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Lymphocyte-nanoparticle biohybrids as a combined nanoimmunotherapy for cancer
S14
Oral Abstracts
suggest that NK cells are active anti-tumor cell therapy agents, the use of HDACi and PD-1/PD-L1 blockade acts to further increase the efficacy of NK cell therapy against NB.
18 CD4 AND CD8 T-CELL POSITIVE SELECTION INCREASES THE ROBUSTNESS OF THE CD22 CAR T-CELL MANUFACTURING PROCESS S.L. Highfill1, J. Jin1, V. Fellowes1, J. Ren1, S. Ramakrishna2, T. Fry2, D. Stroncek1 1 Cell Processing Section, National Institutes of Health, Bethesda, Maryland, United States, 2Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland, United States CAR-T cell therapies targeting the surface markers CD19/CD22 have been very successful in the treatment of B-cell hematologic malignancies. One of the major challenges during CAR T cell manufacturing is low expansion and transduction of autologous CAR T cells. Poor performance is partly, if not entirely, due to the poor quality and quantity of cell components in the apheresis products from patients, particularly patients with high tumor burden. Flask adhesion and elutriation are often utilized as methods to remove monocytes and granulocytes, and anti-CD3/CD28 paramagnetic microparticles are used for T-cell enrichment and activation, but these approaches do not consistently result in an enriched CD3 T-cell population capable of efficient transduction and expansion. Often, residual tumor cells and monocytes remain within the culture and inhibit T-cell expansion. Here, we examine a total of 3 healthy donor samples and 2 B-ALL patient samples. We show that selection with anti-CD4/CD8 microparticles prior to stimulation and transduction with a CD22 CAR lentiviral vector results in increased T-cell expansion versus unselected cells. Moreover, a clinical sample from an ALL patient with high tumor burden that failed to undergo significant expansion while on the CD19 CAR clinical trial regained the ability to proliferate after CD4/CD8 positive selection and transduction with the CD22 CAR (4.9 fold vs. 27 fold expansion). CD3 T-cell percent increased from 7.2% in the starting product of the clinical trial to 50.3% post selection. In addition, tumor cells were efficiently depleted from the starting product as indicated by low percentage of CD22+ cells (from 43% to 0.6%). There were no significant differences when comparing transduction efficiencies from CD4/ CD8 selected versus unselected products in a healthy donor and patient sample. The final product viability was >90% for all products tested. Xenograft models using human NALM6 ALL cell line showed equivalent tumor clearance between selected and unselected T-cell from a healthy donor. In conclusion, purification of T-cells in the starting product using CD4/CD8 positive selection prior to stimulation and transduction significantly enhances expansion of CD22 CAR T cells, especially problematic patient samples with high blast content, and allows for a more robust and consistent manufacturing process.
19 LYMPHOCYTE-NANOPARTICLE BIOHYBRIDS AS A COMBINED NANOIMMUNOTHERAPY FOR CANCER R.A. Burga1,2,3, J. Cano Mejia2, C. Cruz1,2,3, C. Bollard1,3, R. Fernandes1,2 1 George Washington University, Institute for Biomedical Sciences, Washington, District of Columbia, United States, 2Children’s National Health System, Sheikh Zayed Institute for Pediatric Surgical Innovation, Washington, District of Columbia, United States, 3Children’s National Health System, Program for Cell Enhancement and Technologies for Innovation, Washington, District of Columbia, United States T cell therapies have shown promise against leukemias, but little efficacy against solid tumors. Success is limited by an immunosuppressive tumor environment, which precludes effector cell accumulation at the tumor site or renders effector cells dysfunctional preventing tumor clearance. As such, strategies to improve effector cell function at the tumor site have the potential to enhance responses. We have observed that multifunctional nanoparticles can confer additional properties to existing cell-based immunotherapies including ablative heating, magnetic responsiveness, and localized drug delivery. We thus sought to evaluate whether immune cell-nanoparticle biohybrids (ImmunoNPs, Figure 1) could combine the potent cytotoxic capabilities of antigen-specific T cells and ablative therapy from nanoparticles to enhance immune responses within the suppressive tumor microenvironment. We synthesized a robust biohybrid capable of antigen-dependent cytotoxicity, followed by localized ablative therapy to efficiently eliminate residual disease by conjugating T-cells with Prussian blue nanoparticles (which absorb light in the near infrared range). We demonstrated stable T cellnanoparticle conjugation over at least 3 days (51–65.8% by flow cytometry). T-cells within the biohybrid retained their proliferative ability (66.4% for T-cells vs. 66.5% for biohybrid by CFSE dissolution) and effector phenotype (mean 62.7% CD8+ T-cells vs. 55.2% CD8+ biohybrid, n = 7; Figure 2), with no significant increases in markers of exhaustion (PD1, TIM3, LAG3). Furthermore, we demonstrated improved cytotoxicity against tumor antigen-expressing target cells following treatment with ImmunoNPs: each component individually was able to decrease target cell viability from 92.7% (target cells alone) to 46.3% (T-cells alone) or 43.8% (NPs with laser), however maximal eradication occurred with the tandem biohybrid (target cell viability of 28%, Figure 2). Additionally, we found that ablative therapy with non-cellularized Prussian blue nanoparticles was capable of increasing tumor lymphocyte infiltration 3-fold (p < 0.05) compared to untreated tumors in vivo, suggesting that photothermal ablation can augment endogenous immune responses. We believe this work represents a novel modality that combines the strengths of cell-based immunotherapy with nanomedicine in order to achieve maximal therapeutic responses to challenging malignancies and infectious diseases.
Figure 1. Schematic of immune cell-nanoparticle bionconjugates (ImmunoNPs).
Oral Abstracts
suggest that NK cells are active anti-tumor cell therapy agents, the use of HDACi and PD-1/PD-L1 blockade acts to further increase the efficacy of NK cell therapy against NB.
18 CD4 AND CD8 T-CELL POSITIVE SELECTION INCREASES THE ROBUSTNESS OF THE CD22 CAR T-CELL MANUFACTURING PROCESS S.L. Highfill1, J. Jin1, V. Fellowes1, J. Ren1, S. Ramakrishna2, T. Fry2, D. Stroncek1 1 Cell Processing Section, National Institutes of Health, Bethesda, Maryland, United States, 2Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland, United States CAR-T cell therapies targeting the surface markers CD19/CD22 have been very successful in the treatment of B-cell hematologic malignancies. One of the major challenges during CAR T cell manufacturing is low expansion and transduction of autologous CAR T cells. Poor performance is partly, if not entirely, due to the poor quality and quantity of cell components in the apheresis products from patients, particularly patients with high tumor burden. Flask adhesion and elutriation are often utilized as methods to remove monocytes and granulocytes, and anti-CD3/CD28 paramagnetic microparticles are used for T-cell enrichment and activation, but these approaches do not consistently result in an enriched CD3 T-cell population capable of efficient transduction and expansion. Often, residual tumor cells and monocytes remain within the culture and inhibit T-cell expansion. Here, we examine a total of 3 healthy donor samples and 2 B-ALL patient samples. We show that selection with anti-CD4/CD8 microparticles prior to stimulation and transduction with a CD22 CAR lentiviral vector results in increased T-cell expansion versus unselected cells. Moreover, a clinical sample from an ALL patient with high tumor burden that failed to undergo significant expansion while on the CD19 CAR clinical trial regained the ability to proliferate after CD4/CD8 positive selection and transduction with the CD22 CAR (4.9 fold vs. 27 fold expansion). CD3 T-cell percent increased from 7.2% in the starting product of the clinical trial to 50.3% post selection. In addition, tumor cells were efficiently depleted from the starting product as indicated by low percentage of CD22+ cells (from 43% to 0.6%). There were no significant differences when comparing transduction efficiencies from CD4/ CD8 selected versus unselected products in a healthy donor and patient sample. The final product viability was >90% for all products tested. Xenograft models using human NALM6 ALL cell line showed equivalent tumor clearance between selected and unselected T-cell from a healthy donor. In conclusion, purification of T-cells in the starting product using CD4/CD8 positive selection prior to stimulation and transduction significantly enhances expansion of CD22 CAR T cells, especially problematic patient samples with high blast content, and allows for a more robust and consistent manufacturing process.
19 LYMPHOCYTE-NANOPARTICLE BIOHYBRIDS AS A COMBINED NANOIMMUNOTHERAPY FOR CANCER R.A. Burga1,2,3, J. Cano Mejia2, C. Cruz1,2,3, C. Bollard1,3, R. Fernandes1,2 1 George Washington University, Institute for Biomedical Sciences, Washington, District of Columbia, United States, 2Children’s National Health System, Sheikh Zayed Institute for Pediatric Surgical Innovation, Washington, District of Columbia, United States, 3Children’s National Health System, Program for Cell Enhancement and Technologies for Innovation, Washington, District of Columbia, United States T cell therapies have shown promise against leukemias, but little efficacy against solid tumors. Success is limited by an immunosuppressive tumor environment, which precludes effector cell accumulation at the tumor site or renders effector cells dysfunctional preventing tumor clearance. As such, strategies to improve effector cell function at the tumor site have the potential to enhance responses. We have observed that multifunctional nanoparticles can confer additional properties to existing cell-based immunotherapies including ablative heating, magnetic responsiveness, and localized drug delivery. We thus sought to evaluate whether immune cell-nanoparticle biohybrids (ImmunoNPs, Figure 1) could combine the potent cytotoxic capabilities of antigen-specific T cells and ablative therapy from nanoparticles to enhance immune responses within the suppressive tumor microenvironment. We synthesized a robust biohybrid capable of antigen-dependent cytotoxicity, followed by localized ablative therapy to efficiently eliminate residual disease by conjugating T-cells with Prussian blue nanoparticles (which absorb light in the near infrared range). We demonstrated stable T cellnanoparticle conjugation over at least 3 days (51–65.8% by flow cytometry). T-cells within the biohybrid retained their proliferative ability (66.4% for T-cells vs. 66.5% for biohybrid by CFSE dissolution) and effector phenotype (mean 62.7% CD8+ T-cells vs. 55.2% CD8+ biohybrid, n = 7; Figure 2), with no significant increases in markers of exhaustion (PD1, TIM3, LAG3). Furthermore, we demonstrated improved cytotoxicity against tumor antigen-expressing target cells following treatment with ImmunoNPs: each component individually was able to decrease target cell viability from 92.7% (target cells alone) to 46.3% (T-cells alone) or 43.8% (NPs with laser), however maximal eradication occurred with the tandem biohybrid (target cell viability of 28%, Figure 2). Additionally, we found that ablative therapy with non-cellularized Prussian blue nanoparticles was capable of increasing tumor lymphocyte infiltration 3-fold (p < 0.05) compared to untreated tumors in vivo, suggesting that photothermal ablation can augment endogenous immune responses. We believe this work represents a novel modality that combines the strengths of cell-based immunotherapy with nanomedicine in order to achieve maximal therapeutic responses to challenging malignancies and infectious diseases.
Figure 1. Schematic of immune cell-nanoparticle bionconjugates (ImmunoNPs).