CD37 is a safe chimeric antigen receptor target to treat acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells in the bone marrow and the peripheral blood. Nearly half of the AML patients relapse after standard induction therapy, and new forms of therapy are urgently needed. Chimeric antigen receptor (CAR) T therapy has so far not been successful in AML due to lack of efficacy and safety. Indeed, the most attractive antigen targets are stem cell markers such as CD33 or CD123. We demonstrate that CD37, a mature B cell marker, is expressed in AML samples, and its presence correlates with the European LeukemiaNet (ELN) 2017 risk stratification. We repurpose the anti-lymphoma CD37CAR for the treatment of AML and show that CD37CAR T cells specifically kill AML cells, secrete proinflammatory cytokines, and control cancer progression in vivo. Importantly, CD37CAR T cells display no toxicity toward hematopoietic stem cells. Thus, CD37 is a promising and safe CAR T cell AML target.

Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16.

BACKGROUND: Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and the evasion of host immune responses. Currently, debulking surgery and platinum-based chemotherapy are the treatment cornerstones, although recurrence is common. As the clinical efficacy of immune checkpoint blockade is low, new immunotherapeutic strategies are needed. Chimeric antigen receptor (CAR) T cell therapy empowers patients' own T cells to fight and eradicate cancer, and has been tested against various targets in OC. A promising candidate is the MUC16 ectodomain. This ectodomain remains on the cell surface after cleavage of cancer antigen 125 (CA125), the domain distal from the membrane, which is currently used as a serum biomarker for OC. CA125 itself has not been tested as a possible CAR target. In this study, we examined the suitability of the CA125 as a target for CAR T cell therapy. METHODS: We tested a series of antibodies raised against the CA125 extracellular repeat domain of MUC16 and adapted them to the CAR format. Comparisons between these candidates, and against an existing CAR targeting the MUC16 ectodomain, identified K101 as having high potency and specificity. The K101CAR was subjected to further biochemical and functional tests, including examination of the effect of soluble CA125 on its activity. Finally, we used cell lines and advanced orthotopic patient-derived xenograft (PDX) models to validate, in vivo, the efficiency of our K101CAR construct. RESULTS: We observed a high efficacy of K101CAR T cells against cell lines and patient-derived tumors, in vitro and in vivo. We also demonstrated that K101CAR functionality was not impaired by the soluble antigen. Finally, in direct comparisons, K101CAR, which targets the CA125 extracellular repeat domains, was shown to have similar efficacy to the previously validated 4H11CAR, which targets the MUC16 ectodomain. CONCLUSIONS: Our in vitro and in vivo results, including PDX studies, demonstrate that the CA125 domain of MUC16 represents an excellent target for treating MUC16-positive malignancies.

Determination of CAR T cell metabolism in an optimized protocol.

Adoptive transfer of T cells modified to express chimeric antigenic receptors (CAR) has emerged as a solution to cure refractory malignancies. However, although CAR T cell treatment of haematological cancers has now shown impressive improvement in outcome, solid tumours have been more challenging to control. The latter type is protected by a strong tumour microenvironment (TME) which might impact cellular therapeutic treatments. Indeed, the milieu around the tumour can become particularly inhibitory to T cells by directly affecting their metabolism. Consequently, the therapeutic cells become physically impeded before being able to attack the tumour. It is therefore extremely important to understand the mechanism behind this metabolic break in order to develop TME-resistant CAR T cells. Historically, the measurement of cellular metabolism has been performed at a low throughput which only permitted a limited number of measurements. However, this has been changed by the introduction of real-time technologies which have lately become more popular to study CAR T cell quality. Unfortunately, the published protocols lack uniformity and their interpretation become confusing. We herein tested the essential parameters to perform a metabolic study on CAR T cells and propose a check list of factors that should be set in order to draw sound conclusion.

Efficient chimeric antigen receptor targeting of a central epitope of CD22.

Chimeric antigen receptor (CAR) T-cell therapy has had considerable success in the treatment of B-cell malignancies. Targeting the B-lineage marker CD19 has brought great advances to the treatment of acute lymphoblastic leukemia and B-cell lymphomas. However, relapse remains an issue in many cases. Such relapse can result from downregulation or loss of CD19 from the malignant cell population or expression of alternate isoforms. Consequently, there remains a need to target alternative B-cell antigens and diversify the spectrum of epitopes targeted within the same antigen. CD22 has been identified as a substitute target in cases of CD19-negative relapse. One anti-CD22 antibody-clone m971-targets a membrane-proximal epitope of CD22 and has been widely validated and used in the clinic. Here, we have compared m971-CAR with a novel CAR derived from IS7, an antibody that targets a central epitope on CD22. The IS7-CAR has superior avidity and is active and specific against CD22-positive targets, including B-acute lymphoblastic leukemia patient-derived xenograft samples. Side-by-side comparisons indicated that while IS7-CAR killed less rapidly than m971-CAR in vitro, it remains efficient in controlling lymphoma xenograft models in vivo. Thus, IS7-CAR presents a potential alternative candidate for the treatment of refractory B-cell malignancies.

A New Plasmacytoid Dendritic Cell-Based Vaccine in Combination with Anti-PD-1 Expands the Tumor-Specific CD8+ T Cells of Lung Cancer Patients.

The purpose of immune checkpoint inhibitor (ICI)-based therapies is to help the patient's immune system to combat tumors by restoring the immune response mediated by CD8+ cytotoxic T cells. Despite impressive clinical responses, most patients do not respond to ICIs. Therapeutic vaccines with autologous professional antigen-presenting cells, including dendritic cells, do not show yet significant clinical benefit. To improve these approaches, we have developed a new therapeutic vaccine based on an allogeneic plasmacytoid dendritic cell line (PDC*line), which efficiently activates the CD8+ T-cell response in the context of melanoma. The goal of the study is to demonstrate the potential of this platform to activate circulating tumor-specific CD8+ T cells in patients with lung cancer, specifically non-small-cell lung cancer (NSCLC). PDC*line cells loaded with peptides derived from tumor antigens are used to stimulate the peripheral blood mononuclear cells of NSCLC patients. Very interestingly, we demonstrate an efficient activation of specific T cells for at least two tumor antigens in 69% of patients irrespective of tumor antigen mRNA overexpression and NSCLC subtype. We also show, for the first time, that the antitumor CD8+ T-cell expansion is considerably improved by clinical-grade anti-PD-1 antibodies. Using PDC*line cells as an antigen presentation platform, we show that circulating antitumor CD8+ T cells from lung cancer patients can be activated, and we demonstrate the synergistic effect of anti-PD-1 on this expansion. These results are encouraging for the development of a PDC*line-based vaccine in NSCLC patients, especially in combination with ICIs.

How CAR T Cells Breathe.

The manufacture of efficacious CAR T cells represents a major challenge in cellular therapy. An important aspect of their quality concerns energy production and consumption, known as metabolism. T cells tend to adopt diverse metabolic profiles depending on their differentiation state and their stimulation level. It is therefore expected that the introduction of a synthetic molecule such as CAR, activating endogenous signaling pathways, will affect metabolism. In addition, upon patient treatment, the tumor microenvironment might influence the CAR T cell metabolism by compromising the energy resources. The access to novel technology with higher throughput and reduced cost has led to an increased interest in studying metabolism. Indeed, methods to quantify glycolysis and mitochondrial respiration have been available for decades but were rarely applied in the context of CAR T cell therapy before the release of the Seahorse XF apparatus. The present review will focus on the use of this instrument in the context of studies describing the impact of CAR on T cell metabolism and the strategies to render of CAR T cells more metabolically fit.

Recombinant Transcription Factors (TFs) Fused to ZEBRA Minimal Transduction Domain (MD) for Modulation of mRNA Transcripts.

Transcription factors (TFs) are key players in the control of gene expression and consequently all major cellular process, ranging from cell fate determination to cell cycle control and response to the environment.In particular cases, their ectopic expression has shown great promise in cell reprogramming for regenerative medicine, ontogenesis studies, and cell modeling. The current reprogramming methods mainly rely on gene transfer, therefore require technological improvements to limit genetic imprinting and improve safety. Direct protein delivery could represent an attractive alternative. Cell-penetrating peptides (CPPs) fused to recombinant TFs or other proteins involved in the epigenetic definition of cells have great potential in this context. We have thus developed the direct vectorization of Oct4, Sox2, or Nanog TFs and the posttranscriptional regulatory RNA-binding protein Lin28a by using the minimal transduction domain (MD11) of Epstein-Barr virus ZEBRA protein.This section describes the molecular cloning and production of different TFs fused to ZEBRA MD11 domain in the E. coli expression system. We also include the optimized purification conditions for each recombinant protein. The treatment of primary fibroblasts as well as cord blood-derived hematopoietic stem cells is also described. Finally, the transcriptional activation of the target genes following the transfer of TFs analyzed by quantitative PCR is presented.Our work primarily finds applications for advanced medicinal products, an area that requires novel therapy designs and delivery systems devoid of genetic material transfer to improve safety.

Pharmacologic Control of CAR T Cells.

Chimeric antigen receptor (CAR) therapy is a promising modality for the treatment of advanced cancers that are otherwise incurable. During the last decade, different centers worldwide have tested the anti-CD19 CAR T cells and shown clinical benefits in the treatment of B cell tumors. However, despite these encouraging results, CAR treatment has also been found to lead to serious side effects and capricious response profiles in patients. In addition, the CD19 CAR success has been difficult to reproduce for other types of malignancy. The appearance of resistant tumor variants, the lack of antigen specificity, and the occurrence of severe adverse effects due to over-stimulation of the therapeutic cells have been identified as the major impediments. This has motivated a growing interest in developing strategies to overcome these hurdles through CAR control. Among them, the combination of small molecules and approved drugs with CAR T cells has been investigated. These have been exploited to induce a synergistic anti-cancer effect but also to control the presence of the CAR T cells or tune the therapeutic activity. In the present review, we discuss opportunistic and rational approaches involving drugs featuring anti-cancer efficacy and CAR-adjustable effect.

Evaluation of the human type 3 adenoviral dodecahedron as a vector to target acute myeloid leukemia.

Intensive systemic chemotherapy is the gold standard of acute myeloid leukemia (AML) treatment and is associated with considerable off-target toxicities. Safer and targeted delivery systems are thus urgently needed. In this study, we evaluated a virus-like particle derived from the human type 3 adenovirus, called the adenoviral dodecahedron (Dd) to target AML cells. The vectorization of leukemic cells was proved very effective at nanomolar concentrations in a time- and dose-dependent manner, without vector toxicity. The internalization involved clathrin-mediated energy-dependent endocytosis and strongly correlated with the expression of αVß3 integrin. The treatment of healthy donor peripheral blood mononuclear cells showed a preferential targeting of monocytes compared to lymphocytes and granulocytes. Similarly, monocytes but also AML blasts were the best-vectorized populations in patients while acute lymphoid leukemia blasts were less efficiently targeted. Importantly, AML leukemic stem cells (LSCs) could be addressed. Finally, Dd reached peripheral monocytes and bone marrow hematopoietic stem and progenitor cells following intravenous injection in mice, without excessive spreading in other organs. These findings reveal Dd as a promising myeloid vector especially for therapeutic purposes in AML blasts, LSCs, and progenitor cells.

Targeted release of transcription factors for human cell reprogramming by ZEBRA cell-penetrating peptide.

Transcription factors (TFs) are key actors of the control of gene expression and consequently of every major process within cells, ranging from cell fate determination, cell cycle control and response to environment. Their ectopic expression has proven high potential in reprogramming cells for regenerative medicine; ontogenesis studies and cell based modelling. Direct delivery of proteins could represent an alternative to current reprogramming methods using gene transfer but still needs technological improvements. Herein, we set-up an efficient cellular penetration of recombinant TFs fused to the minimal transduction domain (MD) from the ZEBRA protein. We show that ZEBRA MD-fused TFs applied on primary human fibroblasts and cord blood CD34+ hematopoietic stem cells route through the cytoplasm to the nucleus. The delivery of Oct4, Sox2 and Nanog by MD leads to the activation of mRNA transcripts from genes regulated by these TFs. Moreover, the expression of genes involved in the pluripotency network but not directly bound by these TFs, is also induced. Overall, the repeated application of MD-Oct4, MD-Sox2, MD-Nanog TFs and the post-transcriptional regulator RNA-binding protein MD-Lin28a, triggers the rejuvenation of human fibroblasts and CD34+ cells. This study provides powerful tools for cell fate reprogramming without genetic interferences.

Targeted release of transcription factors for cell reprogramming by a natural micro-syringe.

Ectopic expression of defined transcription factors (TFs) for cell fate handling has proven high potential interest in reprogramming differentiated cells, in particular for regenerative medicine, ontogenesis study and cell based modelling. Pluripotency or transdifferentiation induction as TF mediated differentiation is commonly produced by transfer of genetic information with safety concerns. The direct delivery of proteins could represent a safer alternative but still needs significant advances to be efficient. We have successfully developed the direct delivery of proteins by an attenuated bacterium with a type 3 secretion system that does not require challenging and laborious steps for production and purification of recombinant molecules. Here we show that this natural micro-syringe is able to inject TFs to primary human fibroblasts and cord blood CD34+ hematopoietic stem cells. The signal sequence for vectorization of the TF Oct4 has no effect on DNA binding to its nucleic target. As soon as one hour after injection, vectorized TFs are detectable in the nucleus. The injection process is not associated with toxicity and the bacteria can be completely removed from cell cultures. A three days targeted release of Oct4 or Sox2 embryonic TFs results in the induction of the core pluripotency genes expression in fibroblasts and CD34+ hematopoietic stem cells. This micro-syringe vectorization represents a new strategy for TF delivery and has potential applications for cell fate reprogramming.