Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CART cell manufacturing process

Design and Evaluation of TIM-3-CD28 Checkpoint Fusion Proteins to Improve Anti-CD19 CAR T-Cell Function

Therapeutic targeting of inhibitory checkpoint molecules in combination with chimeric antigen receptor (CAR) T cells is currently investigated in a variety of clinical studies for treatment of hematologic and solid malignancies. However, the impact of co-inhibitory axes and their therapeutic implication remains understudied for the majority of acute leukemias due to their low immunogenicity/mutational load. The inhibitory exhaustion molecule TIM-3 is an important marker for the interaction of T cells with leukemic cells. Moreover, inhibitory signals from malignant cells could be transformed into stimulatory signals by synthetic fusion molecules with extracellular inhibitory receptors fused to an intracellular stimulatory domain. Here, we designed a variety of different TIM-3-CD28 fusion proteins to turn inhibitory signals derived by TIM-3 engagement into T-cell activation through CD28. In the absence of anti-CD19 CAR, two TIM-3-CD28 fusion receptors with large parts of CD28 showed strongest responses in terms of cytokine secretion and proliferation upon stimulation with anti-CD3 antibodies compared to controls. We then combined these two novel TIM-3-CD28 fusion proteins with first- and second-generation anti-CD19 CAR T cells and found that the fusion receptor can increase proliferation, activation, and cytotoxic capacity of conventional anti-CD19 CAR T cells. These additionally armed CAR T cells showed excellent effector function. In terms of safety considerations, the fusion receptors showed exclusively increased cytokine release, when the CAR target CD19 was present. We conclude that combining checkpoint fusion proteins with anti-CD19 CARs has the potential to increase T-cell proliferation capacity with the intention to overcome inhibitory signals during the response against malignant cells

The altering cellular components and function in tumor microenvironment during remissive and relapsed stages of anti-CD19 CAR T-cell treated lymphoma mice

Anti-CD19 chimeric antigen receptor (CAR) T cells represent a highly promising strategy for B-cell malignancies. Despite the inspiring initial achievement, remission in a notable fraction of subjects is short-lived, and relapse remains a major challenge. Tumor microenvironment (TME) was proved to be aroused by CAR T cells; however, little is known about the dynamic characteristics of cellular components in TME especially during the different phases of disease after anti-CD19 CAR T-cell treatment. We took advantage of an immunocompetent model receiving syngeneic A20 lymphoma cells to dissect the changes in TME with or without CAR T-cell injection. We found that anti-CD19 CAR T-cell treatment attenuated the symptoms of lymphoma and significantly prolonged mice survival through eradicating systemic CD19+ cells. Increased myeloid subsets, including CD11c+ DCs and F4/80+ macrophages with higher MHC II and CD80 expression in bone marrow, spleen, and liver, were detected when mice reached remission after anti-CD19 CAR T treatment. Compared to mice without anti-CD19 CAR T administration, intrinsic T cells were triggered to produce more IFN-γ and TNF-α. However, some lymphoma mice relapsed by day 42 after therapy, which coincided with CAR T-cell recession, decreased myeloid cell activation and increased Treg cells. Elevated intrinsic T cells with high PD-1 and TIGIT exhaust signatures and attenuated cytotoxicity in TME were associated with the late-stage relapse of CAR T-cell treatment. In summary, the cellular compositions of TME as allies of CAR T cells may contribute to the anti-tumor efficacy at the initial stage, whereas anti-CD19 CAR T-cell disappearance and host response immunosuppression may work together to cause lymphoma relapse after an initial, near-complete elimination phase

Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers.

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 (CART19) 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.post-print1691 K

Treatment of Adult Patients with Relapsed/Refractory B-Cell Philadelphia-Negative Acute Lymphoblastic Leukemia

The majority of adult patients affected by B-cell acute lymphoblastic leukemia (B-ALL) will relapse after an initial response, while approximately 20% will display primary resistant disease. Patients suffering from relapsed/refractory B-ALL have a very poor outcome. Allogeneic hematopoietic cell transplantation (HCT) still represents the only curative approach, but is not so frequently feasible, because of patient’s fitness, donor availability, and the ability to achieve a remission prior to HCT. The estimated remission rates with conventional cytotoxic agents are around 30%, but they are short-lived. These disappointing results led to the introduction of new immunologic-based treatments—blinatumomab and inotuzumab. They produced a substantial improvement in terms of response rates, with the ability, in most cases, to induce a minimal residual disease (MRD)-negative status. Similarly, T cells engineered to express a CD19-specific chimeric antigen receptor (CAR-T) have yielded sensational results among patients with relapsed/refractory B-ALL, with unexpectedly high MRD-negative complete remissions rates. However, the first studies looking at long-term outcomes after CAR-T infusions told us that a significant fraction of such responses are not durable, and may benefit from a consolidation approach such as an allogeneic HCT

Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Non-invasive epigenome editing is a promising strategy for engineering gene expression programs, yet potency, specificity, and persistence remain challenging. Here we show that effective epigenome editing is gated at single-base precision via 'keyhole' sites in endogenous regulatory DNA. Synthetic repressors targeting promoter keyholes can ablate gene expression in up to 99% of primary cells with single-gene specificity and can seamlessly repress multiple genes in combination. Transient exposure of primary T cells to keyhole repressors confers mitotically heritable silencing that persists to the limit of primary cultures in vitro and for at least 4 weeks in vivo, enabling manufacturing of cell products with enhanced therapeutic efficacy. DNA recognition and effector domains can be encoded as separate proteins that reassemble at keyhole sites and function with the same efficiency as single chain effectors, enabling gated control and rapid screening for novel functional domains that modulate endogenous gene expression patterns. Our results provide a powerful and exponentially flexible system for programming gene expression and therapeutic cell products