In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma.

Chimeric antigen receptor T (CAR-T) cell therapy is more effective in relapsed or refractory diffuse large B cell lymphoma (DLBCL) than other therapies, but a high proportion of patients relapse after CAR-T cell therapy owing to antigen escape, limited persistence of CAR-T cells, and immunosuppression in the tumor microenvironment. CAR-T cell exhaustion is a major cause of relapse. Epigenetic modifications can regulate T cell activation, maturation and depletion; they can be applied to reduce T cell depletion, improve infiltration, and promote memory phenotype formation to reduce relapse after CAR-T cell therapy. We propose to develop and validate in vitro the function of novel CAR-T cells for the treatment of DLBCL, which simultaneously express an anti-CD19 CAR with lysine-specific demethylase 1 (LSD1) short hairpin (sh)RNA to prevent depletion and prolong the survival of CAR-T cells. We designed an shRNA sequence targeting LSD1 mRNA, and created a vector with the following elements: the U6 promoter driving expression of the LSD1 shRNA sequence, the EF1a promoter driving a second-generation anti-CD19 CAR sequence encoding an anti-CD19 single-chain variable fragment (FMC63), the CD8 hinge and transmembrane structural domains, the CD28 co-stimulatory structural domain, and the CD3ζ-activating structural domain. The MFG-LSD1 shRNA anti-CD19 CAR plasmid was first constructed, then packaged in retroviral vectors and transduced into human primary peripheral blood mononuclear cell-derived T cells to generate the corresponding CAR-T cells. We examined by flow cytometry the efficiency of two CAR-T cells in killing U-2932 cells (a human DLBCL line) upon co-culture with RNAU6 anti-CD19 CAR-T cells or LSD1 shRNA anti-CD19 CAR-T cells. We analyzed Ki-67 staining of the CAR-T cells by flow cytometry on days 0, 5, and 10, and counted the cells to assess expansion. We also used flow cytometry to detect the central memory T cell (TCM) proportion. We detected the expression of the CAR in the CAR-T cells by flow cytometry, and observed transduction rates of 31.5% for RNAU6 anti-CD19 CAR-T cells and 60.7% for LSD1 shRNA anti-CD19 CAR-T cells. The killing efficiency of LSD1 shRNA anti-CD19 CAR-T cells was significantly higher than that of RNAU6 anti-CD19 CAR-T cells at the low effector target ratio. We further found that LSD1 shRNA anti-CD19 CAR-T cells secreted more IFN-γ and granzyme B than RNAU6 anti-CD19 CAR-T cells. CAR-T cells proliferated after U-2932 cell stimulation and were able to sustain proliferation. After stimulation via U-2932 cell co-culture, both RNAU6 anti-CD19 CAR-T and LSD1 shRNA anti-CD19 CAR-T populations had increased proportions of cells with the TCM phenotype, with a higher percentage among LSD1 shRNA anti-CD19 CAR-T cells. We developed a novel, feasible CD19-LSD1 shRNA CAR-T cell strategy for the treatment of DLBCL. Our in vitro assay results showed that LSD1 shRNA anti-CD19 CAR-T cells more effectively killed target cells than RNAU6 anti-CD19 CAR-T cells, and developed a higher proportion of TCM phenotype cells. LSD1 shRNA anti-CD19 CAR-T cells may represent a potential treatment for DLBCL.

Guo Z ，He M ，Liu N ，Yang Y ，Sun R ，Wang J ，Wang Q ... -  《Frontiers in Immunology》

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

《Jove-Journal of Visualized Experiments》

The impact of CD3ζ ITAM multiplicity and sequence on CAR T-cell survival and function.

Chimeric antigen receptor (CAR) expressing T-cells have shown great promise for the future of cancer immunotherapy with the recent clinical successes achieved in treating different hematologic cancers. Despite these early successes, several challenges remain in the field that require to be solved for the therapy to be more efficacious. One such challenge is the lack of long-term persistence of CD28 based CAR T-cells in patients. Although, CD28 based CAR T-cells elicit a robust acute anti-tumor response, they are more prone to early exhaustion, terminal differentiation and cell death due to their strong signaling patterns. Hence attenuation of signaling strength in CD28 based CARs is an accepted strategy to improve long-term CAR T-cell function and persistence in patients. Previous studies with the conventional T-cell receptor (TCR) have suggested that manipulation of CD3 immunoreceptor tyrosine-based activation motif (ITAM) sequences can alter TCR signaling strength. Based on these studies, we have designed 2nd generation murine anti-CD19 CD28 based CARs with restricted CD3ζ ITAM sequence diversity while maintaining a multiplicity of three. They are called ζAAA, ζBBB and ζCCC based on which CD3ζ ITAM they express. The goal of the study is to understand the non-redundant signaling properties of the individual CD3ζ ITAMs and their effect on CAR T-cell function. We hypothesized that the individual CD3ζ ITAMs will exhibit unique signaling properties in the ITAM restricted CARs which may allow for optimization of CAR signaling and improve CAR T-cell persistence and function. We subjected the ITAM restricted CAR T cells to various conditions of in vitro stimulation using CD19+ tumor cells or CD19-coated magnetic beads. Immunoblotting and flow cytometry based Ca2+ signaling assays were used to quantify signaling differences. Functional differences were studied using in vitro cytotoxicity, degranulation and cytokine expression assays. CAR T cell exhaustion and differentiation were studied using an in vitro exhaustion assay. We observed that ζAAA CARs had stronger signaling strength compared to ζBBB and ζCCC CARs. The signaling differences were reflected in their functional activation profiles with T-cells expressing ζAAA CARs having a strong activation profile and ζCCC CARs having a weak activation profile. ζCCC CAR T cells were less prone to differentiation and exhaustion. Since, weaker signaling ζCCC CARs favored less cell death, exhaustion and differentiation, they might be better candidates for improving long term survival and persistence of CAR T cells in patients.

Majumdar S ，Echelibe H ，Bettini M ，Bettini ML ... -  《Frontiers in Immunology》

Preclinical development of three novel CARs targeting CD79b for the treatment of non-Hodgkin's lymphoma and characterization of the loss of the target antigen.

Infusion of T cells modified with a chimeric antigen receptor (CAR) targeting CD19 has achieved exceptional responses in patients with non-Hodgkin's lymphoma (NHL), which led to the approval of CAR targeting CD19 (CART19) (Axi-cel and Liso-cel) as second line of treatment for adult patients with relapsed/refractory NHL. Unfortunately, 60% of patients still relapse after CART19 due to either a loss of expression of the target antigen (CD19) in the tumor cell, observed in 27% of relapsed patients, a limited CAR-T persistence, and additional mechanisms, including the suppression of the tumor microenvironment. Clinic strategies to prevent target antigen loss include sequential treatment with CARs directed at CD20 or CD22, which have caused loss of the second antigen, suggesting targeting other antigens less prone to disappear. CD79b, expressed in NHL, is a target in patients treated with antibody-drug conjugates (ADC). However, the limited efficacy of ADC suggests that a CAR therapy targeting CD79b might improve results. We designed three new CARs against CD79b termed CAR for Lymphoma (CARLY)1, 2 and 3. We compared their efficacy, phenotype, and inflammatory profiles with CART19 (ARI0001) and CARTBCMA (ARI0002h), which can treat NHL. We also analyzed the target antigen's expression loss (CD79b, CD19, and B-cell maturation antigen(BCMA)). We found that CARLY2 and CARLY3 had high affinity and specificity towards CD79b on B cells. In vitro, all CAR-T cells had similar anti-NHL efficacy, which was retained in an NHL model of CD19- relapse. In vivo, CARLY3 showed the highest efficacy. Analysis of the loss of the target antigen demonstrated that CARLY cells induced CD79b and CD19 downregulation on NHL cells with concomitant trogocytosis of these antigens to T cells, being most notorious in CARLY2, which had the highest affinity towards CD79b and CD19, and supporting the selection of CARLY3 to design a new treatment for patients with NHL. Finally, we created a CAR treatment based on dual targeting of CD79b and BCMA to avoid losing the target antigen. This treatment showed the highest efficacy and did not cause loss of the target antigen. Based on specificity, efficacy, and loss of the target antigen, CARLY3 represents a potential novel CAR treatment for NHL.

Esquinas E ，Moreno-Sanz A ，Sandá V ，Stodulski-Ciesla D ，Borregón J ，Peña-Blanque V ，Fernández-Calles J ，Fernandez-Fuentes N ，Serrano-Lopez J ，Juan M ，Engel P ，Llamas-Sillero P ，Solán-Blanco L ，Martin-Antonio B ... -  《Journal for ImmunoTherapy of Cancer》

CD19-chimeric antigen receptor-invariant natural killer T cells transactivate NK cells and reduce alloreactivity.

Invariant natural killer T (iNKT) cells are a small fraction of T lymphocytes with strong cytotoxic and immunoregulatory properties. We previously showed that human culture-expanded iNKT cells prevent alloreactivity and lyse primary leukemia blasts. Here, iNKT cells have several advantages over T cells based on their immunoregulatory capabilities. Since chimeric antigen receptors (CARs) increase the benefit of immune effector cells, they play a crucial role in improvement of cytotoxic abilities of novel cellular therapeutics such as iNKT cells. In the present study, we investigated transactivation of NK cells and prevention of alloreactivity through iNKT cells transduced with a CD19-directed CAR. iNKT cells were isolated by magnetic cell separation from peripheral blood mononuclear cells and transduced with a CD19-CAR retrovirus. Transduction efficiency, purity and cell subsets were measured by flow cytometry. Transactivation and cytotoxicity assays have been established to investigate the ability of CD19-CAR-iNKT cells to transactivate primary NK cells. A mixed lymphocyte reaction (MLR) was performed to explore the inhibition of alloreactive CD3+ T cells by CD19-CAR-iNKT cells. CD19-CAR-iNKT cells are able to transactivate NK cells independent of cell contact: The expression of activation marker CD69 was significantly increased and also production of the proinflammatory cytokine interferon-gamma was higher in NK cells pretreated with CD19-CAR-iNKT cells. Consequently, the cytotoxic activity of such NK cells was significantly increased being able to lyse leukemia cells more effectively than without prior transactivation. Adding CD19-CAR-iNKT cells to an MLR resulted in a decreased expression of the T cell activation marker CD25 on alloreactive CD3+ T lymphocytes stimulated with HLA mismatched dendritic cells. Also, the proliferation of alloreactive CD3+ T lymphocytes was significantly reduced in this setting. We demonstrate that CD19-CAR-iNKT cells keep their immunoregulatory properties despite transduction with a CAR making them an attractive effector cell population for application after allogeneic hematopoietic cell transplantation. By transactivating NK cells, increasing their cytotoxic activity and suppressing alloreactive T cells, they might further improve outcomes through prevention of both relapse and graft-versus-host disease.

Wesle A ，Moraes Ribeiro E ，Schairer R ，Keppeler H ，Korkmaz F ，Radszuweit P ，Bieber K ，Lengerke C ，Schneidawind D ，Schneidawind C ... -  《-》