puromycin resistance retroviral vector pbabe encoding sv40 t antigen  (Addgene inc)

Journal: Journal for Immunotherapy of Cancer

Article Title: Format-tuning of in vivo-launched bispecific T cell engager enhances efficacy against renal cell carcinoma

doi: 10.1136/jitc-2023-008733

Figure Lengend Snippet: PMTE restores avidity for target cells from PBTE levels. (A) Graphic of the single-chain BTE, PBTE, and PMTE formats. Gray lines represent GS linkers. (B) Plasmid map for the antibody-encoding pVax1 vectors used for synDNA therapies. (C) Flow cytometry binding curves to ACHN-CA9 (n=3), 293T-CA9 (n=4), and T cells (n=3). An irrelevant IgG1 was used as a negative control. (D) K D calculations from respective cell binding curves were plotted as donor mean±SEM. E) z-Movi workflow depicting insertion of adherent cancer cells, suspended T cells, and compounds to a flow chamber. Ultrasound waves pull T cells from cancer cells toward acoustic nodes. Force required to separate cells is recorded at single cell resolution, and detachment force represents synaptic strength generated by bispecific antibodies. (F) Curves represent the percent of cells remaining bound with increasing detachment force over time. Antibodies were analyzed at 30 nM (n=5), 3 nM (n=3), and 300 pM (n=3). An irrelevant BTE was used as a negative control. (G) Area under curve (AUC) analyses of adjacent force curves at 30 nM (n=5), 3 nM (n=3), and 300 pM (n=3) were plotted as mean±SEM. (H) Single cell resolution of force required to detach T cells from cancer cells for individual antibodies for respective antibody concentrations. Points represent individual T cells and data represent all replications collectively. (I) Representative images of the flow chamber at 1000 pN display localization of detached T cells at acoustic nodes for lower avidity antibodies at 30 nM. BTE, bispecific T cell engager; PBTE, persistent bispecific T cell engager; PMTE, persistent multivalent T cell engager. Significance is assigned as * (p<.05), ** (p<0.005), *** (p<0.001), or **** (p<0.0001).

Article Snippet: The plasmid vector used to retrovirally transduce 293 T cells with CA9 was acquired from GenScript.

Techniques: Plasmid Preparation, Flow Cytometry, Binding Assay, Negative Control, Generated

Journal: Journal for Immunotherapy of Cancer

Article Title: Format-tuning of in vivo-launched bispecific T cell engager enhances efficacy against renal cell carcinoma

doi: 10.1136/jitc-2023-008733

Figure Lengend Snippet: PMTE activates T cells with the most potency in vitro. (A) CD69+ and (B) CD25+expression on CD4+ (top row) and CD8+ (bottom row) T cells after 24 hours of co-incubation with antibodies and SKRC-52 cells (n=5). (C) CD69+and D) CD25+expression on CD4+ (top row) and CD8+ (bottom row) T cells after 24 hours of co-incubation with antibodies and 293T-CA9 (n=5). An irrelevant BTE was used as a negative control. Data are expressed as mean±SEM. BTE, bispecific T cell engager; PMTE, persistent multivalent T cell engager. Significance is assigned as * (p<.05), ** (p<0.005), *** (p<0.001), or **** (p<0.0001).

Article Snippet: The plasmid vector used to retrovirally transduce 293 T cells with CA9 was acquired from GenScript.

Techniques: In Vitro, Expressing, Incubation, Negative Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Format-tuning of in vivo-launched bispecific T cell engager enhances efficacy against renal cell carcinoma

doi: 10.1136/jitc-2023-008733

Figure Lengend Snippet: PMTE induces the T cell-mediated cytotoxicity of target cells with the highest potency. (A) Percent of terminal cell population death at increasing concentrations of antibody treatment for the following target cell lines: SKRC-52 (n=4), A-498 (n=3), 786-O (n=2), ACHN-CA9 (n=3), and 293T-CA9 (n=2). Percent cytotoxicity was calculated as (1–(treatment/target cell control))×100) at the terminal experimental time point. (B) Real-time analyses of cell growth over time for 10 nM and 1 pM antibody treatments for the adjacent cell lines illustrate potent cytotoxicity. (C) Representative images taken at the terminal time point of single experiments for each treatment group at 10 nM, illustrating the cytotoxicity of the adjacent target cell lines. PMTE, persistent multivalent T cell engager.

Article Snippet: The plasmid vector used to retrovirally transduce 293 T cells with CA9 was acquired from GenScript.

Techniques: Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Format-tuning of in vivo-launched bispecific T cell engager enhances efficacy against renal cell carcinoma

doi: 10.1136/jitc-2023-008733

Figure Lengend Snippet: PMTE displays improved tumor distribution and tumor control in vivo. (A) Balb/c mice were administered a single 2.5 mg/kg (intravenous) dose of antibodies and circulating antibody was quantified from sera via His-capture ELISA for pharmacokinetic analysis (n=3). Serum concentrations are displayed in both μm/mL and nM units. Corresponding pharmacokinetic calculations are in . (B) The tumor distribution PK analysis workflow began with an ACHN-CA9 xenograft in NSG mice (n=4). Single 0.3 mg/kg (intravenous) doses in PBS were administered when tumors reached 200 mm 3 . Tumors and serum were harvested after 24 hours, tumors homogenized, and antibody concentrations analyzed by His-capture ELISA. An irrelevant BTE was used as negative control. (C) A tumor distribution summary plot of intratumoral concentrations against serum. The tumor/serum concentration ratio (×10 −3 ) is represented by point size. (D) Serum antibody concentrations at 24 hours are given in both ng/mL and nM units. (E) Intratumoral antibody concentrations at 24 are given in both ng/mL and nM units. (F) Relative distribution of antibodies into the tumor compartment is considered as the tumor/serum concentration ratio (×10 −3 ). (G) Nu/J mice harboring subcutaneous ACHN-CA9 tumors were administered 10 6 donor T cells (IP) and 3 doses of recombinant antibodies at 1 mg/kg, once every 5 days (n=5). Arrows represent doses and data are presented individually and as grouped mean. Bar graphs display average tumor volumes (mm 3 ) at days 25 and 30. Data were plotted as mean±SEM. BTE, bispecific T cell engager; PMTE, persistent multivalent T cell engager. Significance is assigned as * (p<.05), ** (p<0.005), *** (p<0.001), or **** (p<0.0001).

Article Snippet: The plasmid vector used to retrovirally transduce 293 T cells with CA9 was acquired from GenScript.

Techniques: Control, In Vivo, Enzyme-linked Immunosorbent Assay, Negative Control, Concentration Assay, Recombinant

Journal: Journal for Immunotherapy of Cancer

Article Title: Format-tuning of in vivo-launched bispecific T cell engager enhances efficacy against renal cell carcinoma

doi: 10.1136/jitc-2023-008733

Figure Lengend Snippet: synDNA-delivered PMTE demonstrates better durability and tumor control in vivo. (A) DNA-based BTEs administered as plasmid DNA with hyaluronidase (50 U/mouse) and electroporation for enhanced cellular uptake. Myocytes synthesize antibodies in situ for secretion into circulation. In tumor models, donor T cells are administered (IP) and treatment efficacy assessed. Serum can be collected from antibody-treated mice, whether naïve or from tumor models, for in vitro functional assessments of in vivo-synthesized antibodies. (B) Naïve, balb/c mice were administered 200 µg of dBTE, dPBTE, or dPMTE DNA (n=5). Sera collected over time were used to stain ACHN and ACHN-CA9 cells by flow cytometry. Untranduced ACHN cells are represented in gray while colored shifts represent ACHN-CA9 cells up to day 21. (C) Sera were also used to stain donor T cells by flow cytometry from days 0 to 21. (D) Sera from synDNA-treated balb/c mice (n=5) were tested for their ability to induce cytotoxicity of SKRC-52 cells in vitro, using an xCELLigence system. Diminished cell indices represent target cell death. Negative controls included sera from empty pVax1-treated mice while recombinant BTE (10 nM) was used as a positive control. (E) NSG mice harboring subcutaneous A-498 tumors were administered 10 6 donor T cells (IP) and a single 10 ug dose of synDNA on day 0, represented by arrow (n=5). Mice are line-plotted individually while bar graph displays tumor volumes (mm 3 ) at day 33 as mean±SEM. (F) NSG mice harboring subcutaneous SKRC-52 tumors were administered 10 6 donor T cells (intraperitoneally) and a single 10 ug dose of synDNA, represented by arrow (n=4–5). Mice are line-plotted individually while bar graph displays tumor volumes (mm 3 ) at day 30 as mean±SEM. BTE, bispecific T cell engager; dBTE, delivery of BTE; dPBTE, delivery of persistent BTE; dPMTE, delivery of persistent multivalent T cell engager. Significance is assigned as * (p<.05), ** (p<0.005), *** (p<0.001), or **** (p<0.0001).

Article Snippet: The plasmid vector used to retrovirally transduce 293 T cells with CA9 was acquired from GenScript.

Techniques: Control, In Vivo, Plasmid Preparation, Electroporation, In Situ, In Vitro, Functional Assay, Synthesized, Staining, Flow Cytometry, Recombinant, Positive Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Format-tuning of in vivo-launched bispecific T cell engager enhances efficacy against renal cell carcinoma

doi: 10.1136/jitc-2023-008733

Figure Lengend Snippet: FSHR-targeted BTEs follow similar formatting trends for binding and potency in vitro. (A) Anti-CA9 scFvs were exchanged for anti-FSHR scFvs in BTE, PBTE, and PMTE formats. Anti-CD3 scFvs were unchanged. (B) Flow cytometry binding curves to OVCAR3-FSHR and T cells. An irrelevant IgG1 was used as negative control. Data are presented as mean±SEM. (C) The concentration-dependent degree of OVCAR3-FSHR cytotoxicity is expressed as the percent of healthy cell control after 5 days. An irrelevant BTE was used as negative control. Data are presented as mean of duplicates. (D) K D values from flow cytometry binding analyses for OVCAR-FSHR and T cells. (E) EC 50 calculations from the OVCAR-FSHR killing assay. BTEs, bispecific T cell engagers; FSHR, follicle-stimulating hormone receptor.

Article Snippet: The plasmid vector used to retrovirally transduce 293 T cells with CA9 was acquired from GenScript.

Techniques: Binding Assay, In Vitro, Flow Cytometry, Negative Control, Concentration Assay, Control