Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Timeline of T cell knock-in electroporation workflow, knock-in strategy and designs for BCMA-CAR (1.6 kb integration) across the series of DNA HDR template formats tested. (b) Comparison of BCMA-CAR HDRTs at concentrations 5nM-160nM or 0-100E3 MOI in terms of knock-in efficiency, (c) live cell count per 1e6 edited cells, and (d) knock-in cell count per 1e6 edited cells measured 7 days post electroporation. (e) Knock-in of a logic-gated synNotch circuit (5.6 kb integration) using linear ssDNA + tCTS, circular cssDNA + CTS, and nanoplasmid + CTS templates with corresponding knock-in efficiency, live cell count and knock-in cell count 7 days post electroporation using Cas9 mRNA. Circular cssDNA was produced and provided by Kano Therapeutics. (f) Knock-in strategy and designs for a logic-gated synNotch circuit (5.6 kb integration) at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation using Cas9 RNP or Cas9 mRNA. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. CTS, Cas9 target site. RNP, ribonucleoprotein. MOI, multiplicity of infection.

Article Snippet: Knockout and knock-in efficiency were evaluated by staining for the TCR with an anti-TCRα/β antibody (Miltenyi Biotec) and staining for the CAR with recombinant BCMA protein (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems BCA-H82E4_200ug).

Techniques: Knock-In, Electroporation, Comparison, Cell Characterization, Produced, Infection

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Knock-in strategy and designs for BCMA-CAR (1.6 kb integration) at concentrations 5nM-160nM with corresponding (b) knock-in efficiency, (c) live cell count, and (d) knock-in cell count 7 days post electroporation for the following templates respectively: dsDNA, dsDNA + tCTS, ssDNA, ssDNA + tCTS, circular ssDNA + CTS, circular ssDNA + mutated v1 CTS v1, circular ssDNA + mutated v2 CTS. Circular cssDNA was produced and provided by Touchlight. (e) Knock-in strategy and designs for CAR-CARD-11 (3.5 kb integration) using linear ssDNA + tCTS template or a nanoplasmid + CTS template at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation.

Article Snippet: Knockout and knock-in efficiency were evaluated by staining for the TCR with an anti-TCRα/β antibody (Miltenyi Biotec) and staining for the CAR with recombinant BCMA protein (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems BCA-H82E4_200ug).

Techniques: Knock-In, Cell Characterization, Electroporation, Produced

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Designs for BCMA-CAR (1.6 kb integration) HDRTs variants tested and corresponding template copies per genome measured at 4 hours and 3 days post electroporation (b) and half-life measured by qPCR. (c) Template copies per genome measured over time at 4 hours, 2 days, 3 days, 5 days, 7 days, and 12 days with R2 and half-life values corresponding to each donor. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. tCTS, truncated Cas9 target site. CTS, Cas9 target site. NP, nanoplasmid. cssDNA, circular cssDNA.

Article Snippet: Knockout and knock-in efficiency were evaluated by staining for the TCR with an anti-TCRα/β antibody (Miltenyi Biotec) and staining for the CAR with recombinant BCMA protein (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems BCA-H82E4_200ug).

Techniques: Electroporation

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Timeline of T cell knock-in electroporation workflow, knock-in strategy and designs for BCMA-CAR (1.6 kb integration) across the series of DNA HDR template formats tested. (b) Comparison of BCMA-CAR HDRTs at concentrations 5nM-160nM or 0-100E3 MOI in terms of knock-in efficiency, (c) live cell count per 1e6 edited cells, and (d) knock-in cell count per 1e6 edited cells measured 7 days post electroporation. (e) Knock-in of a logic-gated synNotch circuit (5.6 kb integration) using linear ssDNA + tCTS, circular cssDNA + CTS, and nanoplasmid + CTS templates with corresponding knock-in efficiency, live cell count and knock-in cell count 7 days post electroporation using Cas9 mRNA. Circular cssDNA was produced and provided by Kano Therapeutics. (f) Knock-in strategy and designs for a logic-gated synNotch circuit (5.6 kb integration) at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation using Cas9 RNP or Cas9 mRNA. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. CTS, Cas9 target site. RNP, ribonucleoprotein. MOI, multiplicity of infection.

Article Snippet: To stain for BCMA-CAR, cells were incubated with 0.3 μg BCMA recombinant protein conjugated to biotin (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems, BCA-H82E4_200ug) for 15 minutes at room temperature prior to surface cell staining.

Techniques: Knock-In, Electroporation, Comparison, Cell Characterization, Produced, Infection

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Knock-in strategy and designs for BCMA-CAR (1.6 kb integration) at concentrations 5nM-160nM with corresponding (b) knock-in efficiency, (c) live cell count, and (d) knock-in cell count 7 days post electroporation for the following templates respectively: dsDNA, dsDNA + tCTS, ssDNA, ssDNA + tCTS, circular ssDNA + CTS, circular ssDNA + mutated v1 CTS v1, circular ssDNA + mutated v2 CTS. Circular cssDNA was produced and provided by Touchlight. (e) Knock-in strategy and designs for CAR-CARD-11 (3.5 kb integration) using linear ssDNA + tCTS template or a nanoplasmid + CTS template at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation.

Article Snippet: To stain for BCMA-CAR, cells were incubated with 0.3 μg BCMA recombinant protein conjugated to biotin (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems, BCA-H82E4_200ug) for 15 minutes at room temperature prior to surface cell staining.

Techniques: Knock-In, Cell Characterization, Electroporation, Produced

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Designs for BCMA-CAR (1.6 kb integration) HDRTs variants tested and corresponding template copies per genome measured at 4 hours and 3 days post electroporation (b) and half-life measured by qPCR. (c) Template copies per genome measured over time at 4 hours, 2 days, 3 days, 5 days, 7 days, and 12 days with R2 and half-life values corresponding to each donor. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. tCTS, truncated Cas9 target site. CTS, Cas9 target site. NP, nanoplasmid. cssDNA, circular cssDNA.

Article Snippet: To stain for BCMA-CAR, cells were incubated with 0.3 μg BCMA recombinant protein conjugated to biotin (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems, BCA-H82E4_200ug) for 15 minutes at room temperature prior to surface cell staining.

Techniques: Electroporation

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: Schematic of BCMA-Nb radiolabeling with (A) [ 18 F]FPy and (B) [ 131 I]I.

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Radioactivity

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Kinetic binding assay of BCMA nanobody to human BCMA protein measured by biolayer interferometry using 100nM anti-BCMA-nanobody (B) Representative flow cytometry histograms of BCMA expression in MC38-BCMA and MC38-WT cells (C) Immunohistochemistry and Immunofluorescent staining of BCMA in excised tumors (D) Saturation binding assays of [ 18 F]FPy-BCMA Nb in MC38-BCMA cells (E) Representative PET/CT coronal images of MC38-BCMA and MC38-WT tumor model (150-250 mm 3 ), injected with 3.7 MBq [ 18 F]FPy-BCMA-Nb at 2 h post-injection. PET scans are normalized to the same scale. Data are presented as mean ± SD (n = 6 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Binding Assay, Flow Cytometry, Expressing, Immunohistochemistry, Staining, Positron Emission Tomography-Computed Tomography, Injection

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Flow cytometry analysis of BCMA expression in H929 and RPMI8226 cell lines. (B) Immunofluorescent staining of H929 cells using anti-BCMA antibody (green) and DAPI (blue). Saturation binding assay to assess the specific binding and dissociation constant of [ 18 F]FPy-BCMA-Nb in (C) H929 and (D) RPMI8226 cells. (E) Internalization of [ 18 F]FPy-BCMA-Nb in H929 cells over 180 minutes. (F) 3 h post-injection uptake of [ 18 F]FPy-BCMA-Nb in the femur/knee region and (G) spine of the human H929 and RPMI8226 MM model compared to healthy mice and blocking. White arrows indicate tumor cells in the bone marrow. Relative quantitative uptake of the tracer in the H929 human MM model with higher BCMA expression in spine, femur, arm, skull bone marrow, and kidney under (H) pre-blocking and (I) post-blocking conditions . Data are presented as mean ± SD (n = 6 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Flow Cytometry, Expressing, Staining, Saturation Assay, Binding Assay, Injection, Blocking Assay

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Schematic illustration of the timeline, treatment regimen and efficacy study for the radiotherapy study. (B) Representative BLI images of mice pre- and post-treatment regimen and (C) overall survival percentage across all groups, including Control (PBS), 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb, compared to 18.5 MBq of [¹³¹I]I-non-specific-Nb treated cohorts, as determined by Kaplan-Meier analysis. Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Control

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Representative Sagittal [ 18 F]FDG PET images of healthy mice compared to treatment groups (PBS, 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb,) acquired pre- and post-treatment regimen. Quantitative analysis of [ 18 F]FDG uptake in bone marrow of the (B) spine, (C)femur, (D) arm, and (E) skull across all groups pre-, during and post-treatment. Gray areas are defined as baseline based on [ 18 F]FDG of healthy mice. Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques:

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: Changes in (A) body weight during the treatment regimen. (B) Complete cell blood counts for mice pre- and post-treatment in all groups including PBS, injected with 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb and 18.5 MBq of [ 131 I]I-non-specific-Nb. Orange-shaded cells indicate values significantly different from standard NSG mouse ranges (p<0.05). Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Injection

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Immunohistochemistry staining and (B) quantitative analysis of BCMA, (C) immunofluorescence staining for and (D) quantitative analysis of Ki67 proliferation marker in femur and spine bone marrow sections of H929 human MM-bearing mice, before and after treatment, compared with healthy mice.

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Marker