EP-4737576-A1 - CONSTRUCTION AND USE OF MOUSE MODEL FOR EFFICIENTLY OBTAINING TUMOR ANTIGEN-SPECIFIC B CELLS

Abstract

The present invention relates to the field of tumor immunology, and particularly relates to a gene editing-based animal model, for efficiently and rapidly screening for tumor antigen-specific B cells, obtaining antibody information, and providing a new target for clinical personalized treatment.

Inventors

• ZHAO, Tongbiao
• ZHU, YINGJIE
• ZHANG, LIN
• CHENG, Chenxi
• CAO, Jiani
• LI, XIAOYAN

Assignees

• Beijing Institute for Stem Cell and Regenerative Medicine

Dates

Publication Date

20260506

Application Date

20230727

Claims (11)

1. A method for constructing an animal model, wherein the method comprises: knocking in a synthetic receptor gene into the B cell activation gene Nur77 and knocking in a screening and detection gene into the conserved sequence of gene Rosa26 of genome.
2. The method of claim 1, wherein the synthetic receptor comprises a leader sequence, a tag sequence, an extracellular receptor sequence, a transmembrane region, and an intracellular activation reporter.
3. The method of claim 1, wherein the screening and detection gene comprises a binding domain, a promoter, and a marker gene.
4. The method of claim 1, further comprising: adding cleavage sites before and after synthetic receptor gene SynNotch, and then inserting the synthetic receptor gene SynNotch and the cleavage sites between upstream and downstream homologous arms before stop codon of the gene Nur77; and adding cleavage sites before and after screening and detection gene mCherry, and then inserting the screening and detection gene mCherry, and cleavage sites between upstream and downstream homologous arms of intron of gene Rosa26.
5. The method of any one of claims 1-4, wherein the animal model is a mouse, a rat, a guinea pig, a hamster, a gerbil, a cotton rat, a pig, a cow, sheep, a rabbit, a cat, a dog, a rhesus monkey, an chimpanzee, a baboon, a marmoset, a cynomolgus monkey, or an alpaca, preferably a mouse.
6. The method of any one of claims 1-4, the method further comprising: 1) construction of transgenic vectors: adding cleavage sites before and after SynNotch receptor expression sequence, inserting the SynNotch receptor expression sequence and the cleavage sites between upstream and downstream homologous arms before stop codon of the gene Nur77, and then ligating the gene Nur77, inserted with the SynNotch receptor expression sequence and the cleavage sites, into plasmid pBluescript II SK to obtain a transgenic vector pBluescript II SK-Nur77SynNotch; adding cleavage sites before and after mCherry selection sequence, inserting the mCherry selection sequence and the cleavage sites between the upstream and downstream homologous arms of the intron of the Rosa26 gene, and then ligating the Rosa26 gene, inserted with the mCherry selection sequence and the cleavage sites, into plasmid pBluescript II SK to obtain a transgenic vector pBluescript II SK-Rosa26mCherry; constructing sgRNA-knockout plasmids for both the SynNotch and mCherry sequences, respectively; 2) acquisition of gene-edited animal model: combining the transgenic vector pBluescript II SK-Nur77SynNotch and the sgRNA-knockout plasmid for SynNotch, and combining the pBluescript II SK-Rosa26mCherry plasmid and the sgRNA-knockout plasmid for mCherry to obtain combinations, and introducing the combinations into a fertilized animal ovum via microinjection, respectively to obtain a F0 generation Nur77 SynNotch - or Rosa26 mCherry -knockin animal; and further carrying out crossing to obtain a homozygous Nur77 SynNotch Rosa26 mCherry double knock-in animal model.
7. An animal model obtained according to the method of any one of claims 1-6.
8. A method for obtaining a tumor antigen-specific B cell, wherein the method comprises: immunizing the animal model obtained by any one of claims 1-6 or the animal model of claim 7 with a tumor cell, and sorting a tumor antigen-specific CD19+IgD-mCherry+B cell.
9. A tumor antigen-specific B cell obtained according to the method of claim 8.
10. Use of the tumor antigen-specific B cell of claim 9 in screening of a tumor antigen-specific antibody.
11. A transgenic vector combination for use in the preparation of an animal model, comprising: transgenic vector 1, obtained by a construction method comprising: adding cleavage sites before and after SynNotch receptor expression sequence, inserting the SynNotch receptor expression sequence and the cleavage sites between upstream and downstream homologous arms before stop codon of gene Nur77, and then ligating same into a plasmid to obtain the transgenic vector 1; and transgenic vector 2, obtained by a construction method comprising: adding cleavage sites before and after mCherry selection sequence, inserting the mCherry selection sequence and the cleavage sites between upstream and downstream homologous arms of intron of gene Rosa26, and then ligating same into a plasmid to obtain the transgenic vector 2.

Description

TECHNICAL FIELD The present disclosure belongs to the field of tumor immunotherapy and relates to a gene editing-based animal model, which is used for rapidly and efficiently sorting tumor antigen-specific B cells; further screening of the sorted B cells enables the acquisition of tumor immunotherapy antibodies or engineered therapeutic cells. In particular, the present disclosure relates to a method for preparing a transgenic C57BL/6 mouse model having a Nur77SynNotchRosa26mCherry dual-signal sensing system capable of labeling B cells specifically activated by tumor antigens and the use thereof. BACKGROUND Cancer poses a serious threat to human life and health worldwide. In recent years, tumor immunotherapy has achieved significant outcomes in clinical treatment, and was ranked by Science as the first of the top ten scientific breakthroughs in 2013. Chimeric Antigen Receptor T (CAR-T) cell immunotherapy targeting CD19 in particular demonstrated significant remission rates in the clinical treatment of hematological tumors. However, the lack of tumor-specific antigens has severely restricted the further application of tumor immunotherapy in the clinical treatment of solid tumors due to off-target toxic and side effects. Achieving precise recognition of tumor cells has become one of the important strategies for optimizing tumor immunotherapy at present. The rapid development of high-throughput sequencing technology has made it possible to conduct in-depth genomic and transcriptomic analyses of tumor tissues. These analyses facilitate the further characterization and classification of tumors in clinical setting and may also enable the screening of tumor-specific antigens. Nevertheless, firstly, the complexity and heterogeneity of solid tumor tissues render the screening of tumor antigen targets challenging; secondly, comprehensive genomic profiling of solid tumor tissues may not directly yield antibody sequences applicable to corresponding immunotherapies. These limit the clinical advancement of tumor immunotherapy. The immune system's surveillance function ensures immune homeostasis in the organism. B cells may produce specific antibodies against specific antigen epitopes, and such specific recognition is reflected in the unique variable region sequences of B cell receptors. In recent years, an increasing number of studies have revealed the important role of tertiary lymphoid structures formed with the participation of B cells in anti-tumor processes. Currently, the acquisition of most antigen-specific monoclonal antibodies (mAbs) still relies on conventional hybridoma technology and phage display technology. With the rapid development of single-cell sequencing technology, mAb sequences that recognize specific protein antigens may be rapidly and efficiently obtained by sorting and sequencing B cells that bind to specific modified antigens. However, the method for capturing antigen-specific B cells relies on the in vitro expression, purification and modification of known protein antigens, and may not be applied to the screening of B cells that recognize unknown antigens (uAgs) on the surface of tumor cell membranes. Therefore, developing a method for the rapid identification and acquisition of tumor antigen-specific B cells is one of the important strategies to accelerate the clinical application of antibody-mediated tumor immunotherapy. Nur77 (NR4A1), as an orphan nuclear receptor, may be rapidly upregulated following induction by T/B cell antigen receptor (TCR/BCR) activation signals. Additionally, Nur77 has been reported to serve as a reporter gene for specific TCR or BCR activation signals. The SynNotch receptor is a customizable molecular sensor developed by the Lim laboratory in 2016, which may achieve the programming of cells. Cells may recognize a specific antigen structure via the SynNotch receptor and execute a series of pre-designed responses. Through combinatorial expression applications, cells may be engineered to recognize multiple target antigens and their output responses may be comprehensively programmed. This allows the SynNotch system in the T cells to be activated by the first type of antigen on the surface of tumor cell membranes, which in turn activates the transcription of CAR that recognizes the second type of antigen on the surface of tumor cell membranes. Ultimately, the CAR-T cells may only be activated when both antigens are expressed on tumor cells, thereby enabling precise recognition and killing of tumor cells and reducing damage to normal tissue cells. To capture tumor antigen-specific B cells, we designed a Nur77SynNotchRosa26mCherry dual-signal sensing system. When the B cell receptor (BCR) recognizes and is activated by the unknown antigen (uAg) on the surface of tumor cell membranes, the orphan nuclear receptor Nur77 is upregulated and drives the localized expression of the SynNotch receptor on the surface of B cell membranes. After the nanobody of the SynNotch receptor outs