and establish an antibody sequence set;
  • amino acid sequence of the heavy chain CDR3 of the novel coronavirus-specific antibody is shown in SEQ ID NO. 1-39.
  • a system for screening specific BCR/TCR comprising a sequencing unit, the sequencing unit is used to perform sequence determination on the BCR/TCR variable region of the lymphocytes of the immunized sample, so as to obtain sequencing data;
  • a sequence set establishment unit the sequence set establishment unit is used to detect all the BCR/TCR sequence quantities, and establish a corresponding sequence set according to the detection results;
  • a sequence alignment unit which aligns and deduplicates the sequence set established by the sample after immunization with the sequence set established by the control group, and obtains the corresponding characteristic BCR/TCR sequence set.
  • the system can use the above method to screen the characteristic BCR/TCR sequence set to obtain the specific BCR/TCR sequence.
  • the present invention compares a huge number of BCR/TCR sequences by comparing the big data of the immune map, which has higher specificity and accuracy, and can greatly improve the efficiency of screening and identification of monoclonal antibodies.
  • the present invention can use any sample containing lymphocytes, including but not limited to: peripheral blood, spleen tissue, tumor tissue, lymphocytes cultured in vitro. Samples can be from animals or from humans. It can be active immunization or from patients.
  • a large number of candidate BCRs/TCRs for complex antigens can be obtained at one time, and after relatively simple identification, they can be used for subsequent research and even clinical trials.
  • Figure 1 is a control diagram for the screening of the heavy chain sequence of the novel coronavirus characteristic antibody.
  • Example 1 Obtaining the characteristic antibody heavy chain sequence set in peripheral blood after novel coronavirus (COVID-19) infection by high-throughput sequencing
  • the lysed peripheral blood samples can be stored in -20°C or -80°C refrigerator, or sent by cold chain.
  • RNA precipitate Dissolve the RNA precipitate with 30 ⁇ L of DEPC/enzyme-free water. After a small amount of aliquots, make a mark and store in a -80°C refrigerator.
  • RNA electrophoresis Take 2 ⁇ L of RNA, use a micro UV spectrophotometer to detect the concentration and OD value, take 2 ⁇ L of RNA electrophoresis to detect the band, use 2% gel, DL2000Marker, and perform reverse transcription experiment immediately.
  • the detection result is that the total amount of RNA is >5 ⁇ g, the volume is >20 ⁇ L, and the OD260/280 ratio is above 1.8.
  • 2% agarose electrophoresis is detected, and three bands appear, which are 28sRNA, 18sRNA, and 5sRNA, of which 28S is 1 to 2 of the brightness of the 18S band. times, the 5S strip is the shallowest.
  • the reverse transcription reaction was performed using the TonkBio TM First chain cDNA synthesis kit.
  • the reaction system and reaction conditions are shown in Table 1:
  • BRTmix primers are shown in Table 2, the primer concentrations are mixed at 1:1:1, and the total concentration is 20 ⁇ M.
  • a specific amplification reaction was performed on the BCR gene of the cDNA, and the PCR enzyme used was Taq enzyme (Item No. TK01015) of Tongke Company, and the reaction system and conditions were shown in Table 3 and Table 4:
  • VHmix primer 20uM
  • JHmix(10uM) 1 ⁇ L 10 ⁇ Buffer
  • MgCl 2 25mM
  • dNTP(10mMeach) 0.8 ⁇ L Taq enzyme 1.2 ⁇ L ddH 2 O 10.9 ⁇ L total 20 ⁇ L
  • VHmix primers are 5'-end multiplex PCR primers.
  • the primer names and sequences are shown in Table 5. They are mixed at 1:1 and the total concentration is 20 ⁇ M.
  • JHmix primers are 3'-end multiplex PCR primers.
  • the names and sequences of the primers are shown in Table 6. They are mixed at 1:1 and the total concentration is 10uM.
  • the lowercase part is paired with the protection sequence of the second round of 3'-end primer barcode.
  • the second round of PCR reaction was carried out using Taq enzyme (product number TK01015) from Tongke Company.
  • the reaction system and reaction conditions are shown in Table 7 and Table 8:
  • the VHmix primer is the 5'-end multiplex PCR primer, that is, the VHmix in the first round of PCR.
  • JHxBCX is a primer at the 3' end.
  • the names and sequences of the primers are shown in Table 9. There are 10 primer sequences with different barcodes.
  • the primer sequences include barcode sequence (underlined part), barcode protection recognition sequence (lowercase part) and specific amplification sequence , a JHxBCX primer is selected for a sample, which corresponds to a unique barcode, so as to distinguish different samples according to the barcode sequence.
  • the amplification effect was detected by electrophoresis, and the target band was more than 400 bp.
  • Magnetic bead purification One sample can be purified after 1-2 tubes of the second round of PCR. Add an equal volume of DNA fragment separation and purification magnetic beads (BMW Catalog No. BMSX) to the PCR product, and elute with 30 ⁇ L of water. The concentration of the purified product was detected using a microspectrophotometer.
  • BMW Catalog No. BMSX DNA fragment separation and purification magnetic beads
  • kits including Ion Plus Fragment Library Kit (Cat. No. 4471252), Ion Xpress Barcode Adaptors 1-16Kit (Cat. No. 4471250), Agencourt AMPure XP (Cat. No. A63881), and the instruments include PCR instrument, Qubit quantifier and nucleic acid quantification reagents.
  • Ion chef-automatic template preparation instrument after installing the reagents and consumables, the experiment can be automatically performed, and the library plus ISP magnetic beads, enrichment, and loading chip work can be completed.
  • Reagents used include Ion 520/530 ExT- chefs-4rxns&4Init NEW-For 600bp (Cat. No. A30670), Ion 530 Chip Kit (Cat. No. A27764).
  • the FQ file for data analysis, and perform IgBLAST on the sequence.
  • the nucleic acid and amino acid sequences of the antibody heavy chain are determined to ensure the functionality of each sample as much as possible.
  • the total number of antibody heavy chain sequences should not be less than 30,000.
  • Example 2 Screening of the most potential new coronavirus antibody heavy chain sequences from the characteristic antibody heavy chain sequences in peripheral blood after new coronavirus infection
  • step 1 Sort the antibody heavy chain sequences screened in step 1 according to "the sum of the number of repetitions of any unique antibody heavy chain CDR3 sequence in all samples from patients with new coronavirus infection" from high to low, and select the top 100 or the top 10%. Since there are few sequences selected in step 1, this step is all reserved.
  • step 3 Screen the antibody heavy chain CDR3 sequence according to the characteristics of the antigen protein: Because the new coronavirus S protein receptor binding region (RBD) contains more positive charges, therefore, in the sequence selected in step 2, select the antibody heavy chain CDR3 sequence that contains more positive charges. A sequence of multiple (1 and more) net negative charges. In this step, a total of 39 sequences were selected, that is to say, they were the CDR3 sequences of the novel coronavirus-specific antibody, and their amino acid sequences were shown in SEQ ID NO. 1 to 39 in Table 10.
  • RBD new coronavirus S protein receptor binding region

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Abstract

L'invention concerne un procédé de criblage d'un BCR/TCR spécifique, et un système associé. Le procédé de criblage d'un BCR/TCR spécifique comprend les étapes suivantes : (1) extraction de l'ARNm des lymphocytes d'un échantillon immunisé à tester ; (2) réalisation d'un traitement de transcription inverse sur l'ARNm obtenu à l'étape (1), puis réalisation d'une amplification PCR multiplex sur un gène d'une région variable BCR/TCR, puis réalisation d'un séquençage à haut débit, et détermination d'une séquence d'acides nucléiques et d'acides aminés de la région variable BCR/TCR ; (3) comparaison de la séquence obtenue à partir du séquençage et d'un résultat de séquençage d'un lymphocyte BCR/TCR d'un échantillon n'ayant pas subi de stimulation antigénique, de manière à obtenir une séquence immunitaire spécifique correspondante. Dans la présente invention, la comparaison est effectuée au moyen des macrodonnées de l'atlas immunitaire, et un grand nombre de séquences BCR/TCR sont comparées. Ainsi, la présente solution est spécifique et précise, peut améliorer l'efficacité du criblage et de l'identification des anticorps et des récepteurs de lymphocytes T, et a de faibles coûts de criblage.
PCT/CN2022/080277 2021-03-15 2022-03-11 Procédé de criblage de bcr/tcr spécifiques, et système associé WO2022194032A1 (fr)

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CN113122617B (zh) * 2021-03-15 2023-07-14 成都益安博生物技术有限公司 一种筛选特异bcr/tcr的方法及其系统
CN114349847B (zh) * 2022-02-08 2023-07-21 深圳市因诺转化医学研究院 靶向新型冠状病毒rna依赖性rna聚合酶的特异性tcr

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