No abstract

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19- leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.

Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML.

Adoptive cell therapy with genetically modified T cells has generated exciting outcomes in hematologic malignancies, but its application to solid tumors has proven challenging. This gap has spurred the investigation of alternative immune cells as therapeutics. Macrophages are potent immune effector cells whose functional plasticity leads to antitumor as well as protumor function in different settings, and this plasticity has led to notable efforts to deplete or repolarize tumor-associated macrophages. Alternatively, macrophages could be adoptively transferred after ex vivo genetic modification. In this review, we highlight the role of macrophages in solid tumors, the progress made with macrophage-focused immunotherapeutic modalities, and the emergence of chimeric antigen receptor macrophage cell therapy.

Epstein-Barr Virus (EBV), which is associated with multiple human tumors, persists as a minichromosome in the nucleus of B-lymphocytes and induces malignancies through incompletely understood mechanisms. Here, we present a large-scale functional genomic analysis of EBV. Our experimentally generated nucleosome positioning maps and viral protein binding data were integrated with over 700 publicly available high-throughput sequencing data sets for human lymphoblastoid cell lines mapped to the EBV genome. We found that viral lytic genes are coexpressed with cellular cancer-associated pathways, suggesting that the lytic cycle may play an unexpected role in virus-mediated oncogenesis. Host regulators of viral oncogene expression and chromosome structure were identified and validated, revealing a role for the B-cell-specific protein Pax5 in viral gene regulation and the cohesin complex in regulating higher order chromatin structure. Our findings provide a deeper understanding of latent viral persistence in oncogenesis and establish a valuable viral genomics resource for future exploration.

The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.

Some patients with otherwise treatment-resistant Hodgkin lymphoma (HL) could benefit from chimeric antigen receptor T cell (CART) therapy. However, HL lacks CD19 and contains a highly immunosuppressive tumor microenvironment (TME). We hypothesized that in HL, CART should target both malignant cells and the TME. We demonstrated CD123 on both HL cells and TME, including tumor-associated macrophages (TAM). In vitro, HL cells convert macrophages towards immunosuppressive TAM that inhibit T cell proliferation. In contrast, anti-CD123 CART recognized and killed TAM thus overcoming immunosuppression. Finally, we showed in immunodeficient mouse models that CART123 eradicate HL, and establish long-term immune memory. A novel platform that targets malignant cells and the microenvironment may be needed to successfully treat malignancies with an immunosuppressive milieu.