anti-cd19-anti-cd3 bispecific antibody  (BPS Bioscience)

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: Optimization of γδ T cell electroporation with bicistronic CD19 CAR-GFP mRNA (A) γδ T cells express GFP after mRNA electroporation using the BioRad Gene Pulser Xcell Electroporator. γδ T cell electroporation was optimized by testing varying cell numbers and mRNA amounts in each reaction. (B) Cell yield, calculated by determining the proportion of live cells remaining 24 h after electroporation to the starting number of cells used for the electroporation reaction, was calculated for all reaction conditions and increased as the cell number increased. (C) GFP mean fluorescence intensity (MFI) was determined by flow cytometry and increased with increasing amounts of mRNA. (D) The percentage of live cells expressing GFP and the CD19 CAR was similar for all conditions and was found to be about 90% and 60%, respectively. (E) γδ T cell cytotoxicity was determined by flow cytometry to test two promising electroporation reaction conditions and found no difference when comparing different cell numbers in each reaction.

Article Snippet: To perform the western blot, γδ T cell conditioned media and anti-CD19-anti-CD3 bispecific antibody standards (BPS Biosciences) were prepared under reducing conditions.

Techniques: Electroporation, Fluorescence, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: Electroporation of γδ T cells before freezing results in lower CAR expression and reduced cytotoxicity γδ T cells were electroporated on day 12 of expansion and were analyzed before and after freezing. (A) While GFP expression (circles) remained constant at around 90% before and after freezing, the CAR percentage (triangles) decreased from about 60% before freezing to about 30%–40% after thawing. Closed data points denote before freezing and open data points denote after thawing. (B) The cytotoxicity of CD19 CAR-expressing γδ T cells before and after freezing was also measured to determine if a freeze/thaw cycle effects the cytotoxicity of the engineered cells. Similar cytotoxicity was observed at low effector to target (E:T) ratios; however, there was a reduction at higher E:T ratios for the thawed engineered cells.

Article Snippet: To perform the western blot, γδ T cell conditioned media and anti-CD19-anti-CD3 bispecific antibody standards (BPS Biosciences) were prepared under reducing conditions.

Techniques: Electroporation, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: CD19 CAR- and CD22 CAR-expressing γδ T cells enhances cytotoxicity against two B-ALL cell lines Effector and target cells were cocultured at the specified E:T ratio for 4 h and the percent cytotoxicity was determined by flow cytometry. Target cells were stained with VPD450 to differentiate effector and target cell death. Mock-electroporated, CD19 CAR-, and CD22 CAR-expressing γδ T cells were tested against the B-ALL cell lines 697 (A and B) and Nalm6 (C). While the cytotoxicity of mock-electroporated γδ T cells remained constant over all E:T ratios, CD19 CAR- and CD22 CAR-expressing γδ T cells exhibited a dose-dependent increase in cytotoxicity.

Article Snippet: To perform the western blot, γδ T cell conditioned media and anti-CD19-anti-CD3 bispecific antibody standards (BPS Biosciences) were prepared under reducing conditions.

Techniques: Expressing, Flow Cytometry, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: γδ T cells express and secrete CD19 sBite after mRNA electroporation (A) γδ T cells were electroporated with 3 μg, 7.5 μg, and 15 μg of mRNA and the amount of CD19 sBite in the conditioned media was determined using an ELISA. (B) Unmodified and CD19 sBite-modified γδ T cells were cocultured with several CD19 + cancer cells lines to test their cytotoxic capabilities. sBite-modified γδ T cells showed increased cytotoxicity at all E:T ratios and reached about 90% at the 5:1 ratio. (C) A CD19KO 697 cell line was generated using CRISPR to test the specificity of the secreted CD19 sBite. As expected, the sBite-modified γδ T cells showed improved cytotoxicity against the naive 697 cell line; however, no increase in cytotoxicity was seen when the CD19KO 697 cell line was used as target cells. (D) To test whether the CD19 sBite can induce killing of unmodified cells, conditioned media from unmodified and sBite-modified γδ T cells was collected after 16 h of culture and mixed with either unmodified or sBite-modified γδ T cells. As expected, the sBite-modified cells showed improved cytotoxicity, regardless of the conditioned media. The unmodified cells cultured with the sBite-conditioned media showed improved cytotoxicity, compared with unmodified cells with unmodified conditioned media.

Article Snippet: To perform the western blot, γδ T cell conditioned media and anti-CD19-anti-CD3 bispecific antibody standards (BPS Biosciences) were prepared under reducing conditions.

Techniques: Electroporation, Enzyme-linked Immunosorbent Assay, Modification, Generated, CRISPR, Cell Culture

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: γδ T cells are not able to kill cancer cells once they extravasate from circulation (A) In this cancer model, cancer cells are injected i.v. and gradually leave the circulation and form non-vascularized nodules in various organs and other compartments, leaving few cancer cells in circulation by days 3 and 7. Based on this model, it can be predicted that the timing for γδ T cell treatment is important in treating mice bearing the 697 cancer cell line. (B) Tissue samples from blood, bone marrow, and spleen were collected 3 weeks after cancer cell injection to detect the presence of cancer cells in each compartment (left flow plots are representative). A substantial number of CD45 + CD3 − 697 cells were detected in the bone marrow, while limited numbers were found in the blood and spleen. (C) Representative hematoxylin and eosin staining images showing the presence of cancer cells with no vasculature around the cancer cells. Histopathological analysis revealed the presence of cancer cells in the brain, liver, lungs, and kidneys, with avascularized nodules found within the liver.

Article Snippet: To perform the western blot, γδ T cell conditioned media and anti-CD19-anti-CD3 bispecific antibody standards (BPS Biosciences) were prepared under reducing conditions.

Techniques: Injection, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: Engineered γδ T cells expressing a CD19 CAR reduce tumor burden and improve survival in the 697 model (A) NSG mice were injected with 2 × 10 6 luciferase-expressing 697 cells and bioluminescence images were captured during the course of the experiment. Mice treated with CD19 CAR-expressing γδ T cells on day 1 of the experiment showed a reduction in tumor burden compared with control mice. (B) Raw total flux values were calculated and showed delayed tumor progression and significantly reduced tumor burden for mice treated with the CD19 CAR-expressing γδ T cells (triangles), compared with the control mice (circles). Statistics were performed using a 2-tailed Student’s t test to compare experimental groups at each given time point. (C) Kaplan-Meier survival curves showed significantly increased survival in mice treated with CD19 CAR-expressing γδ T cells (dashed line), compared with control mice (p = 0.02 by log rank test). Control: n = 4; CD19 CAR: n = 3; error bars indicate standard deviation; ∗∗p < 0.01.

Article Snippet: To perform the western blot, γδ T cell conditioned media and anti-CD19-anti-CD3 bispecific antibody standards (BPS Biosciences) were prepared under reducing conditions.

Techniques: Expressing, Injection, Luciferase, Control, Standard Deviation

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: Engineering γδ T cells with CD19 CAR or sBite mRNA reduces tumor burden and improves survival in the Nalm6 model NSG mice were injected with 2 × 10 6 luciferase-expressing Nalm6 cells and were treated with unmodified, CD19 CAR-modified, or CD19 sBite-modified γδ T cells on day 1 of the experiment with a treatment regimen of twice a week for 2 weeks. (A) Before injection, unmodified or modified γδ T cells were analyzed for CAR expression and MFI using flow cytometry. CAR expression was about 60% for CD19 CAR-expressing γδ T cells and, interestingly, the CD19 sBite-modified γδ T cells bound to the CD19Fc, with an average of about 40% CD19Fc positive (left graph). Despite the CD19 sBite-modified γδ T cell binding to the CD19Fc, the MFI was minimal compared with the CD19 CAR (right graph). (B) The cytotoxicity of the unmodified and modified γδ T cells were also examined before injection at E:T ratios of 1:2 and 2:1. The CD19 CAR- and sBite-modified γδ T cells exhibited increased cytotoxicity compared with the unmodified γδ T cells. (C) Bioluminescent imaging was performed during the experiment and mice treated with unmodified γδ T cells showed a high tumor burden as early as 1 or 2 weeks after cancer cell injection. (D) Raw total flux was determined for each image and graphed over time to compare treatment with unmodified γδ T cells and CD19 CAR-expressing (top graph) or CD19 sBite-expressing γδ T cells (bottom graph). Treatment with modified γδ T cells resulted in delayed tumor progression and reduced tumor burden. Statistics were performed using a 2-tailed Student’s t test to compare experimental groups at each given time point. (E) Kaplan-Meier survival curves were generated to compare survival for each treatment group to treating with unmodified γδ T cells. Treatment with CD19 CAR- and CD19 sBite-expressing γδ T cells resulted in a significant survival benefit compared with treating with unmodified γδ T cells (p = 0.01 for CAR and sBite by log rank test). n = 5; error bars indicate standard deviation; ∗p < 0.05.

Article Snippet: To perform the western blot, γδ T cell conditioned media and anti-CD19-anti-CD3 bispecific antibody standards (BPS Biosciences) were prepared under reducing conditions.

Techniques: Injection, Luciferase, Expressing, Modification, Flow Cytometry, Binding Assay, Imaging, Generated, Standard Deviation

Journal: Molecular Therapy Oncolytics

Article Title: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

doi: 10.1016/j.omto.2023.05.007

Figure Lengend Snippet: Longer treatment regimen does not lengthen survival benefit for Nalm6 model Despite being significant, the survival benefit for the previous in vivo experiments was not as robust as the in vitro data would suggest. (A) To test whether a more extensive treatment regimen of three doses for the first 2 weeks, two doses for the next 2 weeks, and one dose for the final 2 weeks could further improve the survival benefit. (B) NSG mice were injected with 2 × 10 6 luciferase-expressing Nalm6 cells and treated with CD19 sBite-modified γδ T cells using the more extensive treatment regimen. Bioluminescent images were taken and again showed reduced tumor burden for the sBite-treated group, compared with the control group. (C) Graph of raw total flux shows the more extensive treatment regimen delayed tumor progression and reduced tumor burden, compared with control mice. The inset shows an expansion of the first 20 days of treatment. Statistics were performed using a 2-tailed Student’s t test. (D) Kaplan-Meier survival curves were generated for the control group and more extensive treatment regimen of CD19 sBite-expressing γδ T cells. As expected, the more extensive treatment regimen resulted in a significant survival benefit compared with the control group (p = 0.01 by log rank test); however, there was no difference in survival when comparing the more extensive treatment regimen with the previous regimen of twice a week for 2 weeks (p = 0.39 by log rank test). Control: n = 3; sBite: n = 4; error bars indicate standard deviation; ∗p < 0.05.

Article Snippet: To perform the western blot, γδ T cell conditioned media and anti-CD19-anti-CD3 bispecific antibody standards (BPS Biosciences) were prepared under reducing conditions.

Techniques: In Vivo, In Vitro, Injection, Luciferase, Expressing, Modification, Control, Generated, Standard Deviation