Cell banks of diverse antibody-producing cells and uses thereof
A cell and antibody technology, applied in the direction of genetically modified cells, cells modified by introducing foreign genetic material, microbial libraries, etc., can solve the problems of unsatisfactory pharmaceutical research and development, low efficiency, and restrictions on antigen types
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Embodiment 1
[0141] Example 1, Construction of a Ramos Cell-Based Diversified Recombinant Antibody Heavy Chain Cell Bank
[0142] Antibody diversity consists of a combination of antibody gene recombination in pre-B cells and somatic mutation in mature B cells. Millions of years of evolution have built up a basic antibody repertoire in the genome that can respond to different antigens, and these basic antibody repertoires can further generate high-affinity antibodies through somatic mutation. Although the natural Ramos cell antibody heavy chain genes are only 4-34, genetic recombination technology can bring any other genome-encompassed heavy chain antibody genes into Ramos cells. The following is an in situ knock-in experiment using the 1-69 gene as an example. Similar methods can be used to establish Ramos cells starting with any genomic antibody sequence as the initial starting point for the subsequent antibody evolution screening process. There are various gene in situ knock-in methods ...
Embodiment 2
[0155] Example 2. Spontaneous somatic mutation of natural Ramos cell antibody heavy chain gene to generate antigen-specific antibody (specific experimental method reagent description is similar to (Nature Biotechnology 20, 1129-1134 (2002)):
[0156] In this example, the streptavidin model antigen is used as an example, and the antibody gene with high affinity for the model antigen was successfully evolved from the antibody gene with no affinity for the model antigen initially through a series of point mutations by Ramos cells, indicating that the Cell lines have the cellular and molecular biological basis for the in vitro evolution of human antibodies.
[0157] experiment procedure:
[0158] 1. Take 100,000 Ramos cells for expansion, inoculate them into 6-well plates, T-25, and finally culture in T-175 culture flasks;
[0159] 2. When the cell concentration reaches 1.5 million per ml, collect all about 50-100 ml of the culture system;
[0160] 3. Contact and label 75 millio...
Embodiment 3
[0167] Example 3, using the technical solution disclosed in US20180179516A1 to improve the evolution speed of Ramos cell antibody gene diversity, the production speed and mutation rate of antigen-specific antibodies
[0168] Exogenous genes or shRNAs were delivered into Ramos cells by lentivirus-mediated transduction using electroporation. For the construction of the foreign gene, the pCDH vector was used, and the gene was cloned into the pCDH vector by inserting the PCR product of the foreign gene into multiple cloning sites in the vector. The shRNA construct of human Spt5 is as in (A source of the single-stranded DNA substrate for activation-induced deaminase during somatichypermutation. Wang X, Fan M, Kalis S, Wei L, Scharff M D. Nat Commun. 2014 Jun. 13; 5: 4137.) described in. For the exogenous expression of Tat family members and their variants, lentiviral particles were used to introduce gene fragments encoding the corresponding proteins into Ramos reporter cells. eGF...
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