Academic literature on the topic 'Adoptive Neoplasms Receptors'

✓ The use of tumor-infiltrating lymphocytes in the treatment of central nervous system (CNS) neoplasms has met with serious obstacles due to difficulty of culture and poor characterization. Since in other tumors the therapeutic effects of tumor-infiltrating lymphocytes have been shown to rely on T-cell receptor engagement, the authors addressed the question as to whether expression of T-cell receptor variable (V) domains in cultured tumor-infiltrating lymphocytes from CNS is different from that of autologous cultured peripheral blood mononuclear cells. Infiltrating lymphocytes from CNS neoplasms, including primary malignancies, metastatic cancers, and meningiomas, were cultured in the presence of interleukin-2 and anti-CD3 monoclonal antibodies (MoAb's) in order to obtain optimum growth of T cells. Autologous peripheral blood mononuclear cells from the same patients were similarly cultured. After 4 to 5 weeks of culture, 97.3% ± 2.6% (mean ± standard deviation) of the resulting cell populations were CD3-positive lymphocytes. The expression of T-cell receptor V domains was then studied by using a panel of 12 MoAb recognizing gene products from T-cell receptor V-α 2, V-β 5, 6, 8, and 12, V-γ 4 and 9 families, and from two subfamilies of V-δ 2. Remarkably, in over 70% of all paired measurements, percentages of T cells expressing discrete T-cell receptor V-gene products were found to be virtually identical in tumor- and peripheral blood-derived cultured cell populations, with differences never exceeding 1%. In contrast, a different expression of individual V-gene products, concerning both α/β and γ/δ T-cell receptors, could be detected between cultured tumor-infiltrating lymphocytes and autologous peripheral blood-derived T lymphocytes in seven of 12 patients. In two cases, significant differences between the two populations were also observed in the proliferative responses obtained upon stimulation with staphylococcal enterotoxins that trigger defined V-β T-cell receptors. Altogether, these data suggest that the T-cell receptor repertoire of cultured tumor-infiltrating lymphocytes from CNS tumors, suitable for use in adoptive immunotherapies, differs from that of autologous cultured peripheral blood mononuclear cells.

Introduction: Elevated pro-inflammatory cytokines are a hallmark feature of myeloproliferative neoplasms (MPNs). The pro-inflammatory cytokine interleukin-8 (IL8) is increased in patients with myelofibrosis (MF) and correlates with adverse outcome, including overall survival. Previously, the Levine/Fang labs identified increased IL8 secretion from individual CD34+ stem cells in a subset of MF patients. The role of IL8 and its cognate receptors CXCR1/2 in MF pathogenesis has not been delineated. Methods: Single-cell cytokine assays were performed on isolated CD34+ cells from 60 clinically annotated MPN patients (20 MF, 20 PV, 20 ET) using a previously described micro-chip platform (Kleppe et al, Can Disc 2013). 10 healthy donors (CD34+ cells from hip replacements) were used as controls. Integrated RNA-Seq and Assay for Transposase-Accessible Chromatin followed by next-generation sequencing (ATAC-Seq) was performed on CD34+ cells from MPN patients with and without expanded IL8 secreting clones for gene expression/chromatin accessibility analysis. To model the role of IL8-CXCR2 on fibrosis in vivo, the human MPLW515L transplant model (hMPLW515L) of MF was used. Specifically, wild-type (WT) murine bone marrow (Creneg-Cxcr2f/f; Cxcr2WT) or marrow lacking the CXCR2 receptor (VavCre-Cxcr2f/f; Cxcr2KO)were retrovirally infected with MSCV-hMPLW515L-IRES-GFP and transplanted into lethally irradiated WT recipient mice and monitored for disease. Blood counts, chimerism, and flow cytometry were assayed. Moribund mice were sacrificed and assayed for grade reticulin fibrosis and overall survival. Results: Single-cell cytokine assays confirmed an increased proportion of IL8-secreting CD34+ cells in MF patients (40%) in comparison to other MPN sub-types (10% PV/0% ET) (Figure 1A). MF patients with expanded IL8 secreting clones (defined as >50% of total CD34+ cells) had increased leukocytosis (p<0.0001), larger spleen sizes (p=0.0004), greater prevalence of constitutional symptoms (p=0.0084), and higher-grade reticulin fibrosis in marrow (Figure 1B) in comparison to MF patients without prevalent IL8 clones. IHC confirmed increased IL8 expression in marrow biopsies from 8/15 MF patients in comparison to 0/4 normal controls (Figure 1C), and high IL8 expression was also observed in MF splenic megakaryocytes (MKs) as well as in splenic stromal/endothelial cells not seen in normal spleen (Figure 1D). Integrated RNA-Seq/ATAC-Seq analysis of IL8-high MF patients confirmed up-regulation of IL8-CXCR2 signaling and enrichment in pro-inflammatory pathways (i.e TNFa, NFkB, etc) by GSEA, as well as increased expression/accessibility of pro-inflammatory genes S100A8 and S100A9-previously implicated in fibrosis development. Flow analysis of IL8-high MF CD34+ cells revealed enhanced surface expression of CXCR2 and its analog CXCR1, such that MF was characterized by increased IL8 ligand and receptor expression (Figure 1E) and coincided with enhanced NFkB pathway activity (Figure 1F). Consistent with this, colony forming assays of cultured MF CD34+ cells revealed enhanced colony output when cultured with IL8 compared to WT CD34+ cells-an effect ameliorated by co-treatment with the CXCR1/2 antagonist Reparixin (Figure 1G). In vivo, hMPLW515L adoptive transplant with Cxcr2KO hematopoietic donor cells demonstrated improved leukocytosis, thrombocytosis (Figure 2A) and splenomegaly in comparison to Cxcr2WT hMPLW515L recipient mice. Pathologic analysis revealed a reduction in reticulin fibrosis in bone marrow (Figure 2B) and spleen, translating into an improvement in overall survival (Figure 2C). Notably, a significant reduction in dysplastic MKs-a hallmark feature of MF-was also observed in Cxcr2KO hMPLW515L mice (Figure 2D) supporting a role for CXCR2 signaling in MK proliferation. Conclusion: IL8 secreting clones are associated with increased symptom severity and fibrosis grade in MF. Gene expression of MF CD34+ IL8 secreting clones shows up-regulation of inflammatory genes S100A8/A9, implicated in myofibroblast proliferation. Cxcr2 KO abrogates fibrosis formation and prolongs survival in the hMPLW515L model, and CXCR1/2 inhibition impairs colony forming capacity of MF CD34+ cells. These data suggest pharmacologic inhibition of this pathway should be investigated as potential therapy in MF and in PV/ET patients at high risk of fibrotic transformation. Disclosures Fan: IsoPlexis: Current Employment, Current equity holder in private company; Singleron Biotechnologies: Current Employment, Current equity holder in private company. Levine:Morphosys: Consultancy; Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Honoraria; Astellas: Consultancy; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Hoffman:Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy.

Abstract Abstract 3449 Background and Aims. The thrombopoietin receptor (TpoR, c-Mpl) plays a dual role in hematopoiesis. Beside the fact that it is one of the main regulators of megacaryopoiesis and platelet formation, TpoR is also expressed in HSCs and maintains them quiescent. TpoR signals as a homodimer after ligand binding in a 1:2 (ligand to receptor) ratio. Similar to other single pass membrane receptor, it contains an extracellular ligand binding domain, one helical transmembrane domain (TMD) and a cytoplasmic part important for JAKs binding and to mediate signaling. Unlike other cytokine receptors, TpoR contains a unique amphipathic motif (RW515QFP) at the junction between the TMD and the cytoplasmic domain. Mutations within this motif, namely W515K or W515L, lead to activation of the receptor and are associated with JAK2 V617F negative myeloproliferative neoplasms, specifically essential thrombocytopenia and primary myelofibrosis. We asked i) how a tryptophan at position 515 prevents autonomous activation of TpoR and ii) exactly how mutants at W515 impact on TpoR function and lead to oncogenesis. Methods. A combination of site-directed mutagenesis, biochemical and signaling assays, and spectroscopy analysis were employed. We made several specific mutations at position 515 to see if Trp515 is regulatory because of it size and/or aromatic character. We then tested the effect of mutations at adjacent positions in the wild type or mutated W515 constructs. Data were recorded from transiently transfected g2A cells and the results were confirmed in stably transduced BaF3 cells using dual luciferase and growth assays in the presence or absence of ligand. Adoptive transfer in lethally irradiated mice was used for assessing in vivo effects of TpoR mutants. The oligomerization status of the full length TpoR and some of our mutants was tested by employing the Gaussia princeps luciferase complementation assay, where Gaussia princeps luciferase fragments were fused in frame to the carboxyl terminus of TpoR or TpoR mutants. Recombination of the luciferase fragments results in a positive signal and reveals dimerization. Deuterium magic angle spinning (MAS) NMR spectroscopy and analytical ultracentrifugation were used to assess dimerization of the region encompassing the TMD. The tilt angle of this segment was assessed by FTIR spectroscopy. Results. Tryptophan seems to be absolutely required at position 515 to maintain the receptor inactive in absence of ligand. We identified secondary mutations (to tryptophan) that can prevent W515L/K/A activation of TpoR. The doubly mutated receptors behave like wild type receptors exhibiting no activation in absence of TPO and a normal response to TPO. These data suggest that W515 mutants may be specifically targeted for therapy. Mutations at W515 induced an increase in full length TpoR dimerization, and promoted dimerization of the segment comprising the TMD. However, our secondary site mutations showed differences between the mechanisms by which K or L versus A activate at W515. Overall, W515 impairs TpoR dimerization in a conformation productive for JAK2 activation. Measurements of the dichroic ratio by FTIR indicated that W515 increases the tilt angle of the TMD, thus reducing the interface required for TMD dimerization. We further explored the role of W515 in receptor activation by using a model where coiled coils are used to induce distinct dimeric conformations when fused to the TMD-cytosolic domains. W515 was essential for imparting differential signaling to the different dimers, both in cell lines and in vivo, where phenotypes in reconstituted mice no longer differ between receptors that have different dimeric orientations. Conclusions. Tryptophan 515 prevents TpoR dimerization in a productive orientation for JAK2 activation. This effect is reversible when secondary site mutations to tryptophan are introduced at certain positions around W515. Dimerization of TMD induced by ligand rotates W515 so that it is displaced from the headgroup region of the membrane to the dimer interface. This is crucial for imparting specific signaling as a function of dimeric orientation. Our results indicate that the region around W515 is a major switch for receptor function and could be exploited therapeutically. Disclosures: Constantinescu: Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees.

Background: Autologous T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have shown rapid and durable responses in B cell malignancies. Although CD19 CAR-T cells have demonstrated remarkable success, CD19-negative relapses occur in 30-45% of patients, highlighting the need for adoptive immunotherapies with alternative targeting approaches. B-cell activating factor (BAFF) is a critical B cell survival factor. Receptors of BAFF (BAFF-R, TACI and BCMA) are expressed by a wide range of B cell neoplasms, including ALL, CLL, NHL and MM, making them attractive therapeutic targets. We developed a novel ligand-based CAR that when expressed in T cells, targets and eliminates malignant B cells expressing BAFF receptors (BAFF CAR-T). This approach has several potential advantages over CD19 targeting CAR-T therapy: CD19 is expressed on all B cells, but BAFF receptors are expressed only on mature B cells, making it a more specific antigen for targeting and potentially narrowing down the side effect profile. BAFF CAR-T cells are a potential therapeutic strategy to treat CD19 CAR-T relapsed patients as well as chemotherapy resistant patients. Methods: BAFF ligand was fused to a second generation CAR backbone containing 4-1BB costimulatory and CD3ζ intracellular signaling domains. T cells were isolated from human blood, activated and transduced with BAFF-CAR lentiviral particles. In vitro tumor cell killing was analyzed using calcein-AM cytotoxicity assay. For in vivo testing of BAFF CAR-T cytotoxicity, we used mantle cell lymphoma (MCL) Jeko-1 xenograft model. Immunocompromised NSG mice were subcutaneously injected with human MCL cell line Jeko-1 (10.106 cells at day 0). Once these mice developed measurable tumors, we injected T cells transduced with empty vector (control T cells) or BAFF-CAR T cells (10 x 106 cells) or PBS intra-tumorally as a one-time injection. Tumor volumes were measured every other day using calipers. Results: BAFF CAR-T cells showed significant cytotoxicity in vitro (not shown) and in vivo against human MCL cell line Jeko-1. Mice treated with BAFF-CAR-T showed significant reduction in tumor volume compared to mice treated with control T cells and PBS (Figure 1A, B). Tumor progression was observed after control T cell and PBS treatment, whereas the cohort treated with BAFF CAR-T did not show any tumor progression, and with complete or near-complete tumor eradication. Survival analysis showed the BAFF CAR-T treated cohort had significantly longer survival compared to control-T cell and PBS treated cohorts (Figure 1C). Mice were sacrificed when tumor volume reached 2 cm3. Conclusion: Our data suggest that targeting BAFF receptors with a novel, ligand-based BAFF-CAR-T is a feasible and effective immunotherapeutic strategy to eliminate malignant B cells, warranting further development. BAFF-CAR-T cells have therapeutic potential against a wide spectrum of B cell malignancies, including CD19 negative relapsed disease. Clinical grade expansion and clinical trials are in development for BAFF CAR-T therapy non Hodgkin lymphoma patients. Disclosures Parameswaran: Luminary Therapeutics: Consultancy; Luminary therapeutics: Research Funding. de Lima:Kadmon: Other: Personal Fees, Advisory board; BMS: Other: Personal Fees, advisory board; Incyte: Other: Personal Fees, advisory board; Celgene: Research Funding; Pfizer: Other: Personal fees, advisory board, Research Funding. Caimi:Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Verastem: Other: Advisory Board; Kite pharmaceuticals: Other: Advisory Board; ADC therapeutics: Other: Advisory Board, Research Funding; Celgene: Speakers Bureau.

Chimeric antigen receptor (CAR)-T-cell therapy is an innovative form of adoptive cell therapy that has revolutionized the treatment of certain hematological malignancies, including B-cell non-Hodgkin lymphoma (NHL) and B-cell acute lymphoblastic leukemia (ALL). The treatment is currently also being studied in other B-cell neoplasms, including multiple myeloma (MM) and chronic lymphocytic leukemia (CLL). CD19 and B-cell maturation antigen (BCMA) have been the most popular target antigens for CAR-T-cell immunotherapy of these malignancies. This review will discuss the efficacy and toxicity data from the pivotal clinical studies of CD19- and BCMA-targeted CAR-T-cell therapies in relapsed/refractory B-cell malignancies (NHL, ALL, CLL) and MM, respectively.

The adoptive transfer of the chimeric antigen receptor (CAR) expressing T-cells has produced unprecedented successful results in the treatment of B-cell malignancies. However, the use of this technology in other malignancies remains less effective. In the setting of solid neoplasms, CAR T-cell metabolic fitness needs to be optimal to reach the tumor and execute their cytolytic function in an environment often hostile. It is now well established that both tumor and T cell metabolisms play critical roles in controlling the immune response by conditioning the tumor microenvironment and the fate and activity of the T cells. In this review, after a brief description of the tumoral and T cell metabolic reprogramming, we summarize the latest advances and new strategies that have been developed to improve the metabolic fitness and efficacy of CAR T-cell products.

SummaryBlastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy characterized by recurrent skin nodules, an aggressive clinical course with rapid involvement of hematological organs, and a poor prognosis with poor overall survival. BPDCN is derived from plasmacytoid dendritic cells (pDCs) and its pathogenesis is unclear. The tumor cells show aberrant expression of CD4, CD56, interleukin-3 receptor alpha chain (CD123), blood dendritic cell antigen 2 (BDCA 2/CD303), blood dendritic cell antigen 4 (BDCA4) and transcription factor (E protein) E2-2 (TCF4). The best treatment drugs are based on experience by adopting those used for either leukemia or lymphoma. Relapse with drug resistance generally occurs quickly. Stem cell transplantation after the first complete remission is recommended and tagraxofusp is the first targeted therapy. In this review, we summarize the differentiation of BPDCN from its cell origin, its connection with normal pDCs, clinical characteristics, genetic mutations and advances in treatment of BPDCN. This review provides insights into the mechanisms of and new therapeutic approaches for BPDCN.

El campo de la inmunoterapia en el tratamiento del cáncer se ha acelerado en los últimos años y ha entrado en primer plano como un área líder de investigaciones en curso y terapias prometedoras cambiando el panorama del tratamiento para una variedad de tipos de cáncer. Anteriormente, la inmunoterapia en el tratamiento de neoplasias malignas hematológicas. Sin embargo, hoy en día, este tipo de terapias biológicas es utilizada para estimular las defensas naturales del cuerpo en aras de combatir el cáncer a través de sustancias que produce el mismo cuerpo o que se procesan en laboratorios para mejorar o restaurar el sistema inmunológico del paciente, actuando en el retraso del crecimiento de las células cancerígenas, impidiendo la diseminación hacia otros órganos y ayudando al sistema inmunitario a la hora de destruir las células malignas. En el siguiente proceso investigativo se enmarca una revisión de varias formas de terapias basadas en el sistema inmunológico, que han demostrado ser muy prometedoras en pacientes con neoplasias hematológicas, incluidas las terapias monoclonales convencionales como rituximab; anticuerpos monoclonales diseñados mediante ingeniería denominados engagers de células T biespecíficas; anticuerpos monoclonales y fármacos que bloquean las vías inhibidoras de las células T (es decir, PD-1, CTLA-4 e IDO); terapia de transferencia celular adoptiva con células T diseñadas para expresar receptores de antígenos quiméricos o receptores de células T. También se evalúa la idea de usar estas terapias en combinación sugiriendo enfoques de múltiples puntas para mejorar los resultados del tratamiento y las respuestas curativas en los pacientes.

El campo de la inmunoterapia en el tratamiento del cáncer se ha acelerado en los últimos años y ha entrado en primer plano como un área líder de investigaciones en curso y terapias prometedoras cambiando el panorama del tratamiento para una variedad de tipos de cáncer. Anteriormente, la inmunoterapia en el tratamiento de neoplasias malignas hematológicas. Sin embargo, hoy en día, este tipo de terapias biológicas es utilizada para estimular las defensas naturales del cuerpo en aras de combatir el cáncer a través de sustancias que produce el mismo cuerpo o que se procesan en laboratorios para mejorar o restaurar el sistema inmunológico del paciente, actuando en el retraso del crecimiento de las células cancerígenas, impidiendo la diseminación hacia otros órganos y ayudando al sistema inmunitario a la hora de destruir las células malignas. En el siguiente proceso investigativo se enmarca una revisión de varias formas de terapias basadas en el sistema inmunológico, que han demostrado ser muy prometedoras en pacientes con neoplasias hematológicas, incluidas las terapias monoclonales convencionales como rituximab; anticuerpos monoclonales diseñados mediante ingeniería denominados engagers de células T biespecíficas; anticuerpos monoclonales y fármacos que bloquean las vías inhibidoras de las células T (es decir, PD-1, CTLA-4 e IDO); terapia de transferencia celular adoptiva con células T diseñadas para expresar receptores de antígenos quiméricos o receptores de células T. También se evalúa la idea de usar estas terapias en combinación sugiriendo enfoques de múltiples puntas para mejorar los resultados del tratamiento y las respuestas curativas en los pacientes.

Abstract Patients with B-cell lymphomas bearing MYC translocation combined with an additional translocation involving other genes, such as BCL2, BCL3, or BCL6, whose category is defined as double-hit lymphoma (DHL), have dismal prognosis, because these cells are refractory to conventional immunochemotherapy. Recent studies have expanded the concept of a such disease entity to include double-expressing lymphomas (DEL) that co-overexpress MYC protein with those proteins, which have also poor prognosis. An adoptive T-cell immunotherapy with a chimeric antigen receptor (CAR) is clinically shown to have a powerful cytotoxicity in refractory neoplasias. Especially, several results showed that T cells transduced with an anti-CD19-CAR have successfully worked well in patients with refractory acute B-cell lymphoblastic leukemia and B-cell lymphoma as well as chronic B-cell lymphocytic leukemia. Thus, CAR T-cell therapy is a clinically promising tool for various refractory hematopoietic disorders. Accordingly, we examined whether anti-CD19- and/or anti-CD38-CAR-T cells, both of which we previously developed, could abrogate DHL cells. Here, we revealed that the remarkable cytotoxicity of anti-CD19- and/or anti-CD38-CAR T cells against DHL cells from patients. Firstly, DHL cell line cells (KPU-H1, a generous gift form Dr. Junya Kuroda, Kyoto Prefectural University of Medicine) were co-cultured with anti-CD19- and/or anti-CD38-CAR T cells at an effector (E) target (T) ratio of 1: 2 for three days. Cells harvested and stained with anti-CD19 and/or anti-CD38 antibody-APC or -PE were subjected to flow cytometry. Flow cytometric analysis showed that anti-CD19- or anti-CD38-CAR T cells almost killed KPU-H1 cells, respectively (specific cytotoxicity was >90%). Intriguingly, T cells expressing anti-CD19-CAR exerted a collaborative cytotoxicity against KPU-H1 cells with anti-CD38-CAR T cells in vitro. CD38-specific T cells were co-cultured with cytogenetic DHL (n=3) or DEL (n=2) cells from five patients carrying a poor prognosis for 3 days. We examined whether T cells retrovirally transduced with anti-CD19- and/or anti-CD38-CAR vector could show cytotoxicity against primary DHL cells obtained from patients. Anti-CD19 and/or ant-CD38-CAR T cells were co-cultured with primary DHL cells at an E: T ratio of 1: 2 for 3 days. Interestingly, anti-CD19 and anti-CD38-CAR T cells completely abolished these DHL cells from patients, respectively. Additionally, anti-CD19- and anti-38-CAR T cells were synergistically effective to eliminate DHL cells. These results showed that DHL cells, which are refractory or resistant to existing chemotherapeutic agents, can be efficiently abrogated by a clinical use of T cells with anti-CD19- and/or anti-CD38-CAR. These results might warrant adoptive immunotherapy with autologous T cells transduced with anti-CD19 and anti-CD38-CAR for patients with refractory DHL. Disclosures No relevant conflicts of interest to declare.