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WO2021208035A1 - Procédés et réactifs pour la détection à haut débit d'une séquence d'acide nucléique d'un seul récepteur de surface de lymphocyte t - Google Patents

Procédés et réactifs pour la détection à haut débit d'une séquence d'acide nucléique d'un seul récepteur de surface de lymphocyte t Download PDF

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Publication number
WO2021208035A1
WO2021208035A1 PCT/CN2020/085185 CN2020085185W WO2021208035A1 WO 2021208035 A1 WO2021208035 A1 WO 2021208035A1 CN 2020085185 W CN2020085185 W CN 2020085185W WO 2021208035 A1 WO2021208035 A1 WO 2021208035A1
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sequence
tcr
primer
cell
cdna
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PCT/CN2020/085185
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English (en)
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Wenqi ZHU
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Singleron Nanjing Biotechnologies Ltd
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Singleron Nanjing Biotechnologies Ltd
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Priority to PCT/CN2020/085185 priority Critical patent/WO2021208035A1/fr
Priority to US17/996,195 priority patent/US20230193355A1/en
Priority to PCT/CN2021/087517 priority patent/WO2021209009A1/fr
Priority to EP21787533.5A priority patent/EP4136255A4/fr
Priority to CN202180047351.6A priority patent/CN115956115A/zh
Publication of WO2021208035A1 publication Critical patent/WO2021208035A1/fr
Anticipated expiration legal-status Critical
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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present disclosure involves methods and reagents for high-throughput detection of nucleic acid sequences of single cell T cell receptor.
  • T lymphocytes and T lymphocytes participate in the acquired immune responses [1] .
  • Human T cells develop in the thymus from progenitors originating in hematopoietic tissues. During their development, T cells acquire the ability to recognize foreign antigens and provide protection against many different types of pathogens. This functional flexibility is facilitated by the expression of highly polymorphic surface receptors called T cell receptors (TCRs) [2] .
  • TCRs highly polymorphic surface receptors
  • the diversity of T cell receptors (TCRs) , B cell receptors (BCRs) and secreted antibodies constitutes the core of a complex immune system and serves as a key defense component that protects the body from invasion by viren, bacteria and other foreign substances [3] .
  • TCR is a heterodimer-- ⁇ chain ( ⁇ 95%, TRA, TRB) or ⁇ chain ( ⁇ 5%) .
  • Each chain can be divided into variable and constant domains [4] .
  • Each peptide chain can be divided into variable region (V region) , constant region (C region) , transmembrane region and cytoplasmic region.
  • the variable region of ⁇ chain is encoded by V and J gene fragments.
  • the variable region of the ⁇ chain is encoded by three gene segments: V, D, and J.
  • V regions (V ⁇ , V ⁇ ) of the two peptide chains, ⁇ and ⁇ have three hypervariable regions: CDR1, CDR2, and CDR3, of which the CDR3 region (also called hypervariable region) has the largest variation, which directly determines the antigen binding specificity of TCR [5, 6] .
  • TCR profiles are extremely diverse. In humans, it is theoretically estimated that the diversity of TCR- ⁇ receptors exceeds 10 12 in the thymus, and the diversity directly determines the antigen binding specificity of TCR [7] .
  • these CDR3 sequences can be used as Biomarker representing the disease and can be found in peripheral blood; Research on autoimmune diseases such as rheumatoid arthritis, can identify potential autologous clones by high-throughput sequencing to quantify the T cell repertoire of peripheral blood of early or diagnosed rheumatoid arthritis, as a basis for the early diagnosis of medication. It can promote the development of vaccines for different populations by analyzing the effects of people of different ages after injection of vaccines. For tumor research, disease guidance can be monitored by comparing changes in the immune repertoire of patients before and after medication to prevent tumor recurrence.
  • RNA-seq measures the average expression level of tissue samples or cell populations, which makes the difference between cells likely masked by the average value, and cannot specifically describe the diversity of lymphocytes or clonotypes that constitute the immune response.
  • bulk RNA-seq cannot determine which TCRA and TCRB chains combine to form a specific TCR, which is essential for many functional and therapeutic applications [7] . Therefore, the establishment of a method for detecting the diversity of TCR at single cell level is particularly important for promoting the application of immune receptors sequencing in early clinical diagnosis, efficacy evaluation, and prognosis judgment.
  • Clontech generally relies on plate-or well-based microfluidics and is therefore limited in the number of cells that can be processed, typically 10–100. Additionally, a large number of sequencing reads are generally required to computationally reconstruct paired antigen receptors [13] . As such, the cost per cell is relatively high, estimated at $50–$100 USD [14] .
  • Chromium Single Cell V (D) J Reagent Kits launched by 10X Genomics has greatly improved the detection throughput compared to Clontech's products.
  • TCR Chromium Single Cell V
  • hydrogel beads containing cell barcode By encapsulating single cells and hydrogel beads containing cell barcode in individual droplets, TCR from thousands of single cells can be processed and then detected in parallel.
  • the disadvantages of Clontech are as follows: The mapping rate of TCR sequencing is relatively low, the Median UMI detection value of TCR a chain is relatively low resulting the Low detection rate of TCR a chain.
  • the purpose of the present disclosure is to provide a reagent and method for high-throughput detection of the TCR sequence at single cell level.
  • the probe and oligo-dT contain the same PCR handle sequence, so that TCR can be amplified by multiplex PCR.
  • the probe and oligo-dT can be combined with a oligonucleotide sequence that can act as cell barcode to distinguish each single cell from other cells, so that thousands or more of single cells can be analyzed in parallel.
  • This method can also be used in combination with a microfluidic system where each cell in a sample can be partitioned to individual micro-chambers. Single cells can be lyzed in the micro-chambers; mRNA and TCR sequences can be captured at the same time.
  • FIG. 1 Schematic diagram of the present disclosure.
  • Figure 2 Schematic diagram of the embodiment of the present disclosure where cell barcoding capture magnetic bead is used to capture mRNA and TCR sequence.
  • Figure 3 shows the amplified cDNA map.
  • FIG. 4 shows the TCR target enrichment 1 map.
  • Figure 5 shows the TCR target enrichment 2 map.
  • Figure 6 shows the TCR libray map.
  • probe binding to TCR sequence combine with oligo-dT to capture mRNA while improving the capture efficiency of TCR sequences.
  • the probe and polyT oligo contain the same PCR handle sequence, which can act as priming site for RT reactions and TCR Target enrichment reactions.
  • One embodiment of the present disclosure is to add probe binding to TCR sequence to the 3’ end of oligo-dT. This way one can capture and reverse transcribe both mRNA and TCR sequence captured by probe.
  • the resulting cDNA can be used as template to enrich TCR sequence by multiplex PCR.
  • unique cell barcodes in conjunction with the oligo-dT sequence cDNA molecules from the same single cell can be labeled and a group of single cells can be processed in parallel.
  • TCR sequences which can reveal information about T-cell ancestry and antigen specificity
  • Integrating these two types of information can allow one to comprehensively profile a given T cell.
  • GEXSCOPE Single Cell RNAseq Library Construction kit (Singleron Biotechnologies) was used to demonstrate the technical feasibility and the utility of the present disclosure in massively parallel single cell ncRNA sequencing. The experiment was conducted according to manufactuer’s instructions with modifications described below.
  • the primers on all beads comprise a common sequence used for PCR amplification, a bead-specific cell barcode, a unique 8 molecular identifier (UMI) , a oligo-dT sequence for capturing polyadenylated mRNAs and probe sequence annealing to TCR constant Region for capturing TCR mRNA.
  • UMI unique 8 molecular identifier
  • the sequence of the TCR constant region is as follows:
  • PBMC single cell suspension of PBMC was loaded onto the microchip to partition single cells into individual wells on the chip.
  • Cell barcoding magnetic beads were then loaded to the microchip and washed. Only one bead can fall into each well on the microchip based on the diameters of the beads and well (about 25um and 40um, respectively) .
  • the rest of cDNA were used to enrich TCR sequence.
  • Frist-round of enrichment Take 10ng cDNA as the template for the first round of TCR enrichment by multiplex nested PCR using QIAGEN Multiplex PCR kit.
  • TCR V region primer (TRV Reaction1) combined with the universal sequence (Target 1F) .
  • TCR V region primer (TRV Reaction1) including 38 TRA V regions and 36 TRB V region primers, total 74 primers.
  • each TCR V-region primer is 0.06 ⁇ M
  • Target 1F primer is 0.3 ⁇ M.
  • Second-round of enrichment a 10- ⁇ l aliquot of the first reaction was used as a template for second 50- ⁇ l PCR using QIAGEN Multiplex PCR kit;
  • TCR V region primer (TRV Reaction2) combined with the universal sequence (Target 2F)
  • TCR V region primer (TRV Reaction2) includes 36 TRA V region primers, 36 TRB V region primers, total 72 primers.
  • Amplification and library construction Take 20ng of the second-round enrichment products and use KAPA HiFi PCR kit for amplification and library construction by multiplex PCR
  • RNAseq library was sequenced on Illumina NovaSeq with PE150 mode and analyzed with scopeTools bioinformatics workflow (Singleron Biotechnologies) .
  • Figure 3 shows the amplified cDNA map.
  • FIG. 4 shows the TCR target enrichment 1 map.
  • Figure 5 shows the TCR target enrichment 2 map.
  • Figure 6 shows the TCR libray map.
  • the number of T cells annotated in the transcriptome data is consistent with the number of T cells detected in the TCR enrichment library.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un réactif et un procédé pour la détection à haut débit de la séquence TCR au niveau d'une seule cellule.
PCT/CN2020/085185 2020-04-16 2020-04-16 Procédés et réactifs pour la détection à haut débit d'une séquence d'acide nucléique d'un seul récepteur de surface de lymphocyte t Ceased WO2021208035A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
PCT/CN2020/085185 WO2021208035A1 (fr) 2020-04-16 2020-04-16 Procédés et réactifs pour la détection à haut débit d'une séquence d'acide nucléique d'un seul récepteur de surface de lymphocyte t
US17/996,195 US20230193355A1 (en) 2020-04-16 2021-04-15 Methods and compositions for high-throughput target sequencing in single cells
PCT/CN2021/087517 WO2021209009A1 (fr) 2020-04-16 2021-04-15 Procédés et compositions pour le séquençage de cible à haut débit dans des cellules uniques
EP21787533.5A EP4136255A4 (fr) 2020-04-16 2021-04-15 Procédés et compositions pour le séquençage de cible à haut débit dans des cellules uniques
CN202180047351.6A CN115956115A (zh) 2020-04-16 2021-04-15 用于单细胞高通量靶标测序的方法和组合物

Applications Claiming Priority (1)

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PCT/CN2020/085185 WO2021208035A1 (fr) 2020-04-16 2020-04-16 Procédés et réactifs pour la détection à haut débit d'une séquence d'acide nucléique d'un seul récepteur de surface de lymphocyte t

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114891779A (zh) * 2022-03-31 2022-08-12 立凌生物制药(苏州)有限公司 一种克隆tcr序列的检测方法及其应用
CN115029341A (zh) * 2022-05-23 2022-09-09 立凌生物制药(苏州)有限公司 一种快速克隆配对tcr序列检测方法及其应用
WO2023201235A3 (fr) * 2022-04-12 2024-01-18 10X Genomics, Inc. Compositions et procédés de génération et de caractérisation de molécules de liaison à un antigène recombinant

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019084055A1 (fr) * 2017-10-23 2019-05-02 Massachusetts Institute Of Technology Classification de variation génétique à partir de transcriptomes unicellulaires
CN110675914A (zh) * 2019-09-17 2020-01-10 佛山市第一人民医院（中山大学附属佛山医院） 一种筛选肿瘤特异性t细胞及tcr的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019084055A1 (fr) * 2017-10-23 2019-05-02 Massachusetts Institute Of Technology Classification de variation génétique à partir de transcriptomes unicellulaires
CN110675914A (zh) * 2019-09-17 2020-01-10 佛山市第一人民医院（中山大学附属佛山医院） 一种筛选肿瘤特异性t细胞及tcr的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MANDEEP SINGH , GHAMDAN AL-ERYANI , SHAUN CARSWELL , JAMES M. FERGUSON , JAMES BLACKBURN , KIRSTON BARTON , DANIEL RODEN , FABIO : "High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes", NATURE COMMUNICATIONS, vol. 10, no. 1, 3120, 1 December 2019 (2019-12-01), pages 1 - 13, XP055858175, DOI: 10.1038/s41467-019-11049-4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114891779A (zh) * 2022-03-31 2022-08-12 立凌生物制药(苏州)有限公司 一种克隆tcr序列的检测方法及其应用
WO2023201235A3 (fr) * 2022-04-12 2024-01-18 10X Genomics, Inc. Compositions et procédés de génération et de caractérisation de molécules de liaison à un antigène recombinant
CN115029341A (zh) * 2022-05-23 2022-09-09 立凌生物制药(苏州)有限公司 一种快速克隆配对tcr序列检测方法及其应用

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