Characterization of atypical T cells generated during ex vivo expansion process for T cell-based adoptive immunotherapy.

Engineered T cell-based adoptive immunotherapies met promising success for the treatment of hematological malignancies. Nevertheless, major hurdles remain to be overcome regarding the management of relapses and the translation to solid tumor settings. Properties of T cell-based final product should be appropriately controlled to fine-tune the analysis of clinical trial results, to draw relevant conclusions, and finally to improve the efficacy of these immunotherapies. For this purpose, we addressed the existence of atypical T cell subsets and deciphered their phenotypic and functional features in an HPV16-E7 specific and MHC II-restricted transgenic-TCR-engineered T cell setting. To note, atypical T cell subsets include mismatched MHC/co-receptor CD8 or CD4 and miscommitted CD8+ or CD4+ T cells. We generated both mismatched and appropriately matched MHC II-restricted transgenic TCR on CD8 and CD4-expressing T cells, respectively. We established that CD4+ cultured T cells exhibited miscommitted phenotypic cytotoxic pattern and that both interleukin (IL)-2 or IL-7/IL-15 supplementation allowed for the development of this cytotoxic phenotype. Both CD4+ and CD8+ T cell subsets, transduced with HPV16-E7 specific transgenic TCR, demonstrated cytotoxic features after exposure to HPV-16 E7-derived antigen. Ultimately, the presence of such atypical T cells, either mismatched MHC II-restricted TCR/CD8+ T cells or cytotoxic CD4+ T cells, is likely to influence the fate of patient-infused T cell product and would need further investigation.

IL-7 producing immunotherapy improves ex vivo T cell functions of immunosenescent patients, especially post hip fracture.

Following acute stress such as trauma or sepsis, most of critically ill elderly patients become immunosuppressed and susceptible to secondary infections and enhanced mortality. We have developed a virus-based immunotherapy encoding human interleukin-7 (hIL-7) aiming at restoring both innate an adaptative immune homeostasis in these patients. We assessed the impact of this encoded hIL-7 on the ex vivo immune functions of T cells from PBMC of immunosenescent patients with or without hip fracture. T-cell ex vivo phenotyping was characterized in terms of senescence (CD57), IL-7 receptor (CD127) expression, and T cell differentiation profile. Then, post stimulation, activation status, and functionality (STAT5/STAT1 phosphorylation and T cell proliferation assays) were evaluated by flow cytometry. Our data show that T cells from both groups display immunosenescence features, express CD127 and are activated after stimulation by virotherapy-produced hIL-7-Fc. Interestingly, hip fracture patients exhibit a unique functional ability: An important T cell proliferation occurred compared to controls following stimulation with hIL-7-Fc. In addition, stimulation led to an increased naïve T cell as well as a decreased effector memory T cell proportions compared to controls. This preliminary study indicates that the produced hIL-7-Fc is well recognized by T cells and initiates IL-7 signaling through STAT5 and STAT1 phosphorylation. This signaling efficiently leads to T cell proliferation and activation and enables a T cell "rejuvenation." These results are in favor of the clinical development of the hIL-7-Fc expressing virotherapy to restore or induce immune T cell responses in immunosenescent hip fracture patients.

Umbilical Cord Blood as a Source of Less Differentiated T Cells to Produce CD123 CAR-T Cells.

Chimeric Antigen Receptor (CAR) therapy has led to great successes in patients with leukemia and lymphoma. Umbilical Cord Blood (UCB), stored in UCB banks, is an attractive source of T cells for CAR-T production. We used a third generation CD123 CAR-T (CD28/4-1BB), which was previously developed using an adult's Peripheral Blood (PB), to test the ability of obtaining CD123 CAR-T from fresh or cryopreserved UCB. We obtained a cell product with a high and stable transduction efficacy, and a poorly differentiated phenotype of CAR-T cells, while retaining high cytotoxic functions in vitro and in vivo. Moreover, CAR-T produced from cryopreserved UCB are as functional as CAR-T produced from fresh UCB. Overall, these data pave the way for the clinical development of UCB-derived CAR-T. UCB CAR-T could be transferred in an autologous manner (after an UCB transplant) to reduce post-transplant relapses, or in an allogeneic setting, thanks to fewer HLA restrictions which ease the requirements for a match between the donor and recipient.

Homeostatic cytokines tune naivety and stemness of cord blood-derived transgenic T cells.

Engineered T-cell therapies have proven to be successful in cancer and their clinical effectiveness is directly correlated with the infused T-cell differentiation profile. Indeed, stem cell memory and central memory T cells proliferate and persist longer in vivo compared with more-differentiated T cells, while conferring enhanced antitumor activity. Here, we propose an optimized process using cord blood (CB) to generate minimally differentiated T-cell products in terms of phenotype, function, gene expression, and metabolism, using peripheral blood (PB)-derived T cells cultured with IL-2 as a standard. Phenotypically, CB-derived T cells, particularly CD4 T cells, are less differentiated than their PB counterparts when cultured with IL-2 or with IL-7 and IL-15. Furthermore, culture with IL-7 and IL-15 enables better preservation of less-differentiated CB-derived T cells compared with IL-2. In addition, transcriptomic and metabolic assessments of CB-derived transgenic T cells cultured with IL-7 and IL-15 point out their naivety and stemness signature. These relatively quiescent transgenic T cells are nevertheless primed for secondary stimulation and cytokine production. In conclusion, our study indicates that CB may be used as a source of early differentiated T cells to develop more effective adoptive cancer immunotherapy.

An unmet need: Harmonization of IL-7 and IL-15 combination for the ex vivo generation of minimally differentiated T cells.

T cell-based adoptive cell transfer therapy is now clinically used to fight cancer with CD19-targeting chimeric antigen receptor T cells. The use of other T cell-based immunotherapies relying on antigen-specific T cells, genetically modified or not, is expanding in various neoplastic diseases. T cell manufacturing has evolved through sophisticated processes to produce T cells with improved therapeutic potential. Clinical-grade manufacturing processes associated with these therapies must meet pharmaceutical requirements and therefore be standardized. Here, we focus on the use of cytokines to expand minimally differentiated T cells, as well as their standardization and harmonization in research and clinical settings.

Validation of a method evaluating T cell metabolic potential in compliance with ICH Q2 (R1).

BACKGROUND: Metabolic cell features are able to give reliable information on cell functional state. Thus, metabolic potential assessment of T cells in malignancy setting represents a promising area, especially in adoptive cell therapy procedures. Easy to set up and convenient Seahorse technology have recently been proposed by Agilent Technologies and it could be used to monitor T cells metabolic potential. However, this method demonstrates an inter-assay variability and lacks practices standardization. RESULTS: We aimed to overcome these shortcomings thanks to a lymphoblastic derived JURKAT cell line seeding in each experiment to standardize the Seahorse process. We used an adapted XF Cell MitoStress Kit protocol, consisting in the evaluation of basal, stressed and maximal glycolysis and oxidative phosphorylation related parameters, through sequential addition of oligomycin and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) to a glucose containing medium. Data were acquired and analyzed through Agilent Seahorse XFe96 analyzer. Indeed, we validated this method in the light of ICH Q2 (R1) guidelines. We were able to confirm the specificity and accuracy of the method. We also demonstrated the precision, linearity and range of the method in our experimental conditions. CONCLUSION: The validation of the method consisting in a JURKAT cell line experimental incorporation as a control material contributes to improve the Seahorse technology's robustness. These results lay the groundwork for the implementation of this technology to optimize T cell based cellular therapy products production process and monitoring.

Isolation and Characterization of an HLA-DRB1*04-Restricted HPV16-E7 T Cell Receptor for Cancer Immunotherapy.

High-risk human papillomavirus (HPV) infection is a causal factor in oropharyngeal and gynecological malignancies, and development of HPV-targeted immunotherapy could be used to treat patients with these cancers. T cell-mediated adoptive immunotherapy targeting E6 and E7, two HPV16 proteins consistently expressed in tumor cells, appears to be both attractive and safe. However, isolation of HPV-specific T cells is difficult owing to the low frequency of these cell precursors in the peripheral blood. In addition, HPV-positive cancer cells often down-regulate major histocompatibility complex (MHC) class I expression ex vivo, limiting the efficacy of MHC class I-restricted approaches. Of particular interest is that both CD4 and CD8 T cells can mediate the responses. Given that CD4 T cells play a critical role in coordinating effective antitumor responses, the generation of a T helper response in patients with HPV16-associated malignancies would unleash the ultimate potential of immunotherapy. In this view, T-cell receptor (TCR) gene transfer could be a relevant strategy to generate HPV16-E7-specific and MHC class II-restricted T cells in sufficient numbers. An HPV16-E7/HLA-DRB1*04 TCR has been isolated from a cancer patient with complete response, and retroviral particles encoding this TCR have been produced. The transgenic TCR is highly expressed in transduced T cells, with a functional inducible caspase-9 suicide gene safety cassette. TCR transgenic T cells are HPV16-E770-89 specific and HLA-DRB1*04 restricted, as determined by interferon (IFN)-γ secretion. CD8 and CD4 T cells are equivalently transduced and secrete interleukin-2 and IFN-γ when cultured with appropriate targets. We also demonstrate that TCR transgenic T cells recognize the endogenously processed and presented HPV16-E770-89 peptide. In conclusion, our data indicate that the production of MHC class II-restricted HPV16-E7-specific T cells is feasible through TCR gene transfer and could be used for immunotherapy.