Materials and methods for engineering cells and uses thereof in immuno-oncology

A method for producing an engineered human T cell, the method comprising

• delivering to a human T cell:
• (a) a RNA-guided nuclease;
• (b) a guide RNA (gRNA) targeting a site in a T cell receptor alpha chain constant region (TRAC) gene (TRAC gRNA);
• (c) a gRNA targeting a site in a beta-2-microglobulin (B2M) gene;
• and
• (d) a vector comprising a nucleic acid comprising a nucleotide sequence that comprises (i) a first segment that is homologous to the TRAC gene locus left to the site targeted by the TRAC gRNA, (ii) a second segment comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), and (iii) a third segment that is homologous to the TRAC gene locus right to the site targeted by the TRAC gRNA,
• wherein the CAR comprises (i) an ectodomain that comprises an anti-CD19 antibody single-chain variable fragment (scFv), wherein the anti-CD19 scFv comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
• 1595, and a variable light chain comprising the amino acid sequence of SEQ ID NO:
• 1596, (ii) a CD8 transmembrane domain, and (iii) an endodomain that comprises a CD28 or 41BB co-stimulatory domain and optionally a CD3z co-stimulatory domain;
• wherein the gRNA targeting the TRAC gene comprises a nucleotide sequence, which targets the nucleotide sequence of SEQ ID NO:
• 76;
• and
• wherein the gRNA targeting the B2M gene comprises a nucleotide sequence, which targets the nucleotide sequence of SEQ ID NO:
• 417;
• thereby producing an engineered human T cell.