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Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor-CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance.

Allogeneic cell therapies hold promise to be cost effective with scaled manufacturing for multi-dosing and on-demand off-the-shelf availability. A critical consideration for allogeneic cell products is their ability to persist, maintain function and avoid rejection by the patient's immune system. Genetic knockout (KO) of beta-2-microglobulin (B2M) leads to complete loss of cell-surface human leukocyte antigen (HLA) class I expression and efficiently abrogates CD8+ T-cell reactivity. However, loss of HLA class I triggers NK cell-mediated missing-self recognition and manipulation of B2M must therefore be combined with other immune-modulating strategies to limit recipient NK cell reactivity.We hypothesized that rejection by the patient's immune system can be diminished in primary CAR T cells, iPSC-derived T (iT) and NK (iNK) cells by reverse-engineering common tumor escape mechanisms. The adhesion molecules CD54 and CD58 are both present at the target cell side of the immune synapse, and loss of either of these molecules have previously been reported to elicit immune escape. Here, we show that the combined deletion of CD54 and CD58 in allogeneic immune effector cells makes them resistant to rejection by recipient immune cells through unidirectional reduced synapse formation (Figure 1A).HLA class I down-regulation by B2M silencing in primary T and NK cells triggered potent cytotoxicity by resting allogeneic NK cells. This response was mostly driven by educated NK cells expressing either NKG2A or killer cell immunoglobulin-like receptors (KIR) binding to HLA-E and HLA-C, respectively. However, over-expression of HLA-E or single HLA-C ligands in a K562 screening model only shut down the specific response of the NK cell subset carrying the cognate inhibitory receptor, resulting in only partial resistance to NK cells at the bulk level. Notably, the introduction of HLA-E was particularly detrimental in donors with expanded NKG2C+ NK cell subsets, due to its stimulatory effec, QC 20230316

On August 30, 2017, the U.S. Food and Drug Administration (US-FDA) approved tisagenlecleucel (KYMRIAH, Novartis, Basel, Switzerland), a synthetic bioimmune product of anti-CD19 chimeric antigen receptor-T cells (CAR-T), for the treatment of children and young adults with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). With this new era of personalized cancer immunotherapy, multiple challenges are present ranging from implementation of a CAR-T program to safe delivery of the drug, long-term toxicity monitoring and disease assessments. To address these issues, experts representing the American Society for Blood and Marrow Transplant (ASBMT), the European Group for Blood and Marrow Transplantation (EBMT), the International Society of Cell and Gene Therapy (ISCT), and the Foundation for the Accreditation of Cellular Therapy (FACT), formed a global CAR-T task force to identify and address key questions pertinent for hematologists and transplant physicians regarding the clinical use of anti CD19 CAR-T therapy in patients with B-ALL. This article presents an initial roadmap for navigating common clinical practice scenarios that will become more prevalent now that the first commercially available CAR-T product for B-ALL has been approved.

On August 30, 2017 the US Food and Drug Administration approved tisagenlecleucel (Kymriah; Novartis, Basel, Switzerland), a synthetic bioimmune product of anti-CD19 chimeric antigen receptor T cells (CAR-T), for the treatment of children and young adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). With this new era of personalized cancer immunotherapy, multiple challenges are present, ranging from implementation of a CAR-T program to safe delivery of the drug, long-term toxicity monitoring, and disease assessments. To address these issues experts representing the American Society for Blood and Marrow Transplant, the European Society for Blood and Marrow Transplantation, the International Society of Cell and Gene Therapy, and the Foundation for the Accreditation of Cellular Therapy formed a global CAR-T task force to identify and address key questions pertinent for hematologists and transplant physicians regarding the clinical use of anti CD19 CAR-T therapy in patients with B-ALL. This article presents an initial roadmap for navigating common clinical practice scenarios that will become more prevalent now that the first commercially available CAR-T product for B-ALL has been approved.

While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with high spatiotemporal precision for therapeutic applications.

While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with high spatiotemporal precision for therapeutic applications.

There remains an urgent need for the noninvasive tracking of transfused chimeric antigen receptor (CAR) T cells to determine their biodistribution, viability, expansion, and antitumor functionality. DOTA antibody reporter 1 (DAbR1) comprises a single-chain fragment of the antilanthanoid-DOTA antibody 2D12.5/G54C fused to the human CD4-transmembrane domain and binds irreversibly to lanthanoid (S)-2-(4-acrylamidobenzyl)-DOTA (AABD). The aim of this study was to investigate whether DAbR1 can be expressed on lymphocytes and used as a reporter gene as well as a suicide gene for therapy of immune-related adverse effects. Methods: DAbR1 was subcloned together with green fluorescent protein into an SFG-retroviral vector and used to transduce CD3/CD28-activated primary human T cells and second-generation 1928z (CAR) T cells. Cell surface expression of DAbR1 was confirmed by cell uptake studies with radiolabeled AABD. In addition, the feasibility of imaging of DAbR1-positive T cells in vivo after intravenous injection of 86Y/177Lu-AABD was studied and radiation doses determined. Results: A panel of DAbR1-expressing T cells and CAR T cells exhibited greater than 8-fold increased uptake of 86Y-AABD in vitro when compared with nontransduced cells. Imaging studies showed 86Y-AABD was retained by DAbR1-positive T cells while it continuously cleared from normal tissues, allowing for in vivo tracking of intravenously administered CAR T cells. Normal-organ dose estimates were favorable for repeated PET/CT studies. Selective T cell ablation in vivo with 177Lu-AABD seems feasible for clustered T-cell populations. Conclusion: We have demonstrated for the first time that T cells can be modified with DAbR1, enabling their in vivo tracking via PET and SPECT. The favorable biodistribution and high image contrast observed warrant further studies of this new reporter gene.

CD19 chimeric antigen receptors (CARs) have demonstrated great efficacy against a range of B cell malignancies. However, antigen escape and, more generally, heterogeneous antigen expression pose a challenge to applying CAR therapy to a wide range of cancers. We find that low-dose radiation sensitizes tumor cells to immune rejection by locally activated CAR T cells. In a model of pancreatic adenocarcinoma heterogeneously expressing sialyl Lewis-A (sLeA), we show that not only sLeA+ but also sLeA- tumor cells exposed to low-dose radiation become susceptible to CAR therapy, reducing antigen-negative tumor relapse. RNA sequencing analysis of low-dose radiation-exposed tumors reveals the transcriptional signature of cells highly sensitive to TRAIL-mediated death. We find that sLeA-targeted CAR T cells produce TRAIL upon engaging sLeA+ tumor cells, and eliminate sLeA- tumor cells previously exposed to systemic or local low-dose radiation in a TRAIL-dependent manner. These findings enhance the prospects for successfully applying CAR therapy to heterogeneous solid tumors. Local radiation is integral to many tumors standard of care and can be easily implemented as a CAR conditioning regimen.

Chimeric antigen receptor (CAR) therapy targeting CD19 has yielded remarkable outcomes in patients with acute lymphoblastic leukemia. To identify potential CAR targets in acute myeloid leukemia (AML), we probed the AML surfaceome for overexpressed molecules with tolerable systemic expression. We integrated large transcriptomics and proteomics datasets from malignant and normal tissues, and developed an algorithm to identify potential targets expressed in leukemia stem cells, but not in normal CD34+CD38- hematopoietic cells, T cells, or vital tissues. As these investigations did not uncover candidate targets with a profile as favorable as CD19, we developed a generalizable combinatorial targeting strategy fulfilling stringent efficacy and safety criteria. Our findings indicate that several target pairings hold great promise for CAR therapy of AML.

Globin gene transfer in autologous hematopoietic stem cells offers a potentially curative treatment option for patients suffering from beta-thalassemia major who lack an HLA-matched hematopoietic stem cell donor. Based on extensive preclinical investigation, we are initiating a phase 1 clinical trial using G-CSE mobilized, autologous CD34(+) cells transduced with a vector similar to the original TNS9 vector. Our first mobilizations in adult beta-thalassemic subjects have been well tolerated and yielded the required CD34(+) cell dose. To minimize toxicity to enrolled subjects, and in the absence of a demonstrated requirement for myeloablative conditioning, our trial will use a reduced intensity conditioning regimen. Because low vector titers may adversely affect efficacy and safety, we have focused on vector manufacturing processes. We are now in a position to transfer our globin lentiviral vectors in a clinically relevant dosage (averaging 0.8 vector copy per cell in bulk CD34(+) cells) and to supply clinical grade vector to collaborating centers in the U.S.A. and in Europe. We anticipate that the first U.S. trial of globin gene transfer will start in 2010.

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