scFv construct design and preparation process

IgM-induced signaling elicits cytotoxic response in macrophages and can be integrated to a CAR design

A. illustration of experimental setting. B. B16F10 tumor size (mm2) in mice following prophylactic immunization with MoDC pulsed with tumor cell coated with allogeneic IgG or IgM (n=4). C. Mean percentages of B16F10 melanoma cells stained for Annexin V/PI incubated with allogenic IgG and IgM following incubation with MoDC (n=5). D-E. Mean levels of Granzyme B and NO (D) and proinflammatory cytokines (E) in the supernatants of MoDC following overnight activation with IgG and IgM immune complexes. (n=5). F. Mean fluorescent intensity of MAPK enzymes in MoDC following activation for 20 min with IgG and IgM tumor immune complexes (n=5). G. Illustration representing CAR-macrophage design. H. Confocal microscopy images of HEK293FT cells 24 hours post transfection with CAR plasmids and membranous wasabi. I. Representative FACS analysis of HEK239FT cells 24 hours post transfection with CAR plasmids. Results are from one representative experiment out of at least three performed. Statistical significance was calculated using non-parametric t test (*** denote p<0.001, **** denote p<0.0001).

scFv are not expressed by myeloid cells.

A. Confocal microscopy images of DC 2.4 cells 24 hours post transfection with CAR plasmids and membranous wasabi. B. Representative FACS analysis of DC 2.4 cells 24 hours post transfection with CAR plasmids. C. Percentages of transfected cells 24 hours following transfection (n=4). D. Confocal microscopy images of THP-1 cells 72 hours post infection with CAR-C5α-mCherry and tdTomato plasmids. E. Percentages of transfected human cells 72 hours following transduction (n=4). F-G. Representative confocal microscopy (F) and mean percentages (G) of cells expressing chimeric molecules 24 hours after transfection. H-I. Representative confocal microscopy (H) and mean percentages of cells (I) expressing chimeric molecules 24 hours following transfection (n=4). Results are from one representative experiment out of at least three performed. Statistical significance was calculated using non-parametric t test (*** denote p<0.001, **** denote p<0.0001).

Both VH and VL domains prevent expression of ScFv in myeloid cells.

A. Confocal microscopy images of DC 2.4 cells 24 hours post transfection with αCD19-scFv GFP plasmid. B. Geometric mean of GFP positive cells 24hr post transfection with different αCD19- and TA99-ScFv GFP constructs in DC2.4. (n=3) C. Confocal microscopy images of HEK 293FT and DC 2.4 cells 24 hours post transfection with αCD19-variable light chain GFP plasmid. D. Confocal microscopy images of HEK 293FT and DC 2.4 cells 24 hours post transfection with αCD19-variable heavy chain GFP plasmid E. Mean percentages of cells expressing scFv fragments 24 h post transfection (n=). F. left: illustration of mutated variable light chain. right: confocal microscopy images of HEK 293FT and DC 2.4 cells 24 hours post transfection with αCD19-mutated (linear) variable light chain. G. Representative confocal images of HEK293FT and DC2.4 cells 24 hours post transfection with 1/3 fragments of αCD19-variable light chain GFP plasmid H. Mean percentages of GFP positive cells 24hr post transfection with different fragments of scFv-GFP in DC2.4 and RAW264.7 (n=4). Results are from one representative experiment out of at least three performed. Statistical significance was calculated using non-parametric t test (*** denote p<0.001, **** denote p<0.0001).

ScFv fragments induce ER stress in myeloid cells.

A. Confocal microscopy imaging of RAW 264.7 24hr post transfection with linear mRNA vectors translating to GFP and αCD19-scFv GFP. B. qPCR data showing relative mRNA levels in RAW 264.7 transfected with GFP, Fc receptor-GFP and αCD19-ScFv GFP. (n=3) C. Upper: illustration of plasmid subunits. Lower: confocal microscopy imaging of HEK293FT and RAW 264.7 24hr post transfection with T2A ribosomal skipping plasmid including ScFv. D. Upper: Illustration of plasmid subunits. Lower: confocal microscopy imaging of HEK293FT and RAW 264.7 24hr post transfection with plasmid containing no T2A. E. Confocal microscopy images of RAW264.7 cells at 6 hours and 24 hours post transfection with αCD19-ScFv GFP plasmid. F. Volcano plot showing differentially expressed proteins αCD19 ScFv GFP/GFP in DC 2.4 cells. H. Confocal microscopy images of DC2.4 stained with an ER stain, 24 hours post transfection with GFP, membranous TA99-ScFv GFP. I. Confocal microscopy images of DC2.4 stained for BiP 24 hours post transfection. Mean levels of phospho-JNK 6 hours following transfection. Results are from one representative experiment out of at least three performed. Statistical significance was calculated using non-parametric t test (*** denote p<0.001, **** denote p<0.0001).

FcγRI can provide a scaffold for incorporating IgM-induced signaling in myeloid cells and endows them with tumor cell-specific killing ability.

A. Illustration of chimeric Fcγ receptor design. B-C. Representative FACS plots (B) and mean percentages (C) of RAW264.7 cells expressing chimeric Fcγ receptors 24 h after transfection (n=3). D. Confocal microscopy images of RAW264.7 cells 24 hours post transfection with Fcγ receptors tagged with GFP and membrane tagged tdTomato. E. Mean percentages of BMDC 72 h post lentivirus transduction with Fcγ receptors (n=4). F. Confocal microscopy staining of GrB in RAW 264.7 cells co-cultured overnight with 4T1 cells expressing human HER2+. G. Mean counts of GrB in the synapse between transduced RAW264.7 cells and the tumor cells (n=18). H. IncuCyte analysis of human HER2+ 4T1 cells growth following incubation with transduced RAW 264.7 cells (n=6). I. Super-resolution microscopy of GFP-tagged chimeric FcγR and mCherry-tagged gamma chain. J. Confocal microscopy staining of GrB in RAW 264.7 cells co-cultured overnight with 4T1 cells expressing human HER2+. K. IncuCyte analysis of human HER2+ 4T1 cells growth following incubation with transduced RAW 264.7 cells (n=6). Results are from one representative experiment out of at least three performed. Statistical significance was calculated using non-parametric t test (*** denote p<0.001, **** denote p<0.0001).