Genetic engneering of b cell receptors and uses thereof in antigen-induced antibody secretion

A method of genetically engineering a primary mammalian cell of the B lineage for expression of an engineered B cell receptor (BCR) and for antigen-induced secretion of an antibody of interest or antigen-binding fragment thereof or chimera thereof from said cell and or progeny thereof, said method comprising a step of

• contacting said primary B lineage cell with at least one nucleic acid cassette comprising at least one nucleic acid sequence of interest, or with any vector or vehicle comprising said cassette, wherein said nucleic acid sequence of interest comprises at least one nucleic acid sequence coding 5′ to 3′ for at least one variable domain of an immunoglobulin heavy chain (VH) or any binding fragment or chimera thereof and at least one variable domain of an immunoglobulin light chain (VL) or any binding fragment or chimera thereof, or at least one VL or any binding fragment or chimera thereof and at least one VH or any binding fragment or chimera thereof, of said antibody of interest, and at least one splice donor site (SD), wherein said nucleic acid cassette targets the insertion of said nucleic acid sequence of interest into a target genomic sequence within the immunoglobulin heavy chain (IgH) locus, downstream to the J region and upstream of at least one splice acceptor site of the constant domain of said heavy chain of said BCR, wherein said cassette is flanked on at least one of the 5′ and 3′ ends thereof by at least recognition sites for a site-specific integrase or a site-specific recombinase, wherein the insertion of said nucleic acid sequence of interest into the target genomic locus is mediated by at least one of a class switch recombination (CSR), said method further comprising a step of activating said cells, said activation catalyzed by activation induced cytidine deaminase (AID) and said activation occurring prior to said step of contacting the cell with the cassette;
• and wherein said method genetically engineers the primary cell of the B cell lineage to express an engineered BCR and to secrete the antibody of interest or antigen-binding fragment thereof by antigen-induced activation from said cell and or progeny thereof.

A method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathologic disorder in a mammalian subject, said method comprising steps of

• (i) transducing or transfecting a primary mammalian cell of the B lineage or population of cells thereof, with an at least one nucleic acid cassette, wherein said at least one cassette comprises at least one nucleic acid sequence of interest comprising a nucleic acid sequence coding for 5′ to 3′ at least one variable domain of an immunoglobulin heavy chain (VH) of an antibody of interest or any antigen-binding fragment or chimera thereof and at least one variable domain of an immunoglobulin light chain (VL) of an antibody of interest or any antigen-binding fragment or chimera thereof or at least one VL of an antibody of interest or any antigen-binding fragment or chimera thereof and at least one VH of an antibody of interest or any antigen-binding fragment or chimera thereof, and at least one splice donor site (SD), wherein said cassette targets the insertion of said nucleic acid sequence of interest into a target genomic sequence within the IgH locus, downstream to the J region of the variable domain and upstream of at least one splice acceptor site of a constant domain of an immunoglobulin heavy chain in said cell, wherein said cassette is flanked on at least one of the 5′ and 3′ ends thereof by at least recognition sites for a site-specific integrase or a site-specific recombinase, wherein the insertion of said nucleic acid sequence of interest into the target genomic locus is mediated by a class switch recombination,
• (ii) activating said cells, said activation catalyzed by activation induced cytidine deaminase (AID) and said activation occurring prior to said step of transducing or transfecting the cell with the cassette, and wherein said activation is in vitro comprising activation by at least one of Toll-like receptor (TLR)-mediated activation or cluster of differentiation 40 (CD40)-ligation;
• or said activation is in vivo in a mammalian subject in need, wherein said subject was previous immunized against said antigen or a fragment thereof, infected by an agent comprising said antigen or a fragment thereof, or is suffering a pathologic disease associated with said antigen or a fragment thereof;
• and
• (iii) administering to a subject an effective amount of said activated, transduced or transfected cells or a composition thereof;
• and
• wherein said method treats, prevents, ameliorates, inhibits, or delays the onset of a pathologic disorder in a mammalian subject.