Development of CAR-T cell therapy for B-ALL using a point-of-care approach

Abstract

Recently approved by the FDA and European Medicines Agency, CAR-T cell therapy is a new treatment option for B-cell malignancies. Currently, CAR-T cells are manufactured in centralized facilities and face bottlenecks like complex scaling up, high costs, and logistic operations. These difficulties are mainly related to the use of viral vectors and the requirement to expand CAR-T cells to reach the therapeutic dose. In this paper, by using Sleeping Beauty-mediated genetic modification delivered by electroporation, we show that CAR-T cells can be generated and used without the need for ex vivo activation and expansion, consistent with a point-of-care (POC) approach. Our results show that minimally manipulated CAR-T cells are effective in vivo against RS4;11 leukemia cells engrafted in NSG mice even when inoculated after only 4 h of gene transfer. In an effort to better characterize the infused CAR-T cells, we show that 19BBz T lymphocytes infused after 24 h of electroporation (where CAR expression is already detectable) can improve the overall survival and reduce tumor burden in organs of mice engrafted with RS4;11 or Nalm-6 B cell leukemia. A side-by-side comparison of POC approach with a conventional 8-day expansion protocol using Transact beads demonstrated that both approaches have equivalent antitumor activity in vivo. Our data suggest that POC approach is a viable alternative for the generation and use of CAR-T cells, overcoming the limitations of current manufacturing protocols. Its use has the potential to expand CAR immunotherapy to a higher number of patients, especially in the context of low-income countries.

Keywords: CAR-T cells; immunotherapy; point-of-care; sleeping Beauty.

Figure 1.

Evaluation of the potential antileukemic effect of the point-of-care approach. (a) Timeline of the experiment. NSG mice were inoculated with RS4;11 GFP tumor cells and, after 3 days, treated with CAR T cells electroporated 4 h before treatment. (b) Expression of 19BBz CAR in T cells 24 h after electroporation. Mock condition represents the electroporation of PBMC without 19BBz plasmid. (c) Animal blood was collected on day 41 for analysis of tumor burden of RS4;11 GFP by flow cytometry. (d) Kaplan-Meier plot of survival data (PBS n = 6; Mock n = 5; 19BBz n = 9). (e) After euthanasia, tumor burden in blood, bone marrow, spleen, and liver were analyzed by flow cytometry. The survival curve was analyzed by log-rank test and for organ analysis, the Mann–Whitney test was used for paired comparisons. Consider * p < .05, ** p < .01, *** p < .001.

Figure 2.

Comparison of antitumor activity in treated animals with cells electroporated 4 or 24 h earlier. (a) Timeline of the experiment. Animals were treated with CAR T cells from the same donor, but at different times after its production: 4 or 24 h after electroporation. (b) Immunophenotypic characterization of cells and (c) memory phenotype characterization evaluated in CD4+ and CD8 + T cells and represented by Naive (CD45RO-), Central Memory (CM, CD45RO+ and presence of either CD62L or CCR7 markers) and effector Memory (CD45RO+CD62L-CCR7-) 24 h after electroporation. (d) Expression of 19BBz CAR in T cells 24 h after electroporation. (e) Kaplan-Meier plot of overall survival data (Mock 4 h n = 5; Mock 24 h n = 6; 19BBz 4 h n = 6; 19BBz 24 h n = 6). The survival curve was analyzed by log-rank test.

Figure 3.

Effectiveness of point-of-care (POC) approach in animals engrafted with Nalm-6. (a) Timeline of the experiment. NSG mice were inoculated with 10⁵ Nalm-6 GFP cells and were treated after 2 days with CAR T cells produced 24 h earlier. (b) Expression of 19BBz CAR in T cells 24 h after electroporation. (c) Kaplan-Meier plot of survival data (PBS n = 5; Mock n = 5; 19BBz n = 6). (d) After euthanasia, tumor burden of blood, bone marrow, spleen, and liver were analyzed by flow cytometry. The survival curve was analyzed by log-rank test and for organ analysis, the Mann–Whitney test was used for paired comparisons. Consider * p < .05, ** p < .01, *** p < .001.

Figure 4.

Comparison of the efficiency of antitumor activity between the Point-of-care approach and cells expanded with anti-CD3/CD28 coated beads. (a) Comparison of the expansion of CAR+ cells. Cells from this donor were used to treat NSG mice engrafted with RS4;11 in the POC or expanded cells approach. (b) Memory phenotype was evaluated on day 1 and day 8 after electroporation in CD4+ and CD8+T cells and was represented by Naive (CD45RO-), Central Memory (CM, CD45RO+ and presence of either CD62L or CCR7 markers) and Effector Memory (CD45RO+CD62L-CCR7-). (c) Expression of 19BBz CAR in T cells 24 h after electroporation and on day 8 of the expansion protocol. (d) Evaluation by flow cytometry of the tumor burden of the organs in the groups after euthanasia. (e) Kaplan-Meier plot of survival data (POC group: PBS n = 6; Mock n = 6; 19BBz 1,5x10⁴ n = 7; 19BBz 1,5x10⁵ n = 7. Expansion group: PBS n = 6; Mock n = 6; 19BBz 1,5x10⁴ n = 7; 19BBz 1,5x10⁵ n = 7). The survival curve was analyzed by log-rank test and for organ analysis, the Mann–Whitney test was used for paired comparisons. Consider * p < .05, ** p < .01, *** p < .001.