Synthetic dual co-stimulation increases the potency of HIT and TCR-targeted cell therapies.

Chimeric antigen receptor T cells have dramatically improved the treatment of hematologic malignancies. T cell antigen receptor (TCR)-based cell therapies are yet to achieve comparable outcomes. Importantly, chimeric antigen receptors not only target selected antigens but also reprogram T cell functions through the co-stimulatory pathways that they engage upon antigen recognition. We show here that a fusion receptor comprising the CD80 ectodomain and the 4-1BB cytoplasmic domain, termed 80BB, acts as both a ligand and a receptor to engage the CD28 and 4-1BB pathways, thereby increasing the antitumor potency of human leukocyte antigen-independent TCR (HIT) receptor- or TCR-engineered T cells and tumor-infiltrating lymphocytes. Furthermore, 80BB serves as a switch receptor that provides agonistic 4-1BB co-stimulation upon its ligation by the inhibitory CTLA4 molecule. By combining multiple co-stimulatory features in a single antigen-agnostic synthetic receptor, 80BB is a promising tool to sustain CD3-dependent T cell responses in a wide range of targeted immunotherapies.

Transcatheter aortic valve implantation for aortic regurgitation in HeartMate II supported patient using Myval THV: a case report.

De novo aortic regurgitation (AR) presents a great challenge following left ventricular assist device (LVAD) implantation and requires valve replacement in some cases. Patients with LVAD are frequently those who underwent multiple previous sternotomies or suffer from multiple comorbidities. Thus, they are at high surgical risk for further sternotomy. Transcatheter aortic valve implantation (TAVI) previously approved for treatment of severe aortic stenosis is also used for this category of patients. Here, we report the case of a young female patient supported with heart mate II LVAD who presented with severe de novo AR. The patient was successfully treated with TAVI using Myval trancatheter heart valve (THV) in our center. To our knowledge, our patient is the first to be treated with such type of valve using TAVI procedure in LVAD supported patients.

CD19 CAR antigen engagement mechanisms and affinity tuning.

Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface antigens through an scFv (binder), the affinity of which is central to determining CAR T cell function and therapeutic success. CAR T cells targeting CD19 were the first to achieve marked clinical responses in patients with relapsed/refractory B cell malignancies and to be approved by the U.S. Food and Drug Administration (FDA). We report cryo-EM structures of CD19 antigen with the binder FMC63, which is used in four FDA-approved CAR T cell therapies (Kymriah, Yescarta, Tecartus, and Breyanzi), and the binder SJ25C1, which has also been used extensively in multiple clinical trials. We used these structures for molecular dynamics simulations, which guided creation of lower- or higher-affinity binders, and ultimately produced CAR T cells endowed with distinct tumor recognition sensitivities. The CAR T cells exhibited different antigen density requirements to trigger cytolysis and differed in their propensity to prompt trogocytosis upon contacting tumor cells. Our work shows how structural information can be applied to tune CAR T cell performance to specific target antigen densities.

Programming CAR T Cell Tumor Recognition: Tuned Antigen Sensing and Logic Gating.

The success of chimeric antigen receptor (CAR) T cells targeting B-cell malignancies propelled the field of synthetic immunology and raised hopes to treat solid tumors in a similar fashion. Antigen escape and the paucity of tumor-restricted CAR targets are recognized challenges to fulfilling this prospect. Recent advances in CAR T cell engineering extend the toolbox of chimeric receptors available to calibrate antigen sensitivity and combine receptors to create adapted tumor-sensing T cells. Emerging engineering strategies to lower the threshold for effective antigen recognition, when needed, and enable composite antigen recognition hold great promise for overcoming tumor heterogeneity and curbing off-tumor toxicities. SIGNIFICANCE: Improving the clinical efficacy of CAR T cell therapies will require engineering T cells that overcome heterogeneous and low-abundance target expression while minimizing reactivity to normal tissues. Recent advances in CAR design and logic gating are poised to extend the success of CAR T cell therapies beyond B-cell malignancies.

Generation of T-cell-receptor-negative CD8αß-positive CAR T cells from T-cell-derived induced pluripotent stem cells.

The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T-cell-derived induced pluripotent stem cells (TiPS) are a promising source for the generation of 'off-the-shelf' CAR T cells, but the in vitro differentiation of TiPS often yields T cells with suboptimal features. Here we show that the premature expression of the T-cell receptor (TCR) or a constitutively expressed CAR in TiPS promotes the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive differentiation. Delaying CAR expression and calibrating its signalling strength in TiPS enabled the generation of human TCR- CD8αß+ CAR T cells that perform similarly to CD8αß+ CAR T cells from peripheral blood, achieving effective tumour control on systemic administration in a mouse model of leukaemia and without causing graft-versus-host disease. Driving T-cell maturation in TiPS in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αß+ T cells for a broad range of immunotherapies.

HLA-independent T cell receptors for targeting tumors with low antigen density.

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.

Generation of Pure Highly Functional Human Anti-Tumor Specific Cytotoxic T Lymphocytes With Stem Cell-Like Memory Features for Melanoma Immunotherapy.

Adoptive immunotherapy based on the transfer of anti-tumor cytotoxic T lymphocytes (CTLs) is a promising strategy to cure cancers. However, rapid expansion of numerous highly functional CTLs with long-lived features remains a challenge. Here, we constructed NIH/3T3 mouse fibroblast-based artificial antigen presenting cells (AAPCs) and precisely evaluated their ability to circumvent this difficulty. These AAPCs stably express the essential molecules involved in CTL activation in the HLA-A*0201 context and an immunogenic HLA-A*0201 restricted analogue peptide derived from MART-1, an auto-antigen overexpressed in melanoma. Using these AAPCs and pentamer-based magnetic bead-sorting, we defined, in a preclinical setting, the optimal conditions to expand pure MART-1-specific CTLs. Numerous highly purified MART-1-specific CTLs were rapidly obtained from healthy donors and melanoma patients. Both TCR repertoire and CDR3 sequence analyses revealed that MART-1-specific CTL responses were similar to those reported in the literature and obtained with autologous or allogeneic presenting cells. These MART-1-specific CTLs were highly cytotoxic against HLA-A*0201+ MART-1+ tumor cells. Moreover, they harbored a suitable phenotype for immunotherapy, with effector memory, central memory and, most importantly, stem cell-like memory T cell features. Notably, the cells harboring stem cell-like memory phenotype features were capable of self-renewal and of differentiation into potent effector anti-tumor T cells. These "off-the-shelf" AAPCs represent a unique tool to rapidly and easily expand large numbers of long-lived highly functional pure specific CTLs with stem cell-like memory T cell properties, for the development of efficient adoptive immunotherapy strategies against cancers.

Immunogenicity of CAR T cells in cancer therapy.

Patient-derived T cells genetically reprogrammed to express CD19-specific chimeric antigen receptors (CARs) have shown remarkable clinical responses and are commercially available for the treatment of patients with certain advanced-stage B cell malignancies. Nonetheless, several trials have revealed pre-existing and/or treatment-induced immune responses to the mouse-derived single-chain variable fragments included in these constructs. These responses might have contributed to both treatment failure and the limited success of redosing strategies observed in some patients. Data from early phase clinical trials suggest that CAR T cells are also associated with immunogenicity-related events in patients with solid tumours. Generally, the clinical implications of anti-CAR immune responses are poorly understood and highly variable between different CAR constructs and malignancies. These observations highlight an urgent need to uncover the mechanisms of immunogenicity in patients receiving CAR T cells and develop validated assays to enable clinical detection. In this Review, we describe the current clinical evidence of anti-CAR immune responses and discuss how new CAR T cell technologies might impact the risk of immunogenicity. We then suggest ways to reduce the risks of anti-CAR immune responses to CAR T cell products that are advancing towards the clinic. Finally, we summarize measures that investigators could consider in order to systematically monitor and better comprehend the possible effects of immunogenicity during trials involving CAR T cells as well as in routine clinical practice.

Brucella prosthetic valve endocarditis with septic and cardiogenic shock.

A young man with aortic prosthetic valve replacement, presented with prolonged fever and diagnosed with brucella endocarditis based on positive transthoracic echo findings with high titer positive brucellacapt serology. He was started on medical treatment with doxycycline and rifampin to which gentamicin and ceftriaxone were added and he was planned for surgical intervention. Unfortunately, the patient developed cardiogenic with septic shock before performing surgery and died within 24 h soon after admission.

Presence of T cells directed against CD20-derived peptides in healthy individuals and lymphoma patients.

Preclinical and clinical studies have suggested that cancer treatment with antitumor antibodies induces a specific adaptive T cell response. A central role in this process has been attributed to CD4+ T cells, but the relevant T cell epitopes, mostly derived from non-mutated self-antigens, are largely unknown. In this study, we have characterized human CD20-derived epitopes restricted by HLA-DR1, HLA-DR3, HLA-DR4, and HLA-DR7, and investigated whether T cell responses directed against CD20-derived peptides can be elicited in human HLA-DR-transgenic mice and human samples. Based on in vitro binding assays to recombinant human MHC II molecules and on in vivo immunization assays in H-2 KO/HLA-A2+-DR1+ transgenic mice, we have identified 21 MHC II-restricted long peptides derived from intracellular, membrane, or extracellular domains of the human non-mutated CD20 protein that trigger in vitro IFN-γ production by PBMCs and splenocytes from healthy individuals and by PBMCs from follicular lymphoma patients. These CD20-derived MHC II-restricted peptides could serve as a therapeutic tool for improving and/or monitoring anti-CD20 T cell activity in patients treated with rituximab or other anti-CD20 antibodies.

CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape.

Chimeric antigen receptors (CARs) are synthetic antigen receptors that reprogram T cell specificity, function and persistence1. Patient-derived CAR T cells have demonstrated remarkable efficacy against a range of B-cell malignancies1-3, and the results of early clinical trials suggest activity in multiple myeloma4. Despite high complete response rates, relapses occur in a large fraction of patients; some of these are antigen-negative and others are antigen-low1,2,4-9. Unlike the mechanisms that result in complete and permanent antigen loss6,8,9, those that lead to escape of antigen-low tumours remain unclear. Here, using mouse models of leukaemia, we show that CARs provoke reversible antigen loss through trogocytosis, an active process in which the target antigen is transferred to T cells, thereby decreasing target density on tumour cells and abating T cell activity by promoting fratricide T cell killing and T cell exhaustion. These mechanisms affect both CD28- and 4-1BB-based CARs, albeit differentially, depending on antigen density. These dynamic features can be offset by cooperative killing and combinatorial targeting to augment tumour responses to immunotherapy.

Publisher Correction: Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency.

In the version of this article originally published, there was an error in the legend for Extended Data Fig. 7. The legend for panel f was originally: "f, FACS analysis of IL7R-, CD62L- and CD45RA- expression on TRAC-1928ζ and TRAC-1XX CAR T cells at day 63 post CAR infusion (representative for at least n = 3 mice per group in one independent experiment)." The legend should have been: "f, FACS analysis of IL7R+, CD62L+ and CD45RA+ expression on TRAC-1928ζ and TRAC-1XX CAR T cells at day 63 post CAR infusion (representative for at least n = 3 mice per group in one independent experiment)." The error has been corrected in the HTML and PDF versions of this article.

Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency.

Chimeric antigen receptors (CARs) are synthetic receptors that target and reprogram T cells to acquire augmented antitumor properties1. CD19-specific CARs that comprise CD28 and CD3ζ signaling motifs2 have induced remarkable responses in patients with refractory leukemia3-5 and lymphoma6 and were recently approved by the US Food and Drug Administration7. These CARs program highly performing effector functions that mediate potent tumor elimination4,8 despite the limited persistence they confer on T cells3-6,8. Extending their functional persistence without compromising their potency should improve current CAR therapies. Strong T cell activation drives exhaustion9,10, which may be accentuated by the redundancy of CD28 and CD3ζ signaling11,12 as well as the spatiotemporal constraints imparted by the structure of second-generation CARs2. Thus, we hypothesized that calibrating the activation potential of CD28-based CARs would differentially reprogram T cell function and differentiation. Here, we show that CARs encoding a single immunoreceptor tyrosine-based activation motif direct T cells to different fates by balancing effector and memory programs, thereby yielding CAR designs with enhanced therapeutic profiles.

Low-Dose Radiation Conditioning Enables CAR T Cells to Mitigate Antigen Escape.

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.

Insights into Chimeric Antigen Receptor Therapy for Chronic Lymphoblastic Leukemia.

In a recent study, Fraietta and colleagues identified chimeric antigen receptor (CAR) T cell biomarkers that may predict the success or failure of CAR therapy in patients with refractory chronic lymphoblastic leukemia (CLL). These findings open new prospects for improving T cell product manufacturing and the management of patients with CLL undergoing T cell-based therapies.

CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade.

Chimeric antigen receptor (CAR) therapy targeting CD19 is an effective treatment for refractory B cell malignancies, especially acute lymphoblastic leukemia (ALL) 1 . Although a majority of patients will achieve a complete response following a single infusion of CD19-targeted CAR-modified T cells (CD19 CAR T cells)2-4, the broad applicability of this treatment is hampered by severe cytokine release syndrome (CRS), which is characterized by fever, hypotension and respiratory insufficiency associated with elevated serum cytokines, including interleukin-6 (IL-6)2,5. CRS usually occurs within days of T cell infusion at the peak of CAR T cell expansion. In ALL, it is most frequent and more severe in patients with high tumor burden2-4. CRS may respond to IL-6 receptor blockade but can require further treatment with high dose corticosteroids to curb potentially lethal severity2-9. Improved therapeutic and preventive treatments require a better understanding of CRS physiopathology, which has so far remained elusive. Here we report a murine model of CRS that develops within 2-3 d of CAR T cell infusion and that is potentially lethal and responsive to IL-6 receptor blockade. We show that its severity is mediated not by CAR T cell-derived cytokines, but by IL-6, IL-1 and nitric oxide (NO) produced by recipient macrophages, which enables new therapeutic interventions.

Integrating Proteomics and Transcriptomics for Systematic Combinatorial Chimeric Antigen Receptor Therapy of AML.

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.

The ADAMTS131239-1253 peptide is a dominant HLA-DR1-restricted CD4+ T-cell epitope.

Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4+ T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4+ T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4+ T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4+ T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS131239-1253 peptide as the single immunodominant HLA-DR1-restricted CD4+ T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4+ T cells from Sure-L1 mice and was recognized by CD4+ T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS131239-1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4+ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS131239-1253-loaded HLA-DR tetramers.