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 setting1. (B) Bl6Fl0 tumor size (mm2) in mice following prophylactic immunization with MoDC pulsed with tumor cells coated with allogeneic IgG or IgM (n=4). (C) Mean percentages ofB16Fl0 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 Band NO (D) and proinflammatory cytokines (E) in the supernatants ofMoDC following overnight activation with IgG and IgM immune complexes. (n=5). (F) Mean fluorescent intensity (MFI) of MAPK enzymes in MoDC following activation for 20 min with IgG and IgM tumor immune complexes (n=5). (G) Illustration representing CAR-macrophage design1. (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). 1Illustrations were created using

scFvis not expressed by myeloid cells.

(A) Confocal microscopy images of DC 2.4 cells 24 hours post-transfec­ tion with CAR plasmids and membranous wasabi. (B) Representative FACS analysis of DC 2.4 cells 24 hours post-trans­ fection with CAR plasmids. (C) Percentages of transfected cells 24 hours following transfection (n=4). (D) Confocal microscopy images ofTHP-1 cells 72 hours post lentiviral infection with CAR-C5u-mCherryand tdTomato plasmids. (E) Percentages of transfected human cell lines 72 hours following transduction (n=4). (F-G) Representative confocal micros­ copy (F)1 and mean percentages (G) of cells expressing chimeric molecules 24 hours after transfection. (H-1) Representa­ tive confocal microscopy (H)1 and mean percentages of cells (I) expressing chimeric molecules 24 hours following trans­ fection (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). 11llustrations were created using BioRender.com

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

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 uCD19-scFv GFP plasmid.1 (B) Geometric mean of GFP positive cells 24hr post transfection with different uCD19- 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 uCD19-variable light chain GFP plasmid.1 (D) Confocal microscopy images of HEK 293FT and DC 2.4 cells 24 hours post transfection with uCDl 9-variable heavy chain GFP plasmid1 (E) Mean percentages of cells expressing scFv fragments 24 h post transfection (n=). (F) left: illustra­ tion of mutated variable light chain. right: confocal microscopy images of HEK 293FT and DC 2.4 cells 24 hours post transfection with uCD19-mutated (linear) variable light chain. (G) Representative confocal images of HEK293FT and DC2.4 cells 24 hours post transfection with 1/3 fragments of uCD19-variable light chain GFP plasmid (H) Mean percent­ ages 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). 1Illustrations were created using BioRender.com

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

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 uCD19-scFv GFP. (B) qPCR data showing relative mRNA levels in RAW 264.7 transfected with GFP, Fe receptor-GFP, and uCD19-ScFv GFP. (n=3) (C) Upper: illustration of plasmid subunits. Lower: confocal microscopy imaging ofHEK293FT and RAW 264.7 24hr post-transfectionwith T2A ribosomal skipping plasmidincluding ScFv. (D) Upper: Illustration of plasmid subunits. Lower: confocal microscopy imag­ ing 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 uCDl 9-ScFv GFP plasmid. (F) Volcano plot showing differentially expressed proteins uCD19 ScFv GFP/GFP in DC 2.4 cells. (G) Confocal microscopy images of DC2.4 stained with an ER stain, 24 hours post-transfection with GFP, membranous TA99-ScFv GFP. (H) Confocal micros­ copy images ofDC2.4 stained for BiP 24 hours post-transfection. (I) Mean levels of phospho-JNK 6 hours following trans­ fection. (J) Percentage of cells expressing GFP or TRP-TCRl 24 hours following transfection. Results are from one repre­ sentative experiment out of at least three performed. Statistical significance was calculated using non-parametric t-test (***

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

(A) Illustration of chimeric Fey receptor design.1 (B-C) Representative FACS plots (B) and mean percentages (C) of RAW264.7 cells expressing chimeric Fcyreceptors 24 h after transfection (n=3). (D) Confocal microscopy images of RAW264.7 cells 24 hours post transfection with Fey receptors tagged with GFP and membrane tagged tdTomato. (E) Mean percentages of BMDC 72 h post lentivirus transduction with Fey receptors (n=4). (F) Confocal microscopy staining ofGrB in RAW 264.7 cells co-cultured overnight with 4Tl cells expressing human HERZ+. (G) Mean counts of GrB in the synapse between transduced RAW264.7 cells and the tumor cells (n=18). (H) IncuCyte analysis of human HERZ+ 4Tl cells growth following incubation with transduced RAW 264.7 cells (n=6). (I) Super-resolution micros­ copy of GFP-tagged chimeric FcyR and mCherry-tagged gamma chain. (J) Confocal microscopy staining of GrB in RAW 264.7 cells co-cultured overnight with 4Tl cells expressing human HERZ+. (K) IncuCyte analysis of human HERZ+ 4Tl cells growth following incubation with transduced RAW 264.7 cells (n=6). (L) mean percentages of CD3+ out of CD45+ cells in B16F10 tumors from day 26. (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<0.01, **** denote p<0.0001). 1Illustrations