Development of semi-automated devices that can reduce the hands-on time and standardize the production of clinical-grade CAR T-cells, such as CliniMACS Prodigy from Miltenyi, is key to facilitate the development of CAR T-cell therapies, especially in academic institutions. However, the feasibility of manufacturing CAR T-cell products from heavily pre-treated patients with this system has not been demonstrated yet. Here we report and characterize the production of 28 CAR T-cell products in the context of a phase I clinical trial for CD19+ B-cell malignancies (NCT03144583). The system includes CD4-CD8 cell selection, lentiviral transduction and T-cell expansion using IL-7/IL-15. Twenty-seven out of 28 CAR T-cell products manufactured met the full list of specifications and were considered valid products. Ex vivo cell expansion lasted an average of 8.5 days and had a mean transduction rate of 30.6 ± 13.44%. All products obtained presented cytotoxic activity against CD19+ cells and were proficient in the secretion of pro-inflammatory cytokines. Expansion kinetics was slower in patient's cells compared to healthy donor's cells. However, product potency was comparable. CAR T-cell subset phenotype was highly variable among patients and largely determined by the initial product. T_CM and T_EM were the predominant T-cell phenotypes obtained. 38.7% of CAR T-cells obtained presented a T_N or T_CM phenotype, in average, which are the subsets capable of establishing a long-lasting T-cell memory in patients. An in-depth analysis to identify individual factors contributing to the optimal T-cell phenotype revealed that ex vivo cell expansion leads to reduced numbers of T_N, T_SCM, and T_EFF cells, while T_CM cells increase, both due to cell expansion and CAR-expression. Overall, our results show for the first time that clinical-grade production of CAR T-cells for heavily pre-treated patients using CliniMACS Prodigy system is feasible, and that the obtained products meet the current quality standards of the field. Reduced ex vivo expansion may yield CAR T-cell products with increased persistence in vivo.

In recent years, the relevance of the immune system to fight cancer has led to the development of immunotherapy, including the adoptive cell transfer of immune cells, such as natural killer (NK) cells and chimeric antigen receptors (CAR)-modified T cells. The discovery of donor NK cells’ anti-tumor activity in acute myeloid leukemia patients receiving allogeneic stem cell transplantation (allo-SCT) was the trigger to conduct many clinical trials infusing NK cells. Surprisingly, many of these studies did not obtain optimal results, suggesting that many different NK cell parameters combined with the best clinical protocol need to be optimized. Various parameters including the high array of activating receptors that NK cells have, the source of NK cells selected to treat patients, different cytotoxic mechanisms that NK cells activate depending on the target cell and tumor cell survival mechanisms need to be considered before choosing the best immunotherapeutic strategy using NK cells. In this review, we will discuss these parameters to help improve current strategies using NK cells in cancer therapy. Moreover, the chimeric antigen receptor (CAR) modification, which has revolutionized the concept of immunotherapy, will be discussed in the context of NK cells. Lastly, the dark side of NK cells and their involvement in inflammation will also be discussed.

Chimeric antigen receptor (CAR)–modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.

Multiple myeloma (MM) remains an incurable hematological malignancy characterized by clonal proliferation of malignant plasma cells in bone marrow. In the last 20 years, the introduction of autologous stem cell transplantation, followed by proteasome inhibitors and immunomodulatory agents, increased the survival of MM patients by 50%. However, still a high proportion of patients relapse and become refractory, especially, high-risk patients with adverse cytogenetics where these treatment combinations have shown limited benefit. Therefore, novel strategies, such as immunotherapy, have been developed in the last few years to help improve the survival of these patients. Immunotherapy treatments include a high number of different strategies used to attack the tumor cells by using the immune system. Here, we will review the most successful immunotherapy strategies published up to date in patients with relapsed or refractory (R/R) MM, including monoclonal antibodies targeting specific antigens on the tumor cells, antibodies combined with cytotoxic drugs or Antibodies Drug Conjugates, immune checkpoint inhibitors which eliminate the barriers that damper immune cells and prevent them from attacking tumor cells, bi-specific T-cell engagers antibodies (BiTEs), bi-specific antibodies and the infusion of chimeric antigen receptor-modified T cells. We overview the results of clinical studies that have been presented up to date and also review pre-clinical studies describing potential novel treatments for MM.

Background/Objectives: Potency testing of clinical-grade lentiviral vectors (LVVs) is critical to support a drug’s commercial approval. Careful consideration should be paid to the development of a suitable potency test during the drug’s clinical development. We aimed to develop an affordable, quantitative test for our CAR19-LVV, based on a measure of transgene’s functional activity. Methods: Several indicators of functional activity of CAR19-LVV were explored in a co-culture setting of CAR-transduced Jurkat cells and CD19-expressing target cells. The selected assay was further developed and subjected to validation. Assay’s adaptability to other CAR-encoding LVV and autologous CAR-T cell products was also investigated. Results: Measure of CD69 expression on the membrane of Jurkat-CAR-expressing cells is a specific indicator of CAR functionality. Quantification of CD69 in terms of mean fluorescence intensity (MFI), coupled with an intra-assay standard curve calibration, allows for a quantitative assay with high precision, specificity, robustness, linearity and accuracy. The assay has also shown optimal performance for a CARBCMA-LVV product. Importantly, we show that in primary T cells, CD69 expression reflects CAR-T cell cytotoxicity. After adaptation, we have applied a CD69-based potency test, with simultaneous measurement of CAR-T cell cytotoxicity, to autologous CAR-T cell products, demonstrating the assay’s specificity also in this context. Conclusions: We developed a validated, in vitro cell-based potency test, using a quantitative flow-cytometry method, for our CAR19-LVV. The assay is based on the detection of T-cell activation upon CAR binding to antigen, which is a measure of transgene functionality. The assay was easily adapted to another CAR-encoding LVV, targeting a different molecule. Furthermore, the same assay principle can be applied in the context of autologous CAR-T cell products. The quantitative CD69 potency assay shows reduced variability among autologous products compared to the IFNγ assay and allows for simultaneous evaluation of traditional semi-quantitative cytotoxicity, thereby directly evaluating the drug’s mechanism of action (MoA) in the same assay.

Background: Chimeric antigen receptor (CAR) T cells directed against CD19 have achieved impressive outcomes for the treatment of relapsed/refractory B lineage lymphoid neoplasms. However, CAR-T therapy still has important limitations due to severe side effects and the lack of efficiency in 40-50% of the patients. Most CARs-T products are generated using retroviral vectors with strong promoters. However, high CAR expression levels can lead to tonic signalling, premature exhaustion and over-stimulation of CAR-T cells, reducing efficacy and increasing side effects. TCR-like expression of the CAR through genome editing resulted in enhanced anti-tumour potency, reducing tonic signalling and improving CAR-T phenotype. In this manuscript, we searched for LVs that mimic the TCR expression pattern as a closer-to-clinic alternative for the generation of improved CAR-T cells. Methods: Different LVs containing viral and human promoters were analysed to select those that closely mimic a TCR-like kinetic profile upon T-cell activation. WAS gene proximal promoter-driven LVs (AW-LVs) were selected to express a second generation 4-1BB aCD19 CAR (ARI-0001) into T cells to generate AWARI CAR-T cells. TCR-like AWARI and EF1α-driven ARI CAR T cells were analysed for in vitro and in vivo killing efficiency using leukaemia and lymphoma cellular models. Tonic signalling, exhaustion markers and phenotype were determined by flow cytometry. Large-scale automated manufacturing of AWARI CAR-T cells was performed in a CliniMACs Prodigy bioreactor. Results: Our data showed that LVs expressing the transgene through the WAS gene proximal promoter mimic very closely the TCR (CD3) expression pattern kinetic upon TCR stimulation or antigen encounter. Compared to EF1α-driven ARI CAR-T cells, AWARI CAR-T cells exhibited a higher proportion of naïve/stem cell memory T cells with less exhausted phenotype after efficient killing of CD19+ cells both in vitro and in vivo . AWARI CAR-T cells also showed lower tonic signalling and reduced secretion of pro-inflammatory cytokines and were efficiently manufactured in large-scale GMP-like conditions. Conclusions: WAS-gene -promoter driven LVs can be used to generate physiological 4-1BB-CAR-T cell products with lower tonic signalling, improved phenotype and a safer profile. We propose the use of TCR-like LVs as an alternative to strong-promoter driven LVs for the generation of CAR-T products.