Chimeric Receptor Research Articles

Direct CD32 T-cell cytotoxicity: implications for breast cancer prognosis and treatment.

The FcγRII (CD32) ligands are IgFc fragments and pentraxins. The existence of additional ligands is unknown. We engineered T cells with human chimeric receptors resulting from the fusion between CD32 extracellular portion and transmembrane CD8α linked to CD28/ζ chain intracellular moiety (CD32-CR). Transduced T cells recognized three breast cancer (BC) and one colon cancer cell line among 15 tested in the absence of targeting antibodies. Sensitive BC cell conjugation with CD32-CR T cells induced CD32 polarization and down-regulation, CD107a release, mutual elimination, and proinflammatory cytokine production unaffected by human IgGs but enhanced by cetuximab. CD32-CR T cells protected immunodeficient mice from subcutaneous growth of MDA-MB-468 BC cells. RNAseq analysis identified a 42 gene fingerprint predicting BC cell sensitivity and favorable outcomes in advanced BC. ICAM1 was a major regulator of CD32-CR T cell-mediated cytotoxicity. CD32-CR T cells may help identify cell surface CD32 ligand(s) and novel prognostically relevant transcriptomic signatures and develop innovative BC treatments.

Co-expression of a PD-L1-specific chimeric switch receptor augments the efficacy and persistence of CAR T cells via the CD70-CD27 axis

Co-expression of chimeric switch receptors (CSRs) specific for PD-L1 improves the antitumor effects of chimeric antigen receptor (CAR) T cells. However, the effects of trans-recognition between CSRs and PD-L1 expressed by activated CAR T cells remain unclear. Here, we design a CSR specific for PD-L1 (CARP), containing the transmembrane and cytoplasmic signaling domains of CD28 but not the CD3 ζ chain. We show that CARP T cells enhance the antitumor activity of anti-mesothelin CAR (CARMz) T cells in vitro and in vivo. In addition, confocal microscopy indicates that PD-L1 molecules on CARMz T cells accumulate at cell-cell contacts with CARP T cells. Using single-cell RNA-sequencing analysis, we reveal that CARP T cells promote CARMz T cells differentiation into central memory-like T cells, upregulate genes related to Th1 cells, and downregulate Th2-associated cytokines through the CD70-CD27 axis. Moreover, these effects are not restricted to PD-L1, as CAR19 T cells expressing anti-CD19 CSR exhibit similar effects on anti-PSCA CAR T cells with truncated CD19 expression. These findings suggest that target trans-recognition by CSRs on CAR T cells may improve the efficacy and persistence of CAR T cells via the CD70-CD27 axis.

The new 2022 ESC Guidelines on Cardio-oncology and their impact on the Acute Cardiovascular Care Society.

In this perspective piece on the recently published ESC Guidelines on Cardio-oncology and the Consensus Statements from the Acute Cardiovascular Care Association, we summarize key learning points regarding the management of acute cardiovascular disease in patients with cancer. This document outlines where other pre-existing ESC Guidelines can be applied to the management of acute cardiovascular disease in patients with cancer while simultaneously highlighting important gaps in knowledge that require further research. Cancer and cardiovascular disease share common risk factors and often co-exist, especially in older patients. In addition, patients with cancer undergoing active treatment are exposed to multiple, potentially cardiotoxic drugs, which may manifest as a variety of cardiovascular events, including left-ventricular systolic dysfunction and heart failure, arrhythmias, hypertension, or acute venous and arterial vascular events. Knowledge about potential causative cancer therapeutics is necessary for rapid recognition and management to improve cardiovascular outcomes and guide ongoing cancer treatment. Specifically, the importance of rapidly interrupting culprit cancer drugs is highlighted, as well as instituting standard guideline-based therapies for conditions such as acute heart failure and acute coronary syndromes [ST-elevation myocardial infarction and high-risk non-ST-elevation acute coronary syndrome (ACS)]. Given the high prevalence of thrombocytopenia and increased bleeding risk in patients with cancer, we are provided with platelet cut-offs for the use of different antiplatelet agents and anticoagulants for patients with ACS and atrial arrhythmias. In contrast, given the hypercoagulable milieu of cancer, we are provided information regarding types of anticoagulants, drug-drug interactions, and duration of anticoagulation in patients with acute venous thromboembolism, as well as for atrial fibrillation. They also discuss the diagnostic and treatment strategies for the unique cardiotoxicities seen with novel cancer therapeutics such as immune checkpoint inhibitors and chimeric receptor antigen T-cell therapy. Last, but not least, the authors emphasize that the care of these patients requires close collaboration between cardiology and oncology to maximize both cardiovascular and cancer outcomes.

Human Melanocortin-2 Receptor: Identifying a Role for Residues in the TM4, EC2, and TM5 Domains in Activation and Trafficking as a Result of Co-Expression with the Accessory Protein, Mrap1 in Chinese Hamster Ovary Cells.

Human melanocortin-2 receptor (hMC2R) co-expressed with the accessory protein mouse (m)MRAP1 in Chinese Hamster Ovary (CHO) cells has been used as a model system to investigate the activation and trafficking of hMC2R. A previous study had shown that the N-terminal domain of mMRAP1 makes contact with one of the extracellular domains of hMC2R to facilitate activation of hMC2R. A chimeric receptor paradigm was used in which the extracellular domains of hMC2R were replaced with the corresponding domains from Xenopus tropicalis MC1R, a receptor that does not interact with MRAP1, to reveal that EC2 (Extracellular domain 2) is the most likely contact site for hMC2R and mMRAP1 to facilitate activation of the receptor following an ACTH binding event. Prior to activation, mMRAP1 facilitates the trafficking of hMC2R from the ER to the plasma membrane. This process is dependent on the transmembrane domain (TM) of mMRAP1 making contact with one or more TMs of hMC2R. A single alanine substitution paradigm was used to identify residues in TM4 (i.e., I163, M165), EC2 (F167), and TM5 (F178) that play a role in the trafficking of hMC2R to the plasma membrane. These results provide further clarification of the activation mechanism for hMC2R.

Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo.

Chimeric receptor T cells (CAR-T) can effectively cure leukemia; however, there are two limitations: a complicated preparation process ex vivo and cytokine release syndrome (CRS). In this study, we constructed a lipid nanoparticle system modified by CD3 antibody on the surface, loading with the plasmid containing the combination gene of interleukin 6 short hairpin RNA (IL-6 shRNA) and CD19-CAR (AntiCD3-LNP/CAR19+shIL6). The system targeted T cells by the mediation of CD3 antibody and stably transfected T cells to transform them into CAR-T cells with IL-6 knockdown, thus killing CD19-highly expressed leukemia tumor cells and reducing CRS caused by IL-6. In vivo experiments showed that AntiCD3-LNP/CAR19+shIL6 could stably transfect T cells and produce CAR-T within 90days to kill the tumor. This significantly prolonged the survival time of leukemia model mice and demonstrated the prepared LNP exhibited the same anti-tumor effect as the traditional CAR-T cells prepared ex vivo. In this study, CAR-T cells were directly produced in vivo after intravenous injection of the lipid nanoparticles, without the need of using the current complex process ex vivo. Additionally, IL-6 expression was silenced, which would be helpful to reduce the CRS and improve the safety of CAR-T therapy. This method improves the convenience of using CAR-T technology and is helpful in further promoting the clinical application of CAR-T.

HIV-Specific CAR T Cells with CD28 or 4-1BB Signaling Domains Are Phenotypically and Functionally Distinct and Effective at Suppressing HIV and Simian Immunodeficiency Virus.

Despite mounting a robust antiviral CD8 T cell response to HIV infection, most infected individuals are unable to control HIV viral load without antiretroviral therapy (ART). Chimeric Ag receptor (CAR) T cell treatment is under intensive investigation as an alternative therapy for ART-free remission of chronic HIV infection. However, achieving durable remission of HIV will require a successful balance between CAR T cell effector function and persistence. CAR T cells with CD28 costimulatory domains have robust effector function but limited persistence in vivo, whereas CAR T cells with 4-1BB costimulatory domains present a more undifferentiated phenotype and greater in vivo persistence. We compared the in vitro phenotype and function of rhesus macaque and human CAR T cells that contained either the CD28 or 4-1BB costimulatory domain; both constructs also included CARs that are bispecific for gp120 of HIV or SIV and the CXCR5 moiety to promote in vivo homing of CAR/CXCR5 T cells to B cell follicles. Cells were transduced using a gammaretroviral vector and evaluated using flow cytometry. 4-1BB-CAR/CXCR5 T cells were phenotypically distinct from CD28-CAR/CXCR5 T cells and showed increased expression of CAR and CD95. Importantly, both CD28- and 4-1BB-CAR/CXCR5 T cells retained equal capacity to recognize and suppress SIV in vitro. These studies provide new insights into rhesus macaque and human 4-1BB- and CD28-bearing CAR T cells.

Antibiotics versus no treatment for asymptomatic bacteriuria in residents of aged care facilities: a systematic review and meta-analysis

l-Glutamate (Glu) activates at least eight different G protein-coupled receptors known as metabotropic glutamate (mGlu) receptors, which mostly act as regulators of synaptic transmission. These receptors consist of two domains: an extracellular domain in which agonists bind and a transmembrane heptahelix region involved in G protein activation. Although new mGlu receptor agonists and antagonists have been described, few are selective for a single mGlu subtype. Here, we have examined the effects of a novel compound, BAY36-7620 [(3a<i>S</i>,6a<i>S</i>)- 6a-Naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[<i>c</i>]furan-1-on], on mGlu receptors (mGlu1–8), transiently expressed in human embryonic kidney 293 cells. BAY36-7620 is a potent (IC₅₀ = 0.16 μM) and selective antagonist at mGlu1 receptors and inhibits &gt;60% of mGlu1a receptor constitutive activity (IC⁵⁰ = 0.38 μM). BAY36-7620 is therefore the first described mGlu1 receptor inverse agonist. To address the mechanism of action of BAY36-7620, Glu dose-response curves were performed in the presence of increasing concentrations of BAY36-7620. The results show that BAY36-7620 largely decreases the maximal effect of Glu. Moreover, BAY36-7620 did not displace the [³H]quisqualate binding from the Glu-binding pocket, further indicating that BAY36-7620 is a noncompetitive mGlu1 antagonist. Studies of chimeric receptors containing regions of mGlu1 and regions of DmGluA, mGlu2, or mGlu5, revealed that the transmembrane region of mGlu1 is necessary for activity of BAY36-7620. Transmembrane helices 4 to 7 are shown to play a critical role in the selectivity of BAY36-7620. This specific site of action of BAY36-7620 differs from that of competitive antagonists and indicates that the transmembrane region plays a pivotal role in the agonist-independent activity of this receptor. BAY36-7620 will be useful to further delineate the functional importance of the mGlu1 receptor, including its putative agonist-independent activity.

Cancer Immunotherapies Based on Genetically Engineered Macrophages

Anticancer immunotherapies are therapeutics aimed at eliciting immune responses against tumor cells. Immunotherapies based on adoptive transfer of engineered immune cells have raised great hopes of cures because of the success of chimeric antigen receptor T-cell therapy in treating some hematologic malignancies. In parallel, advances in detailed analyses of the microenvironment of many solid tumors using high-dimensional approaches have established the origins and abundant presence of tumor-associated macrophages. These macrophages have an anti-inflammatory phenotype and promote tumor growth through a variety of mechanisms. Attempts have been made to engineer macrophages with chimeric receptors or transgenes to counteract their protumor activities and promote their antitumor functions such as phagocytosis of cancer cells, presentation of tumor antigens, and production of inflammatory cytokines. In this review, we cover current breakthroughs in engineering myeloid cells to combat cancer as well as potential prospects for myeloid-cell treatments.

Fine-tuning T cell function through engineered orthogonal chimeric cytokine receptors.

Fine-tuning T cell function through engineered orthogonal chimeric cytokine receptors.

Antigen-Dependent Inducible T-Cell Reporter System for PET Imaging of Breast Cancer and Glioblastoma.

For the past several decades, chimeric antigen receptor T-cell therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells invivo. Methods: Using synthetic biology, we engineered T cells with a chimeric receptor synthetic intramembrane proteolysis receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette on recognition of specific tumor markers. We then applied a SNIPR-based PET reporter system to 2 cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors, respectively. Results: Antigen-specific reporter induction of the SNIPR PET T cells was confirmed invitro using green fluorescent protein fluorescence, luciferase luminescence, and the HSV-TK PET reporter with 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG). T cells associated with their target antigens were successfully imaged using PET in dual-xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII- animal models, with more than 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. Conclusion: The main innovation found in this work was PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.

A Setmelanotide-like Effect at MC4R Is Achieved by MC4R Dimer Separation.

Melanocortin 4 receptor (MC4R) is part of the leptin-melanocortin pathway and plays an essential role in mediating energy homeostasis. Mutations in the MC4R are the most frequent monogenic cause for obesity. Due to increasing numbers of people with excess body weight, the MC4R has become a target of interest in the search of treatment options. We have previously reported that the MC4R forms homodimers, affecting receptor Gs signaling properties. Recent studies introducing setmelanotide, a novel synthetic MC4R agonist, suggest a predominant role of the Gq/11 pathway regarding weight regulation. In this study, we analyzed effects of inhibiting homodimerization on Gq/11 signaling using previously reported MC4R/CB1R chimeras. NanoBRETTM studies to determine protein–protein interaction were conducted, confirming decreased homodimerization capacities of chimeric receptors in HEK293 cells. Gq/11 signaling of chimeric receptors was analyzed using luciferase-based reporter gene (NFAT) assays. Results demonstrate an improvement of alpha-MSH-induced NFAT signaling of chimeras, reaching the level of setmelanotide signaling at wild-type MC4R (MC4R-WT). In summary, our study shows that inhibiting homodimerization has a setmelanotide-like effect on Gq/11 signaling, with chimeric receptors presenting increased potency compared to MC4R-WT. These findings indicate the potential of inhibiting MC4R homodimerization as a therapeutic target to treat obesity.

DNAM-1-chimeric receptor-engineered NK cells, combined with Nutlin-3a, more effectively fight neuroblastoma cells in vitro: a proof-of-concept study.

Adoptive transfer of engineered NK cells, one of clinical approaches to fight cancer, is gaining great interest in the last decade. However, the development of new strategies is needed to improve clinical efficacy and safety of NK cell-based immunotherapy. NK cell-mediated recognition and lysis of tumor cells are strictly dependent on the expression of ligands for NK cell-activating receptors NKG2D and DNAM-1 on tumor cells. Of note, the PVR/CD155 and Nectin-2/CD112 ligands for DNAM-1 are expressed primarily on solid tumor cells and poorly expressed in normal tissue cells. Here, we generated human NK cells expressing either the full length DNAM-1 receptor or three different DNAM-1-based chimeric receptor that provide the expression of DNAM-1 fused to a costimulatory molecule such as 2B4 and CD3ζ chain. Upon transfection into primary human NK cells isolated from healthy donors, we evaluated the surface expression of DNAM-1 and, as a functional readout, we assessed the extent of degranulation, cytotoxicity and the production of IFNγ and TNFα in response to human leukemic K562 cell line. In addition, we explored the effect of Nutlin-3a, a MDM2-targeting drug able of restoring p53 functions and known to have an immunomodulatory effect, on the degranulation of DNAM-1-engineered NK cells in response to human neuroblastoma (NB) LA-N-5 and SMS-KCNR cell lines. By comparing NK cells transfected with four different plasmid vectors and through blocking experiments, DNAM-1-CD3ζ-engineered NK cells showed the strongest response. Furthermore, both LA-N-5 and SMS-KCNR cells pretreated with Nutlin-3a were significantly more susceptible to DNAM-1-engineered NK cells than NK cells transfected with the empty vector. Our results provide a proof-of-concept suggesting that the combined use of DNAM-1-chimeric receptor-engineered NK cells and Nutlin-3a may represent a novel therapeutic approach for the treatment of solid tumors, such as NB, carrying dysfunctional p53.

LB952 A phase 1 trial of DSG3-CAART cells in mucosal-dominant pemphigus vulgaris (mPV) patients: Preliminary data

mPV is mediated by anti-desmoglein 3 (DSG3) autoantibodies (Abs) and treated with chronic, broad immunosuppressive therapy. Based on the long-lasting remission of B cell cancers with chimeric antigen receptor T (CART) cells, we genetically modified autologous T cells to express chimeric autoantibody receptors comprising the DSG3 autoantigen (DSG3-CAART) to target only anti-DSG3 B cells. In this ongoing open-label study of adults with active, anti-DSG3 Ab-positive mPV, subjects are assigned to receive 2e7,1e8, 5e8, 2.5e9 and 5-7.5e9 DSG3-CAART after discontinuing immunosuppressives or tapering steroids. The primary endpoint is related adverse events (AEs) within 3 months of infusion. Screening characteristics of the 9 subjects in the first 3 cohorts who have completed 3 months (median (range)): 67% female; age 53y (32-70); disease duration 4.0y (0.3-15.4); PDAI 12 (1-20); anti-DSG3 Ab 104 U/mL (51-169). Prior medications: prednisone (9), rituximab (6) and mycophenolate (6). No dose-limiting toxicities or serious AEs were observed over 3 months. Disease was clear or almost clear (PDAI 0-1) in 0, 1, 2, 5 and 3 of the 9 subjects at screening, baseline and 1, 2 and 3 months after treatment, respectively. Anti-DSG3 Ab levels through month 3 were increased, stable (+/-20%) or decreased in 3, 5 and 1 subjects, respectively. There was a dose-dependent increase in DSG3-CAART persistence (rs=0.85) within 29 days, but at much lower levels than for cancer CART therapy. DSG3-CAART from all subjects exhibited specific in vitro lysis of anti-DSG3-expressing cells. The favorable DSG3-CAART safety profile along with the preliminary clinical and biological data from the first 3 cohorts provide rationale to evaluate higher doses and manufacturing enhancements in future cohorts.

Dual CAR-T cells to treat cancers co-expressing NKG2D and PD1 ligands in xenograft models of peritoneal metastasis.

While the expression of either NKG2D ligands or PD-1 ligands has been reported in various types of cancers, the co-expression of the two sets of ligands in the same tumour tissues is still un-investigated. After examining 68 primary ovarian cancer samples, we observed around 80% of the co-expression in low grade serous and endometrioid ovarian cancer samples. We then constructed a dual CAR system that splits the conventional single-input of a 2nd generation CAR into two independent chimeric receptors, one composed of the NKG2D extracellular domain linked with DAP12 for T cell activation and another using the PD-1 extracellular domain linked with 4-1BB for costimulatory signal 2 input. Given the limitation of the low-affinity PD-1 receptor in recognizing cancer cells with low levels of PD-1 ligands, we also used a high-affinity scFv specific to PD-L1 in our combinatorial approach to expand the range of target cancer cells with different expression levels of PD-L1. The two types of dual CAR-T cells were generated through electroporation of non-viral piggyBac transposon plasmids and were effective in eliminating the target cancer cells. Especially, the dual CAR-T cells with anti-PD-L1 scFv were capable of eradicating established tumors in mouse models of peritoneal metastasis of colorectal cancer and ovarian cancer. Since both NKG2D ligands and PD-1 ligands have been marked as favourable cancer therapeutic targets, the new dual CAR-T cells developed in this study hold attractive application potential in treating metastatic peritoneal carcinoma.

Transmembrane Domain 3 Is a Transplantable Pharmacophore in the Photodynamic Activation of Cholecystokinin 1 Receptor.

Cholecystokinin 1 receptor (CCK1R) is activated in photodynamic action by singlet oxygen, but detailed molecular mechanisms are not elucidated. To identify the pharmacophore(s) in photodynamic CCK1R activation, we examined photodynamic activation of point mutants CCK1RM121/3.32A, CCK1RM121/3.32Q, and a chimeric receptor with CCK1R transmembrane domain 3 (TM3) transplanted to muscarinic ACh receptor 3 (M3R) which is unaffected by photodynamic action. These engineered receptors were tagged at the N-terminus with genetically encoded protein photosensitizer miniSOG, and their light-driven photodynamic activation was compared to wild type CCK1R and M3R, as monitored by Fura-2 fluorescent calcium imaging. Photodynamic activations of miniSOG-CCK1RM121/3.32A and miniSOG-CCK1RM121/3.32Q were found to be 55% and 73%, respectively, when compared to miniSOG-CCK1R (100%), whereas miniSOG-M3R was not affected (0% activation). Notably, the chimeric receptor miniSOG-M3R-TM3CCK1R was effectively activated photodynamically (65%). These data suggest that TM3 is an important pharmacophore in photodynamic CCK1R activation, readily transplantable to nonsusceptible M3R for photodynamic activation.

Orthotopic and Heterotopic Murine Models of Pancreatic Cancer Exhibit Different Immunological Microenvironments and Different Responses to Immunotherapy

For decades, tumor-bearing murine models established using tumor cell lines have been the most commonly used models to study human cancers. Even though there are several studies reported that implant sites caused disparities in tumor behaviors, few of them illuminated the positional effect on immunotherapy. Herein, we describe surgical techniques for a novel orthotopic implantation of syngeneic pancreatic ductal adenocarcinoma (PDAC) tissue slices. This method has a high success modeling rate and stable growth kinetics, which makes it useful for testing novel therapeutics. Pathological examination indicated that the orthotopic tumor displayed poor vascularization, desmoplastic stromal reaction, and a highly immunosuppressive tumor microenvironment. This unique microenvironment resulted in limited response to PD1/CTLA4 blockade therapy and anti-MUC1 (αMUC1) CAR-T transfer treatment. To reverse the suppressive tumor microenvironment, we developed gene modified T-cells bearing a chimeric receptor in which activating receptor NKG2D fused to intracellular domains of 4-1BB and CD3ζ (NKG2D CAR). The NKG2D CAR-T cells target myeloid-derived suppressor cells (MDSCs), which overexpress Rae1 (NKG2D ligands) within the TME. Results indicated that NKG2D CAR-T cells eliminated MDSCs and improved antitumor activity of subsequently infused CAR-T cells. Moreover, we generated a bicistronic CAR-T, including αMUC1 CAR and NKG2D CAR separated by a P2A element. Treatment with the dual targeted bicistronic CAR-T cells also resulted in prolonged survival of orthotopic model mice. In summary, this study describes construction of a novel orthotopic PDAC model through implantation of tissue slices and discusses resistance to immunotherapy from the perspective of a PDAC microenvironment. Based on the obtained results, it is evident that elimination MDSCs by NKG2D CAR could rescue the impaired CAR-T cell activity.

STAP-2 Is a Novel Positive Regulator of TCR-Proximal Signals.

TCR ligation with an Ag presented on MHC molecules promotes T cell activation, leading to the selection, differentiation, and proliferation of T cells and cytokine production. These immunological events are optimally arranged to provide appropriate responses against a variety of pathogens. We here propose signal-transducing adaptor protein-2 (STAP-2) as a new positive regulator of TCR signaling. STAP-2-deficient T cells showed reduced, whereas STAP-2-overexpressing T cells showed enhanced, TCR-mediated signaling and downstream IL-2 production. For the mechanisms, STAP-2 associated with TCR-proximal CD3ζ immunoreceptor tyrosine activation motifs and phosphorylated LCK, resulting in enhancement of their binding after TCR stimulation. In parallel, STAP-2 expression is required for full activation of downstream TCR signaling. Importantly, STAP-2-deficient mice exhibited slight phenotypes of CD4+ T-cell-mediated inflammatory diseases, such as experimental autoimmune encephalomyelitis, whereas STAP-2-overexpressing transgenic mice showed severe phenotypes of these diseases. Together, STAP-2 is an adaptor protein to enhance TCR signaling; therefore, manipulating STAP-2 will have an ability to improve the treatment of patients with autoimmune diseases as well as the chimeric Ag receptor T cell therapy.

PB2098: ‘REAL-WORLD’ TREATMENT PATTERNS AND PATHWAYS OF SECOND LINE DIFFUSE LARGE B-CELL LYMPHOMA PATIENTS IN UK, CANADA, FRANCE, GERMANY, ITALY AND SPAIN USING A PROSPECTIVE SURVEY OF PHYSICIANS

Background: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent form of Non-Hodgkin Lymphoma, representing 25% to 35% of cases annually1. Despite this, there is a lack of research into treatment patterns of real-world patients, especially in the 2nd line (2L) setting. Aims: To determine the ‘real-world’ treatment patterns and treatment pathway of DLBCL patients treated at the second line, with a focus on patients who are ‘fast progressors’ as defined below. Methods: In this study ‘real-world’ data were drawn from the Adelphi DLBCL Disease Specific Programme™ (DSP), a point-in-time survey of clinicians and their next 6 presenting patients with DLBCL. The survey was conducted in France, Germany, Italy, Spain, the United Kingdom (UK) and Canada between Jan-May 2021. Patients who were refractory/relapsed <12 months from 1st line (1L) therapy, eligible for a stem cell transplant (SCT) at initial relapse/refractory, had an ECOG performance score of 0-1 and had no history of hepatitis B or C or CNS events were studied. Results: 47 patients met the criteria. Mean age of the patients was 58.0 (SD: 11.7) years, 68% were male and 26% were retired. At 1L, 94% of patients received R-CHOP with a median of 171.2 days (IQR: 61.0-247.5) between the end of their 1L and the start of their 2L treatments. All patients received salvage chemotherapy (SC) in the form of R-ESHAP, R-DHAP or R-GDP in most cases (Figure 1). 49% of the patients had a complete response (CR) to SC, 21% had partial response (PR), 30% stable/no response. In total 26 (55%) patients received high-dose therapy-SCT (HDT-SCT). Figure 1 shows the response to 2L treatments across patients and indicates why some patients did not progress to SCT despite being previously eligible. Patients receiving SCT had a median time from 2nd line SCT to relapse/refractory of 189.0 (IQR: 94.0-333.0) days. 54% of patients received chimeric receptor antigen T-cell (CAR-T) therapies at 3L (Figure 1), with the majority having previously received SCT. CAR-T use was higher in UK (67%), Canada (63%) and Germany (60%) than in other regions. Image:Summary/Conclusion: Despite all patients with DLBCL observed being SCT eligible at their first relapse, only half of these patients went on to receive an SCT. Unacceptable response to SC, progressive disease and patient choice were common reasons for not going on to receive SCT. Despite seemingly favourable responses to SCT, these patients progressed quickly and were likely to receive a CAR-T treatment in 3L, indicating an unmet need and highlighting potentially high resource use and substantial impacts to quality of life.

Evolutionary Analysis and Functional Identification of Ancient Brassinosteroid Receptors in Ceratopteris richardii

Phytohormones play an important role in the adaptive evolution of terrestrial plants. Brassinosteroids (BRs) are essential hormones that regulate multiple aspects of plant growth and development in angiosperms, but the presence of BR signaling in non-seed plants such as ferns remains unknown. Here, we found that BR promotes the growth of Ceratopteris richardii, while the synthetic inhibitor PCZ inhibits the growth. Using full-length transcriptome sequencing, we identified four BRI1-like receptors. By constructing chimeric receptors, we found that the kinase domains of these four receptors could trigger BR downstream signaling. Further, the extracellular domains of two receptors were functionally interchangeable with that of BRI1. In addition, we identified a co-receptor, CtSERK1, that could phosphorylate with CtBRL2s in vitro. Together, these proved the presence of a receptor complex in Ceratopteris richardii that might perceive BR and activate downstream hormone signaling. Our results shed light on the biological and molecular mechanisms of BR signaling in ferns and the role of BR hormone signaling in the adaptive evolution of terrestrial plants.

Abstract 3605: Human chimeric orthogonal IL9 receptor signaling promotes stemness and polyfunctionality for adoptive T cell therapy of cancer

Abstract Background: Orthogonal cytokine-receptor pairs allow selective in vivo manipulation of T cells after adoptive cell therapy (ACT) of cancer. Using mouse models, we previously described a chimeric orthogonal receptor (o9R) comprised of an orthogonal IL-2Rβ extracellular domain (ECD) and an IL-9R intracellular domain (ICD). Adoptive transfer of tumor-specific T cells engineered with o9R showed superior anti-tumor activity in mice treated with orthogonal IL-2 (oIL2). To evaluate the translational potential of o9R signaling, we generated and evaluated a human chimeric orthogonal IL-2Rβ ECD/IL-9R ICD (ho9R). Methods: We hypothesized that ho9R in tumor-specific T cells would result in distinct signaling, phenotypic and functional properties compared to the human orthogonal IL-2Rβ (ho2R), mirroring our observations in the mouse system. Activated T cells were retrovirally transduced with two vectors: one encoding either ho9R or ho2R and another encoding a T cell receptor (TCR) specific for NY-ESO-1 in the context of HLA*0201. Transduced and sorted T cells were exposed to mouse serum albumin-bound human orthogonal IL2 (MSA-hoIL2) or wildtype IL-2 (MSA-hIL2) for 20 mins to evaluate STAT-1, -3 and -5 phosphorylation. We assessed proliferation and memory differentiation of transduced T cells cultured in MSA-hoIL2 or MSA-hIL2 for one week or after repetitive tumor challenge using an HLA*0201+ NY-ESO-1+ melanoma tumor cell line (M407-nRFP) in the presence of MSA-hoIL2 or MSA-hIL2. After four tumor challenges, T cells were restimulated to quantify intracellular cytokine production. Comparisons between ho2R and ho9R T cells were made using unpaired t-tests or 2-way ANOVA. Results: Consistent with the mouse system, human T cells signaling through ho9R activated pSTAT-1, -3 and -5, whereas ho2R signaling primarily activated pSTAT5. Similar to the mouse system, ho9R signaling resulted in weaker proliferation than ho2R or wildtype IL-2 signaling (p&amp;lt;0.001). Despite weaker proliferation, ho9R signaling expanded a population of TSCM cells (CD45RA+CD27+CCR7+CD95+; p&amp;lt;0.0001). The difference in T cell phenotype between ho9R/NYESO1-TCR and ho2R/NYESO1-TCR T cells persisted even after four challenges with M407-nRFP in the presence of MSA-hoIL2: ho9R/NYESO1-TCR T cells retained more CD45RA+CD27+ and TSCM cells (p&amp;lt;0.01), and expressed higher levels of CD62L (p&amp;lt;0.05) and CXCR3 (p&amp;lt;0.001). Upon re-exposure to antigen after four tumor challenges, ho9R/NYESO1-TCR T cells expressed more IFNγ, TNFα, and IL-2 (p&amp;lt;0.0001) and exhibited greater polyfunctionality than ho2R/NYESO1-TCR T cells. Conclusion: In tumor-specific T cells ho9R signaling activates STAT-1, -3, and -5, promotes stemness, and maintains effector polyfunctionality despite repetitive antigen stimulation, providing human evidence for its potential in adoptive T cell therapy of cancer. Citation Format: Mito Tariveranmoshabad, Leon L. Su, Amy L. Sun, Lora K. Picton, Antoni Ribas, K. Christopher Garcia, Anusha Kalbasi. Human chimeric orthogonal IL9 receptor signaling promotes stemness and polyfunctionality for adoptive T cell therapy of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3605.