Application of multimer in detection and preparation of car-expressing cell

A method for specific expansion of chimeric antigen receptor expressing cells, the method comprises the following steps:

• supplementing an expansion reagent to a cultured CAR-T cell population;
• and rendering the cultured CAR-T cell population to expand to meet a preset standard of therapy;
• wherein the expansion reagent comprises a complex of antigens and a multimer;
• the antigen, is expressed on a target cell surface, and is specifically recognized by a chimeric antigen receptor;
• the multimer, comprising:
• a scaffold protein, which is comprised of four protein subunits;
• a scaffold protein is composed of functional protein subunits;
• or a scaffold protein is composed of a mixture of functional and dysfunctional protein subunits;
• the functional protein subunit is derived from a streptavidin and has no biotin-binding activity;
• the scaffold protein has at least one functional protein subunit;
• the functional protein subunit's C-terminus has a cysteine and a biotin tag;
• the amino acid sequence of the functional protein subunit is set forth in SEQ ID NO:
• 1 or SEQ ID NO:2;
• the number of the dysfunctional protein subunit is not greater than 2, and the amino acid sequence is set forth in SEQ ID NO:
• 3;
• a streptavidin, binding to the biotin tag of the scaffold protein, wherein the streptavidin is used to bind 1˜3 of the antigens via biotin;
• or the expansion reagent comprises a streptavidin;
• the streptavidin binds to 2 to 4 antigens via biotin;
• the antigen, is expressed on a target cell surface and is specifically recognized by a chimeric antigen receptor.

A method for detection of chimeric antigen receptor expression, the method comprises the following steps:

• using a detection reagent to stain a chimeric antigen receptor, and an expression of the chimeric antigen receptor is measured with single-cell sequencing;
• wherein the detection reagent is an antigen-multimer which is labeled with oligo nucleotide, the antigen-multimer comprises a complex of antigens and a multimer;
• the antigen, is expressed on a target cell surface, and is specifically recognized by a chimeric antigen receptor;
• the multimer, comprising:
• a scaffold protein, which is comprised of four protein subunits;
• a scaffold protein is composed of functional protein subunits;
• or a scaffold protein is composed of a mixture of functional and dysfunctional protein subunits;
• the functional protein subunit is derived from a streptavidin and has no biotin-binding activity;
• the scaffold protein has at least one functional protein subunit, the functional protein subunit's C-terminus has a cysteine and a biotin tag;
• the amino acid sequence of the functional protein subunit is set forth in SEQ ID NO:1 or SEQ ID NO:2;
• the number of the dysfunctional protein subunit is not greater than 2, and the amino acid sequence is set forth in SEQ ID NO:3;
• a streptavidin, binding to the biotin tag of the scaffold protein, wherein the streptavidin is used to bind 1˜3 of the antigens via biotin;
• the streptavidin has detectable labels;
• or the detection reagent comprises a streptavidin;
• the streptavidin is unlabeled, fluorescence-labeled, or oligo nucleotide-labeled;
• the streptavidin binds to 2 to 4 antigens via biotin;
• the antigen, is expressed at a surface of a target cell, and is specifically recognized by a chimeric antigen receptor.