Engineered multi-component system for identification and characterisation of T-cell receptors and T-cell antigens

A method for preparing an engineered antigen presenting cell p (eAPC-p), comprising:

• combining Component A with at least one Component C in the presence of one or more integration factors, wherein:
• Component A comprises an engineered antigen presenting cell (eAPC) that lacks endogenous surface expression of at least one family of target HLA class I, target HLA class II, or target non-HLA antigen-presenting complex (aAPX) and a target analyte antigenic molecule (aAM), and further comprises a single Component B,
• Component B is a genomic receiver site for integration of at least one open reading frame (ORF) encoding at least one aAPX, and Component B is a synthetic construct designed for recombinase mediated cassette exchange (RMCE),
• Component C is a genetic donor vector for delivery of (i) a single ORF encoding at least one of the target aAPX;
• or (ii) two or more ORFs encoding at least one of the target aAPX, and Component C is matched to Component B;
• and
• preparing the eAPC-p by selecting for one or more of:
• loss of one or more selection markers from the genomic receiver site;
• gain of a surface expression of at least one of the target aAPX;
• and
• gain of one or more selection markers at the genomic receiver site.

A method for preparing an engineered antigen presenting cell a (eAPC-a), comprising:

• combining Component A with at least one Component C in the presence of one or more integration factors, wherein:
• Component A comprises an engineered antigen presenting cell (eAPC) that lacks endogenous surface expression of at least one target HLA class I, target HLA class II, or target non-HLA family of antigen-presenting complex (aAPX) and a target analyte antigenic molecule (aAM), and further comprises a single Component B,
• Component B is a genomic receiver site for integration of at least one open reading frame (ORF) encoding at least one aAM, and Component B is a synthetic construct designed for recombinase mediated cassette exchange (RMCE),
• Component C is a genetic donor vector for delivery of (i) a single ORF encoding at least one of the target aAM;
• or (ii) two or more ORFs encoding at least one of the target aAM, and Component C is matched to Component B;
• and
• preparing the eAPC-a by selecting for one or more of:
• loss of one or more selection markers from the genomic receiver site;
• gain of a surface expression of at least one of the target aAM;
• or
• gain of one or more selection markers at the genomic receiver site.

A method for preparing an engineered antigen presenting cell pa (eAPC-pa), comprising:

• combining an engineered antigen presenting cell a (eAPC-a) with at least one Component C in the presence of one or more integration factors, wherein:
• the eAPC-a comprises at least one genomic receiver site for integration of at least one open reading frame (ORF) encoding at least one aAPX and/or aAM,
• at least one genomic receiver site is a synthetic construct designed for recombinase mediated cassette exchange (RMCE),
• at least one ORF encoding at least one of a target aAM is integrated into at least one genomic receiver site,
• Component C is a genetic donor vector for delivery of (i) a single ORF encoding at least one of a target aAPX;
• or (ii) two or more ORFs encoding at least one of the target aAPX, and Component C is matched to at least one genomic receiver site;
• and
• preparing the eAPC-pa by selecting for one or more of:
• loss of one or more selection markers from the genomic receiver site;
• gain of a surface expression of at least one of the target aAPX or a complex comprising at least one of the target aAPX and the target aAM (aAPX:AM);
• or
• gain of one or more selection markers at the genomic receiver site.

A method for preparing an engineered antigen presenting cell pa (eAPC-pa), comprising:

• combining an engineered antigen presenting cell p (eAPC-p) with at least one Component C in the presence of one or more integration factors, wherein:
• the eAPC-a comprises at least one genomic receiver site for integration of at least one open reading frame (ORF) encoding at least one aAPX and/or aAM,
• at least one genomic receiver site is a synthetic construct designed for recombinase mediated cassette exchange (RMCE),
• at least one ORF encoding at least one of a target aAPX is integrated into at least one genomic receiver site,
• Component C is a genetic donor vector for delivery of (i) a single ORF encoding at least one of a target aAM, or (ii) two or more ORFs encoding at least one of the target aAM, and Component C is matched to at least one genomic receiver site;
• and
• preparing the eAPC-pa by selecting for one or more of:
• loss of one or more selection markers from the genomic receiver site;
• gain of a surface expression of at least one of the target aAM or a complex comprising at least one of the target aAPX and the target aAM (aAPX:AM);
• or
• gain of one or more selection markers at the genomic receiver site.

An analytical device for characterization of the specificity of an expressed analyte antigen to an analyte T cell receptor (TCR), comprising an engineered antigen-presenting cell (eAPC), wherein:

• the eAPC is an engineered cell that lacks endogenous surface expression of at least one family of target HLA class I, target HLA class II, or target non-HLA antigen-presenting complex (aAPX) and a target analyte antigenic molecule (aAM), and further comprises a Component B and Component C, and optionally comprises Component D and Component E,
• Component B and Component D are genomic receiver sites for integration of at least one open reading frame (ORF) encoding at least one aAPX and/or aAM,
• Component B and Component D are synthetic constructs designed for recombinase mediated cassette exchange (RMCE);
• and
• Component C and Component E are genetic donor vectors for delivery of (i) a single ORF encoding at least one of the target aAPX, the target aAM, and/or cargo molecule (CM);
• or (ii) two or more ORFs encoding at least one of the target aAPX, target aAM, and/or CM,
• Component C is matched to recombines with Component Band the ORF(s) of Component C is integrated into Component B,
• Component E is matched to recombines with Component D and the ORF(s) of Component E is integrated into Component D,
• the expressed analyte antigen is expressed on the surface of the eAPC, and
• the expressed analyte antigen is selected from one or more of the target aAM, the target aAPX, a complex comprising the target aAM and the target aAPX (aAPX:aAM), a complex comprising the target aAPX and the CM (aAPX:CM), or an affinity reagent.

An analytical device for characterization of the affinity of an expressed analyte antigen to an analyte TCR, comprising an engineered antigen-presenting cell (eAPC), wherein:

• the eAPC is an engineered cell that lacks endogenous surface expression of at least one target HLA class I, target HLA class II, or target non-HLA family of antigen-presenting complex (aAPX) and a target analyte antigenic molecule (aAM), and further comprises a Component B and Component C, and optionally comprises Component D and Component E,
• Component B and Component D are genomic receiver sites for integration of at least one open reading frame (ORF) encoding at least one aAPX and/or aAM,
• Component B and Component D are synthetic constructs designed for recombinase mediated cassette exchange (RMCE);
• and
• Component C and Component E are genetic donor vectors for delivery of (i) a single ORF encoding at least one of the target aAPX, the target aAM, and/or cargo molecule (CM);
• or (ii) two or more ORFs encoding at least one of the target aAPX, the target aAM, and/or CM,
• Component C is matched to recombines with Component Band the ORF(s) of Component C is integrated into Component B,
• Component E is matched to recombines with Component D and the ORF(s) of Component E is integrated into Component D,
• the expressed analyte antigen is expressed on the surface of the eAPC, and
• the expressed analyte antigen is selected from one or more of the target aAM, the target aAPX, a complex comprising the target aAM and the target aAPX (aAPX:aAM), a complex comprising an the target aAPX and the CM (aAPX:CM), or an affinity reagent.

An analytical device for characterization of a signal response of an expressed analyte antigen to an analyte TCR, comprising an engineered antigen-presenting cell (eAPC), wherein:

• the eAPC is an engineered cell that lacks endogenous surface expression of at least one family of target HLA class I, target HLA class II, or target non-HLA antigen-presenting complex (aAPX) and the target analyte antigenic molecule (aAM), and further comprises a Component B and Component C, and optionally comprises Component D and Component E,
• Component B and Component D are genomic receiver sites for integration of at least one open reading frame (ORF) encoding at least one aAPX and/or aAM,
• Component B and Component D are synthetic constructs designed for recombinase mediated cassette exchange (RMCE);
• and
• Component C and Component E are genetic donor vectors for delivery of (i) a single ORF encoding at least one of the target aAPX, the target aAM, and/or cargo molecule (CM);
• or (ii) two or more ORFs encoding at least one of the target aAPX, the target aAM, and/or the CM,
• Component C is matched to recombines with Component Band the ORF(s) of Component C is integrated into Component B,
• Component E is matched to recombines with Component D and the ORF(s) of Component E is integrated into Component D,
• the expressed analyte antigen is expressed on the surface of the eAPC, and
• the expressed analyte antigen is selected from one or more of the target aAM, the target aAPX, a complex comprising the target aAM and the target aAPX (aAPX:aAM), a complex comprising the target aAPX and CM (aAPX:CM), or an affinity reagent.