scholarly journals The Spatial Heterogeneity in Newly Diagnosed Multiple Myeloma Patients - from Sub-Clonal Architecture to the Immune Microenvironment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 729-729
Author(s):  
Lukas John ◽  
Alexandra Poos ◽  
Stephan M Tirier ◽  
Jan-Philipp Mallm ◽  
Nina Prokoph ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Medicinal Product ◽  
Board Of Directors ◽  
Research Funding ◽  
Tumor Heterogeneity ◽  
Investigational Medicinal Product ◽  
Mutational Signatures ◽  
Advisory Committees ◽  
Cell Clones

Abstract Tumor heterogeneity plays a significant role in the development of therapy resistance in multiple myeloma (MM). Focal lesions (FLs), which are nodular accumulations of MM cells, have been shown to be hotspots of genetic spatial tumor heterogeneity, which is characterized by unique tumor sub-clones at different sites in the bone marrow (BM). However, little is known about the mechanisms leading to mutations in FLs, the architecture of the tumor microenvironment (ME) at these sites, and the link between FL sub-clones and relapse. We applied whole genome sequencing (WGS) to CD138 + MM cells from paired FL and iliac crest random BM aspirates (RBMA) of 15 newly diagnosed MM (NDMM) patients. For 7 of these patients, single cell (sc) analyses were performed, including sc gene expression (scRNA) and T-cell receptor (TCR)-sequencing and sc assay for transposase-accessible chromatin (ATAC)-sequencing for paired BM CD138 + MM and CD138 - ME, as well as peripheral blood mononuclear cells (PBMC). WGS data was analyzed using inhouse pipelines. Mutations, copy-number-variations and mutational signatures were called using mpileup, ACESeq and mmsig. Neoantigen epitopes were predicted using NeoPredPipe. Sc data was generated using the 10X Genomics platform. Pre-processing and analysis of the sc data was performed with CellRanger and the R-packages Seurat, ArchR and inferCNV. In 13/15 patients we found significant differences in chromosomal and mutational profiles between FLs and paired RBMAs, with major unshared mutations (mutation seen in > 60% cells) being enriched at the FL site (mean 310 vs. 123, p<0.05). Mutations in driver genes, such as KRAS, CYLD, CDKN2C and TP53, were site-unique or strongly enriched in FLs in 6/15 patients. To identify the mechanisms underlying heterogeneous mutations, we analyzed mutational signatures and found COSMIC signature SBS18 in these mutations, suggesting a role of reactive oxygen species. Combining WGS and sc sequencing, we observed between 3 and 6 sub-clones per patient. Sub-clones, which dominated in FLs, showed increased regulatory accessibility and expression of genes associated with disease aggressiveness and drug resistance such as CXCR4 and members of the NFKB- and interferon pathways, implying that FLs could play a significant role in the development of treatment resistance. Indeed, comparing sub-clones at baseline and at relapse after high-dose melphalan and autologous stem cell transplantation in one patient, we observed expansions of tumor cells at relapse, which were closely related to the main FL sub-clone at baseline. On average, 23 (range 0-83) site-unique baseline mutations were predicted to be neoantigens. Thus, we hypothesized that spatial tumor heterogeneity could be associated with heterogeneity in the tumor ME. We did not observe expansion of site-unique T cell clones, but some of the clones were enriched up to 10-fold at one of the two sites. These clones were typically seen in the PB at low frequency. Expanded T-cells clones were almost exclusively found in the CD8 +-compartment, with 65% and 27% of expanded T-cell clones being CD45RO +/CD57 +-memory- and CD69 +-effector-T-cells, respectively. Besides differences in the T-cell clonality, we observed changes in proportions of other cell types, including a depletion of CD14+- and CD16+-macrophages in FLs (p<0.05). Furthermore, we observed gene expression differences between FL and RBMA macrophages, especially for genes involved in TNFα, IL-6 and JAK/STAT3 signaling. While CCL2, CD44, CXCL2/3, KLF2/4 and CCR1 were significantly higher expressed in FLs compared to RBMAs, BTG2, DUSP1 and HIF1A were down-regulated. In conclusion, our results strengthen the concept of MM as a spatially heterogeneous disease, suggest that reactive oxygen species result in site-specific mutagenesis, and support the hypothesis that FLs are the origin of aggressive disease. We demonstrate spatial heterogeneity at single-cell level in the BM immune ME for the first time, which implies that understanding the complex biology of FLs could be important in the context of novel immune therapies such as bispecific antibodies and CAR-T-cells. Disclosures John: Janssen: Consultancy. Müller-Tidow: Janssen Cilag: Consultancy, Research Funding; Bioline: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Goldschmidt: Takeda: Consultancy, Research Funding; Sanofi: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Dietmar-Hopp-Foundation: Other: Grant; Novartis: Honoraria, Research Funding; Mundipharma: Research Funding; MSD: Research Funding; Molecular Partners: Research Funding; Johns Hopkins University: Other: Grant; Janssen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Incyte: Research Funding; GSK: Honoraria; Chugai: Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; BMS: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Celgene: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Adaptive Biotechnology: Consultancy; Amgen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding. Raab: Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Weinhold: Sanofi: Honoraria.

scholarly journals Mapping the Multiple Myeloma T Cell Landscape By Immunotherapeutic Perturbation Reveals Mechanism and Determinants of Response to Bispecific T Cell Engagers

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 731-731
Author(s):  
Mirco Friedrich ◽  
Paola Neri ◽  
Noemie Leblay ◽  
Niklas Kehl ◽  
Julius Michel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Medicinal Product ◽  
Board Of Directors ◽  
Research Funding ◽  
Investigational Medicinal Product ◽  
Advisory Committees ◽  
T Cell Clones ◽  
Cell Clones

Abstract Immunotherapies have transformed the clinical care of patients with cancer. Bispecific T cell engagers (TCEs) have recently entered early-phase clinical trials of multiple myeloma (MM) and shown remarkable response rates even in heavily pretreated patients. However, T cells are heterogeneous with respect to phenotype, function and specificity for tumor antigens and currently we have limited understanding how to identify and monitor tumor specific T cells in hematological malignancies. It is furthermore unclear why individual patients fail to elicit an antitumor immune response upon treatment with TCEs and whether a persistent T cell response to TCEs relies on reinvigoration of pre-existing tumor-infiltrating lymphocytes or on recruitment of novel T cells. Here we performed longitudinal paired single-cell RNA and T cell receptor (TCR) sequencing on >100,000 immune cells from patients with MM before, during and after TCE therapy. We defined transcriptional gradients of MM-infiltrating immune cells between n=5 healthy bone marrow donors, n=10 newly diagnosed MM patients and n=11 refractory MM patients undergoing immunotherapy with bispecific BCMA-targeting antibodies. By tracking T cell clones over time using their TCR as individual barcode, we further integrated these longitudinal in vivo data with protein-level analysis and functional validation in MM bone-marrow cultures exposed to TCEs. Refractory MM patients exhibited a highly individual bone-marrow immune composition, that was significantly perturbed compared to healthy or diseased, but therapy-naïve bone marrow. We observed that the inter-patient heterogeneity in the T cell landscape composition is superimposed by conserved TCR repertoire dynamics forming a trajectory between early anti-tumor effector states and exhaustion. In all patients, we observed a dichotomy of TCE-responsive versus TCE-refractory T cell clones. Longitudinal tracking of TCE-responsive T cell clones and their transcriptional phenotypes revealed coupling of tumor recognition, clonal expansion and T cell dysfunction marked by expression of cytotoxicity (GZMB, GNLY) and terminal exhaustion markers, such as TOX and CD39. Significant clonal replacement of T cells was evident in n=5 clinically responding patients with MM throughout continued TCE therapy and driven by a subset of non-exhausted, naïve-like CD8 + T cells. The top 1% TCE-responsive clones were fate-determined and either followed a memory-exhaustion or cytotoxicity trajectory. Patients who did not respond to TCE therapy exhibited a dysfunctional T cell landscape before therapy that limited clonal expansion and TCR persistence. As proof-of-concept, we matched single-cell profiling data of n=10 individual patients with protein-level analysis and functional validation of TCE-driven T cell expansion in vitro, providing the first signals of preferential expansion of specific fate- and avidity-determined clones upon TCE-mediated stimulation. We propose the mode of action of TCE therapy in MM to be driven by pre-existing T cell fate commitments that determine clonotype diversification and persistence, and ultimately, clinical response. Our results further demonstrate that clinical TCE response derives from a distinct repertoire of pre-existing T cell clones, whereas other clonotypes are functionally excluded from the repertoire and subsequently lost during therapy. We define the determinants of response to TCE treatment to be inherent to the individual's T cell repertoire before therapy. Our results provide the rationale for response prediction and monitoring of future immunotherapy approaches in MM patients beyond TCE therapy. Figure 1 Figure 1. Disclosures Neri: BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Goldschmidt: Amgen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Adaptive Biotechnology: Consultancy; Celgene: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; BMS: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Chugai: Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; GSK: Honoraria; Incyte: Research Funding; Janssen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Johns Hopkins University: Other: Grant; Molecular Partners: Research Funding; MSD: Research Funding; Mundipharma: Research Funding; Novartis: Honoraria, Research Funding; Dietmar-Hopp-Foundation: Other: Grant; Sanofi: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Takeda: Consultancy, Research Funding. Weinhold: Sanofi: Honoraria. Raab: Abbvie: Consultancy, Honoraria; Roche: Consultancy; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bahlis: Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genentech: Consultancy; Janssen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

scholarly journals Long-Term Remission of CLL Sustained By Pauciclonal Anti-CD19 Chimeric Antigen Receptor T (CTL019) Cell Clones

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 699-699 ◽  
Author(s):  
J. Joseph Melenhorst ◽  
David L. Porter ◽  
Lifeng Tian ◽  
Simon F Lacey ◽  
Christopher L Nobles ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Integration Site ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Clones ◽  
Car T

Abstract We recently demonstrated that sustained remission in 41 CLL patients treated with the CD19-specific, 4-1BB/CD3zeta-signaling chimeric antigen receptor (CAR19) T-cells correlated strongly with the expansion and persistence of the engineered T cells and that important pathways such as T cell exhaustion, glycolysis and T cell differentiation segregated responders from non-responders (Fraietta et al., 2018, Nature Medicine). We here report two advanced, chemotherapy-resistant CLL patients with the longest (7 years) follow-up on any trial of CART19 cells. Both patients had received five therapies before being treated at the University of Pennsylvania with autologous, murine CTL019 (tisagenlecleucel) cells for their CLL in 2010, receiving 1.1e9 and 1.4e7 CAR19+ T cells, respectively. Both patients have persistence of CAR-engineered T cells and both patients are still in remission as determined by flow cytometry and deep sequencing of IgH rearrangements for 5.5-7 years. Thus, the infused CAR-T cells have maintained these patients in deep molecular remission of their disease for the longest period of time that has been reported to date. To understand the fate of the infused CAR-T cells we determined the phenotype, function, and clonal nature of the persisting CTL019 cells. Flow cytometric CART19 cell analyses demonstrated that early during the anti-leukemia response, activated, HLA-DR-expressing CD8+ CAR-T cells rapidly expanded, followed by similarly activated CD4+ CAR-T cells. With tumor clearance the CAR-T cell population contracted, but an activated CD4+ CAR-T cell population was maintained and was still detectable at the last follow-up of 7 years. The CD8+ CAR-T cell pool remained present at low frequencies. Both populations had acquired and maintained an effector memory phenotype, a phenotype most consistent with active disease control. Furthermore, the analysis of the classical immune checkpoint inhibitory markers PD1, TIM3, LAG3, and CTLA4 showed that only PD1 was expressed from the earliest to the latest time point on >80% of all CAR-T cells, whereas LAG3 and TIM3 were expressed only early on but lost after tumor clearance. These data suggest that the initial tumor clearance was mediated by CD8+ CAR-T cells, but sustained by a CD4+ CAR-T cell population that still actively engages with target cells. To understand the clonal nature of these long-term persisting CAR-T cells we used two complementary methods: a) CAR T cells were sorted from post-infusion aliquots during the first two years for T cell receptor-beta deep-sequencing (TCR-seq); b) the CAR integration sites in the genome were sequenced in the infusion product and in circulating CAR-T cells. TCR-seq analysis of early post-infusion time points demonstrated that the circulating CAR-T cell populations consisted of hundreds to thousands of distinct clones which in patient 1 and 2 displayed clonal focusing by 21 and 1 month post-infusion, respectively, with some clones making up as much as 12% (patient 1) and 48% (patient 2) of the CAR-T cell repertoire. The analysis of clonotype sharing at the various time points via Morisita's overlap index analysis similarly showed repertoire stabilization late (21 months; patient 1) and early (1 month; patient 2) after infusion. Lastly, fate mapping of the infused CART19 cells via CAR integration site analysis in the infusion product until the latest time point indicated that the infusion products for both patients had a very diverse, non-clonal make-up, containing over 8,000 and 3,700 integration sites in patients 1 and 2, respectively. The higher degree of clonality in patient 2 but not 1 CAR-T cells as seen by TCR-seq was confirmed by integration site analysis, as was the sharing of CAR-T cell clones over time. Importantly, whereas the CAR integration site repertoire in patient 1 was diverse in the first two years, it stabilized and trended towards oligoclonality 21 months after infusion. Lastly, CAR integration site analysis revealed a high degree of clonal persistence, suggesting that tumor control and B cell aplasia were maintained by few, highly functional CD4+ CAR-T cell clones. In summary, we demonstrate that in both patients with the longest persistence of CAR-T cells reported thus far, early and late phases of the anti-CLL response are dominated by highly activated CD8+ and CD4+ CAR-T cells, respectively, largely comprised of a small number of persisting CD4+ CAR-T cell clones. Disclosures Melenhorst: Parker Institute for Cancer Immunotherapy: Research Funding; Incyte: Research Funding; Casi Pharmaceuticals: Consultancy; novartis: Patents & Royalties, Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Porter:Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board. Lacey:Novartis Pharmaceuticals Corporation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding. Fraietta:Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Frey:Novartis: Consultancy; Servier Consultancy: Consultancy. Young:Novartis: Patents & Royalties, Research Funding. Siegel:Novartis: Research Funding. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals Synergistic Role of Leukemic and Non-Leukemic Immune Repertoires in CD8+ T-Cell Large Granular Lymphocytic Leukemia As Identified By Single-Cell Transcriptomics

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1318-1318
Author(s):  
Dipabarna Bhattacharya ◽  
Jani Huuhtanen ◽  
Matti Kankainen ◽  
Tapio Lönnberg ◽  
Cassandra M Kerr ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Healthy Controls ◽  
Advisory Committees ◽  
Cell Clones ◽  
Tcr Repertoire

Abstract Background: T-cell large granular lymphocytic leukemia (T-LGLL), a rare lymphoproliferative disorder of mature T cells, is characterized by the accumulation of activated effector T cells leading to a clonally restricted T-cell receptor (TCR) repertoire. Chronic antigen stimulation together with activating somatic STAT3 mutations have been proposed to lead to clonal expansion of leukemic cells. However, no holistic research has been done to show how leukemic and non-leukemic cells liaise to sustain abnormal immune reactivity in T-LGLL. Methods: We investigated the transcriptome and TCR repertoire in T-LGLL using: 1) single-cell RNA and TCR (scRNA+TCRαβ) sequencing from CD45+ sorted blood cells (T-LGLL n=11, healthy n=6), 2) TCRβ sequencing from blood mononuclear cells (T-LGLL n=48, healthy n=823), 3) bulk RNA sequencing (T-LGLL n=15, healthy n=5), 4) plasma cytokine profiling (T-LGLL n=9, healthy n=9), and 5) flow cytometry validations (T-LGLL n=6, healthy n=6) (Figure) Results: ScRNA+TCRαβ-seq data revealed that in healthy controls, hyperexpanded CD8+ T-cell clones (at least 10 cells with identical TCRs) preferentially had an effector memory phenotype, whereas in T-LGLL, the hyperexpanded clonotypes represented a more cytotoxic (increased expression of GZMB, PRF1, KLRB1) and exhausted (LAG3 and TIGIT) phenotype. Using flow cytometry, we confirmed that upon anti-CD3/CD28/CD49 antibody stimulation, T-LGLL clones (CD8+CD57+) expressed higher levels of cytotoxic proteins (GZMA /GZMB , PRF1) but were deficient in degranulation responses and cytokine secretion as measured by expression of CD107a/b and TNFα/IFNγ, respectively. Focused re-clustering of the extracted T-LGLL clones from the scRNA+TCRαβ-seq data revealed considerable heterogeneity among the T-LGLL clones and partly separated the mutated (mt) STAT3 and wild type (wt) STAT3 clones. STAT3wt clones upregulated T-cell activation and TCR signaling pathways, with a higher cytotoxicity and lower exhaustion score as compared to STAT3mt clones. This was validated with bulk RNA-seq data. To understand the antigen specificities of the T-LGLL clones, we combined previously profiled T-LGLL TCRs with our data to form the largest described dataset of 200 T-LGLL clones from 170 patients. Notably, T-LGLL clones were found to be private to each patient. Furthermore, the analysis by GLIPH2 algorithm grouping TCRs did not reveal detectable structural similarities, suggesting the absence of a unifying antigen in T-LGLL. However, in 67% of T-LGLL patients, the TCRs of leukemic clones shared amino acid level similarities with the rest of the non-leukemic TCR repertoire suggesting that the clonal and non-clonal immune repertoires are connected via common target antigens. To analyze the non-clonal immune repertoire in T-LGLL in detail, we compared our data to other published scRNAseq data from solid tumors (n=4) and hematologic cancers (n=8) and healthy controls (n=6). The analysis revealed that in T-LGLL also the non-leukemic CD8+ and CD4+ T cells were more mature, cytotoxic, and clonally restricted. When compared to healthy controls and other cancer patients, in non-leukemic T-LGLL the most upregulated pathway was IFNγ response. Finally, most of the upregulated cytokines in T-LGLL (e.g., CCL2/3/7, CXCL10/11, IL15RA) were secreted predominantly by monocytes and dendritic cells, which also had upregulated HLA class II expression and enhanced scavenging potential in T-LGLL patients. Ligand-receptor analysis with CellPhoneDB revealed that the number of predicted cell-cell interactions was significantly higher in T-LGLL as compared to reactive T-cell clones in healthy controls. The most co-stimulatory interactions (e.g., CD2-CD58, TNFSF14-TNFRSF14) occurred between the IFNγ secreting T-LGLL clones and the pro-inflammatory cytokine secreting monocytes. Conclusions: Our study shows a synergistic interplay between the leukemic and non-leukemic immune cell repertoires in T-LGLL, where an aberrant antigen-driven immune response including hyperexpanded CD8+ T-LGLL cells, non-leukemic CD8+ cells, CD4+ cells, and monocytes contribute to the persistence of the T-LGLL clones. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clones in patients with T-LGLL. Figure 1 Figure 1. Disclosures Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Maciejewski: Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy. Mustjoki: Novartis: Research Funding; BMS: Research Funding; Janpix: Research Funding; Pfizer: Research Funding.

scholarly journals Intra-Tumoral CD8+ T-Cells in Follicular Lymphoma Contain Large Clonal Expansions That Are Amenable to Dual-Checkpoint Blockade

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2793-2793 ◽  
Author(s):  
Karthik Nath ◽  
Soi C. Law ◽  
Muhammed B. Sabdia ◽  
Lilia Merida De Long ◽  
Mohamed Shanavas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Checkpoint Blockade ◽  
T Cell Subsets ◽  
Advisory Committees ◽  
Cell Clones ◽  
Tcr Repertoire

Introduction. Intra-tumoral T-cell infiltration is associated with R-CHOP responsiveness in aggressive B-cell lymphoma (Keane, Lancet Haem 2015). These patients also have a broad (i.e. diverse) intra-tumoral T-cell receptor (TCR) repertoire with a ~20% superior survival compared to those with a narrow (i.e. clonal) repertoire after R-CHOP therapy. Here, the major contributor to the TCR clonal expansion were CD8+ T cells (Keane, CCR 2017). Paradoxically, our recent results in Follicular Lymphoma (FL) (Tobin, JCO in press) found that clonal T-cell expansions were markedly enriched in those patients that experienced progression of disease within 24 months (POD24). Given that FL is a histological subtype associated with a tumor microenvironment distinct from DLBCL including numerous CD4+ T-follicular helper cells (TFH), we now expand upon these findings by comparing TCR repertoires across histological subtypes. We then established whether the TCR repertoire in FL is related to differential TCR clonal expansions between different T-cell subsets and immune checkpoints. Finally, the overlap between tissue and blood TCR repertoires was investigated. Methods. Firstly, unbiased, high-throughput TCRβ sequencing (ImmunoSEQ, Adaptive Biotechnologies) was compared in 164 FFPE tissues (12 healthy nodes, 40 FL, 88 DLBCL, and as a comparator tumor known to be sensitive to checkpoint blockade and to have a high neoantigen burden, 24 melanoma tissues). Next, to determine the contribution of individual T-cell subsets to overall clonality, a further 21 fresh de-aggregated/cryopreserved FL tumor samples were FACS sorted into four T-cell groupings: CD8+ cytotoxic T-lymphocytes (CTLs), CD4+ TFH, CD4+ regulatory T-cells (TREGs) and 'other' (non-TFH/TREG) CD4+ T-cells. Flow cytometry quantified the expression of the checkpoints LAG3, TIM3 and PD1. Then, 5 FL paired tissue/blood samples were tested for shared TCR clones. Results. FL exhibited strikingly reduced TCR repertoire clonality (higher diversity) compared to DLBCL, melanoma and healthy lymph nodes (Fig 1A). Analysis of de-aggregated sorted nodal T-cells revealed a more complex TCR repertoire. The outcome measure was median clonality index (CIx ranging from '0' or minimal, to '1' or maximal clonality). Large T-cell clones in FL (CIx=0.12) predominantly resided within the CTL subset (34% all T-cells). By contrast, there was marked T-cell diversity in TFH (CIx=0.04; 27% all T-cells), TREG (CIx=0.02; 7% all T-cells) and 'other' CD4+ T-cells (CIx=0.02; 32% all T-cells) (Fig 1B). The CTL population had a bimodal expression for PD1 (+51%/-49%), a marker in FL that has been shown to remain functionally active unless co-expressed with LAG3 and/or TIM3 (Yang, Oncotarget 2017). These dual-checkpoint expressing CTLs have reduced capacity to produce cytokines or lytic granules (i.e. they are 'exhausted'). Notably, 54% of the PD1+ CTLs co-expressed either LAG3 or TIM3. Put together, these results are consistent with expanded CTL clones that are frequently functionally exhausted. In contrast, TFH, TREG and 'other' CD4+ T-cells had a low expression of LAG3 and TIM3, although PD1 was frequently found (as expected, particularly in the TFH cells). Finally, in paired tissue/blood samples, there was weak overlap between the circulating and intra-tumoral TCR repertoire in CTLs and TFH T-cells. Conclusion. Although FL has a markedly less clonal TCR repertoire compared to DLBCL, melanoma and even healthy nodes, this result is misleading. Detailed analysis on sorted intra-tumoral T-cell subsets in FL revealed large clonal expansions in CTLs, with approximately half of these classified as functionally exhausted (dual-positive for PD1 and LAG3 and/or TIM3), a state potentially amenable to reversal by dual-checkpoint blockade. The explanation for TCR repertoire diversity lies in CD4+ T-cells (representing approximately two-thirds of T-cells, including the large TFH subset). T-cells in blood did not reflect FL tissue T-cell clones, further highlighting the need for sorted intra-tumoral nodal tissues to accurately assess TCR repertoires in FL. Further characterization of the neo-antigenic targets that CTL clones potentially recognize is required. These results have implications for therapeutic vaccine design and selective recruitment of patients for immune checkpoint blockade. Disclosures Keane: MSD: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Roche: Consultancy, Other: Travel Grant; BMS: Research Funding. Gandhi:Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding.

The Bone Marrow Microenvironment of Multiple Myeloma Long-Term Survivors at Single Cell Resolution

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-33
Author(s):  
Raphael Lutz ◽  
Abdelrahman Mahmoud ◽  
Mohamed H.S. Awwad ◽  
Charles D. Imbusch ◽  
Tobias Boch ◽  
...  
Keyword(s):  
T Cell ◽  
Disease Activity ◽  
Medicinal Product ◽  
Board Of Directors ◽  
Research Funding ◽  
Investigational Medicinal Product ◽  
Initial Diagnosis ◽  
Cell Compartment ◽  
Advisory Committees ◽  
Early Relapse

To date, multiple myeloma (MM) remains an incurable disease with only a minor fraction of patients experiencing long-term remission (LTR) over 7 years after a single therapy line. Myeloma cells strongly depend on the interaction with their bone marrow microenvironment (BMME), but the molecular and cellular adaptations of the BMME to active MM disease and the role of the immune system in patients experiencing LTR remain poorly understood. In order to gain a global and detailed understanding of the BMME, we profiled over 290.000 BM resident cells from 11 MM patients in LTR 7 to 17 years after first-line therapy and 3 healthy donors using droplet-based single-cell RNA sequencing. Paired BM samples collected at initial diagnosis enabled us to analyze the changes from first diagnosis to the state of LTR in individual patients. At initial diagnosis, we observed significant remodeling of the T cell, NK cell and myeloid compartments which was only partially reversible upon LTR. In- depth analysis of the CD8+ T- cell compartment revealed an unknown immunophenotype of myeloma-associated CD8+ T (MAT) cells expressing key mediators of T cell dysfunction such as NR4A2. The amino acid transporter LAT1 (SLC7A5), which is known to be critical to maintain the activation state of T cells, and the surface marker CD6 were specifically expressed by MAT cells. We validated the existence of this novel T cell immunophenotype in an independent group of 30 MM patients using FACS. The number of MAT cells was associated with myeloma cell burden indicating that MAT-cells might be an indirect marker for tumor load within the BM. The clinical and prognostic meaning of this population is currently under investigation. Within the myeloid compartment, we detected myeloma associated myeloid (MAM) cells at initial diagnosis that were only present in case of active disease. These MAM cells shared features of immunosuppression, inflammation and migration as well as chemotaxis hinting towards a phenomenon of immune cell recruitment to the site of disease. At the LTR stage, 6 of 11 patients were still in complete remission (CR), while 5 patients presented with detectable disease activity after having achieved a CR. In the CR-group we observed a healthy-like state in the BMME but still detected a myeloma associated imprint even in minimal residual disease negative patients. Within the CD8+ T cell compartment, this imprint included a higher metabolic activity in the naïve T cell compartment as well as a higher grade of cytotoxicity within the effector T cell and NK cell compartment. These observations might reflect a state of active immunosurveillance in MM patients to maintain CR at the LTR state. In contrast, 5 LTR patients with detectable disease activity lost the CR associated immune signature approaching a BMME remodeling similar to initial diagnosis. Increasing disease activity over the next 2 years within this patient population showed that we captured a state of early relapse. This enabled us to describe programs specific to early relapse in comparison to the full- blown disease state. In this context, an increase in plasmacytoid dendritic cells (pDCs), key players in the production of interferons, was observed at the stage of early relapse hinting towards a role for pDCs in establishing the inflammatory changes in the BMME upon resurgence of disease. Together, this study provides a comprehensive overview of the molecular and cellular patterns within the BMME that underlie active myeloma disease as well as LTR in MM. We describe novel immunophenotypes of T cells and myeloid cells associated with myeloma cell burden within the BM. At the stage of LTR, our results reveal how patients in CR approach a healthy-like state but still preserve an imprint of MM potentially associated with active immunosurveillance. Finally, this study deepens the understanding how BMME remodeling evolves from an early phase of relapse to a full-blown disease. Disclosures Durie: Amgen, Celgene, Johnson & Johnson, and Takeda: Consultancy. Raab:Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding. Müller-Tidow:Pfizer: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; BiolineRx: Research Funding; Janssen-Cilag GmbH: Speakers Bureau. Goldschmidt:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding; Merck Sharp and Dohme (MSD): Research Funding; Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Incyte: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline (GSK): Honoraria; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Molecular Partners: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Mundipharma GmbH: Research Funding.

scholarly journals Safety and Efficacy of Multiantigen-Targeted T Cells for Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1014-1014 ◽  
Author(s):  
Premal Lulla ◽  
Ifigeneia Tzannou ◽  
George Carrum ◽  
Carlos A. Ramos ◽  
Rammurti Kamble ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Advisory Committees ◽  
T Cell Clones ◽  
Cell Clones ◽  
Equity Ownership ◽  
T Cell Lines

Abstract Despite an array of approved agents for the treatment of multiple myeloma (MM), most patients eventually relapse after conventional treatments. The adoptive transfer of tumor-targeted T cells has demonstrated efficacy in the treatment of patients with chemo-refractory hematological malignancies including MM. While the majority of T cell-based immunotherapeutic studies in the clinic explore genetically modified T cells that target a single tumor-expressed antigen, we have developed a strategy to generate non-engineered T cell lines that simultaneously target multiple MM-expressed antigens, thereby reducing the risk of tumor immune evasion. We manufacture multiTAA-specific T cells targeting the tumor-associated antigens PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin by culturing patient-derived PBMCs with autologous DCs loaded with pepmixes (15mer peptides overlapping by 11 aminos acids) spanning all 5 target antigens in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail. In our current clinical trial (NCT02291848), we have successfully generated multi-antigen-targeted lines from 18/ of 19 patients thus far, with one in production. The T cell lines comprise of CD3+ T cells (mean 95.6±2.2%) with a mixture of CD4+ (28.9±7.2%) and CD8+ (56.6±7.2%) T cells, which express central and effector memory markers (CD45RO+/CD62L+/CCR7+ -- 1.21±0.2%; CD45RO+/CD62L+/CCR7- -- 15.16±2.5%; CD45RO+/CD62L-/CCR7- -- 56.9±6.3%). All the expanded lines were specific for two to five target antigens with the majority of lines (13 of 18) specific for ≥3, (PRAME: Mean 45, range: 0 to 205 spot forming units (SFU)/2x105 input cells ; SSX2 mean: 57, 0 to 583, NYESO1: mean: 51, 0 to 125 , MAGE-A4 Mean: 67, 0 to 377 and Survivin mean: 53, 0 to 51), and did not react against non-malignant autologous recipient cells (2±3% specific lysis; E:T 20:1). We assessed the clonal diversity of the clinical product using TCR vβ deep sequencing analysis. We found both polyclonality and that the majority (mean 79%; range: 59 to 95%) represented rare T cell clones that were unique to the ex vivo expanded cell line and below levels of detection in the patients peripheral blood prior to infusion, thereby enabling in vivo tracking studies.. To date we have infused 18 patients with at least 2 infusions, 2 weeks apart of doses ranging from 0.5 to 2x107/m2. These patients had received a median of 4 lines of prior therapy including high dose chemotherapy with autologous stem cell rescue. Ten patients were refractory to their latest therapy and had active MM, while 8 were in remission at the time of infusion. At the 6 week evaluation period, of the 10 patients receiving multiTAA-specific T cells to treat active disease, 1 had a complete remission (CR) by the international myeloma working group (IMWG) response criteria, 1 had a partial remission (PR) and 8 others had stable disease (SD). Seven of these 10 patients were infused more than 1 year ago. Although 2 of the 7 subsequently had disease progression, the remaining 5 continue to respond, with sustained CR (1), PR (2) or SD (2). Of the 8 patients in CR at the time of T cell infusion, all remained in CR at the week 6 disease assessment and of the 6 evaluable patients who are >1 year post T cells, only one patient has relapsed, at 7 months after T cell infusion. These clinical responses correlated with the emergence and persistence (>6 months) of "line-exclusive" tumor-reactive T cells in patient peripheral blood, as assessed by longitudinal tracking of infused T cell clones using TCR deep sequencing. These infused product-derived T cells were detected in both peripheral blood (mean 0.43% ±SD of 0.3 of the total repertoire) and the marrow (mean 0.61%±0.24% of total repertoire). The expansion of product-derived T cell clones was higher among patients with active MM than in patients treated in remission (active: 0.60±0.39%, remission: 0.2±0.08%, p=0.048). Notably, no patient, including the complete responder, had infusion-related systemic- or neuro-toxicity. Thus, autologous multiTAA-targeted T cells directed to PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin can be safely administered to patients with MM, in whom they can subsequently be detected long-term in peripheral blood and marrow, and where they produce sustained tumor responses including CR. It will be of interest to discover whether larger or more frequent doses of these T cells can produce further benefit with maintained safety. Disclosures Brenner: Marker: Equity Ownership. Heslop:Marker: Equity Ownership; Viracyte: Equity Ownership; Cell Medica: Research Funding; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Cytosen: Membership on an entity's Board of Directors or advisory committees. Vera:Marker: Equity Ownership. Leen:Marker: Equity Ownership.

scholarly journals Automated Manufacture of Matched Donor-Derived Allogeneic CD19 CAR T-Cells for Relapsed/Refractory B-ALL Following Allogeneic Stem Cell Transplantation: Toxicity, Efficacy and the Important Role of Lymphodepletion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 776-776
Author(s):  
Claire Roddie ◽  
Maeve A O'Reilly ◽  
Maria A V Marzolini ◽  
Leigh Wood ◽  
Juliana Dias Alves Pinto ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Peak Car ◽  
Matched Donor

Introduction: 2nd generation CD19 CAR T cells show unprecedented efficacy in B-ALL, but several challenges remain: (1) scaling manufacture to meet patient need and (2) feasibility of generating products from lymphopenic patients post allogeneic stem cell transplant (allo-SCT). To overcome these issues we propose: (1) use of the CliniMACS Prodigy (Miltenyi Biotec), a semi-automated cGMP platform that simplifies CAR T cell manufacture and (2) the use of matched donor T cells to overcome the challenge posed by patient lymphopenia, albeit this may come with a heightened risk of graft versus host disease (GvHD). CARD (NCT02893189) is a Phase I study of matched donor derived CD19 CAR T cells generated on the CliniMACS Prodigy in 14 adult patients with relapsed/refractory (r/r) B ALL following allo-SCT. We additionally explore the requirement for lymphodepletion (LD) in the allogeneic CAR T cell setting and report on the incidence of GvHD with this therapy. Methods: Manufacturing: CARD utilises non-mobilised matched donor leucapheresate to manufacture 2nd generation CD19CAR T cells using a closed CliniMACS® Prodigy/ TransACTTM process. Study design: Eligible subjects are aged 16-70y with r/r B ALL following allo SCT. Study endpoints include feasibility of CD19CAR T cell manufacture from allo-SCT donors on the CliniMACS Prodigy and assessments of engraftment and safety including GvHD. To assess the requirement for LD prior to CD19CAR T cells in lymphopenic post-allo-SCT patients, the study is split into Cohort 1 (no LD) and Cohort 2 (fludarabine (30 mg/m2 x3) and cyclophosphamide (300mg/m2 x3)). To mitigate for the potential GvHD risk, cell dosing on study mirrors conventional donor lymphocyte infusion (DLI) schedules and is based on total CD3+ (not CAR T) cell numbers: Dose 1=1x106/kg CD3+ T cells; Dose 2= 3x106/kg CD3+ T cells; Dose 3= 1x107/kg CD3+ T cells. Results: As of 26 July 2019, 17 matched allo SCT donors were leukapheresed and 16 products were successfully manufactured and QP released. Patient demographics are as follows: (1) median patient age was 43y (range 19-64y); (2) 4/17 had prior blinatumomab and 5/17 prior inotuzumab ozogamicin; (3) 7/17 had myeloablative allo SCT and 10/17 reduced intensity allo SCT of which 6/17 were sibling donors and 12/17 were matched unrelated donors. No patients with haploidentical transplant were enrolled. To date, 12/16 patients have received at least 1 dose of CD19CAR T cells: 7/16 on Cohort 1 and 5/16 on Cohort 2 (2/16 are pending infusion on Cohort 2 and 2/16 died of fungal infection prior to infusion). Median follow-up for all 12 patients is 22.9 months (IQR 2.9-25.9; range 0.7 - 25.9). At the time of CAR T cell infusion, 7/12 patients were in morphological relapse with >5% leukemic blasts. Despite this, CD19CAR T cells were administered safely: only 2/12 patients experienced Grade 3 CRS (UPenn criteria), both in Cohort 1, which fully resolved with Tocilizumab and corticosteroids. No patients experienced ≥Grade 3 neurotoxicity and importantly, no patients experienced clinically significant GvHD. In Cohort 1 (7 patients), median peak CAR expansion by flow was 87 CD19CAR/uL blood whereas in Cohort 2 (5 patients to date), median peak CAR expansion was 1309 CD19CAR/uL blood. This difference is likely to reflect the use of LD in Cohort 2. CAR T cell persistence by qPCR in Cohort 1 is short, with demonstrable CAR in only 2/7 treated patients at Month 2. Data for Cohort 2 is immature, but this will also be reported at the meeting in addition to potential mechanisms underlying the short persistence observed in Cohort 1. Of the 10 response evaluable patients (2/12 pending marrow assessment), 9/10 (90%) achieved flow/molecular MRD negative CR at 6 weeks. 2/9 responders experienced CD19 negative relapse (one at M3, one at M5) and 3/9 responders experienced CD19+ relapse (one at M3, one at M9, one at M12). 4/10 (40%) response evaluable patients remain on study and continue in flow/molecular MRD negative remission at a median follow up of 11.9 months (range 2.9-25.9). Conclusions: Donor-derived matched allogeneic CD19 CAR T cells are straightforward to manufacture using the CliniMACS Prodigy and deliver excellent early remission rates, with 90% MRD negative CR observed at Week 6 in the absence of severe CAR associated toxicity or GvHD. Peak CAR expansion appears to be compromised by the absence of LD and this may lead to a higher relapse rate. Updated results from Cohorts 1 and 2 will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Qasim:Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Identification of Two CAR T-Cell Populations Associated with Complete Response or Progressive Disease in Adult Lymphoma Patients Treated with Axi-Cel

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 779-779 ◽  
Author(s):  
Zinaida Good ◽  
Jay Y. Spiegel ◽  
Bita Sahaf ◽  
Meena B. Malipatlolla ◽  
Matthew J. Frank ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Board ◽  
Advisory Committees ◽  
Car T Cells ◽  
Scientific Advisory Board ◽  
Car T ◽  
Scientific Advisory

Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.

scholarly journals Human CD83 Targeted Chimeric Antigen Receptor T Cell for the Prevention of Graft Versus Host Disease and Treatment of Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 196-196
Author(s):  
Bishwas Shrestha ◽  
Kelly Walton ◽  
Jordan Reff ◽  
Elizabeth M. Sagatys ◽  
Nhan Tu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Graft Versus Host ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Fund Research

Distinct from pharmacologic immunosuppression, we designed a programmed cytolytic effector T cell that prevents graft versus host disease (GVHD). CD83 is expressed on allo-activated conventional T cells (Tconv) and pro-inflammatory dendritic cells (DCs), which are implicated in GVHD pathogenesis. Therefore we developed a novel human CD83 targeted chimeric antigen receptor (CAR) T cell for GVHD prophylaxis. Here we demonstrate that human CD83 CAR T cells eradicate cell mediators of GVHD, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide lasting protection from xenogeneic GVHD. Further, we show human, acute myeloid leukemia (AML) expresses CD83 and can be targeted by CD83 CAR T cells. A 2nd generation CD83 CAR was generated with CD3ζ and 41BB costimulatory domain that was retrovirally transduced in human T cells to generate CD83 CAR T cells. The CD83 CAR construct exhibited a high degree of transduction efficiency of about 60%. The CD83 CAR T cells demonstrated robust IFN-γ and IL-2 production, killing, and proliferation when cultured with CD83+ target cells. To test whether human CD83 CAR T cells reduce alloreactivity in vitro, we investigated their suppressive function in allogeneic mixed leukocyte reactions (alloMLR). CD83 CAR T cells were added to 5-day alloMLRs consisting of autologous T cells and allogeneic monocyte-derived DCs at ratios ranging from 3:1 to 1:10. The CD83 CAR T cells potently reduced alloreactive T cell proliferation compared to mock transduced and CD19 CAR T cells. We identified that CD83 is differentially expressed on alloreactive Tconv, compared to Tregs. Moreover, the CD83 CAR T cell efficiently depletes CD83+ Tconv and proinflammatory DCs with 48 hours of engagement. To test the efficacy of human CD83 CAR T cells in vivo, we used an established xenogeneic GVHD model, where mice were inoculated with human PBMCs (25x106) and autologous CD83 CAR (1-10x106) or mock transduced T cells. The CD83 CAR T cells were well tolerated by the mice, and significantly improved survival compared to mock transduced T cells (Figure 1A). Mice treated with CD83 CAR T cells exhibited negligible GVHD target organ damage at day +21 (Figure 1B). Mice inoculated with CD83 CAR T cells demonstrated significantly fewer CD1c+, CD83+ DCs (1.7x106 v 6.2x105, P=0.002), CD4+, CD83+ T cells (4.8x103 v 5.8x102, P=0.005), and pathogenic Th1 cells (3.1x105 v 1.1x102, P=0.005) at day +21, compared to mice treated with mock transduced T cells. Moreover, the ratio of Treg to alloreactive Tconv (CD25+ non-Treg) was significantly increased among mice treated with CD83 CAR T cells (78 v 346, P=0.02), compared to mice injected with mock transduced T cells. Further, CD83 appears to be a promising candidate to target myeloid malignancies. We observed CD83 expression on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, the CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Thus, we evaluated for stem cell killing in human colony forming unit (CFU) assays, which demonstrated negligible on-target, off-tumor toxicity. Therefore, the human CD83 CAR T cell is an innovative cell-based approach to prevent GVHD, while providing direct anti-tumor activity against myeloid malignancies. Figure Disclosures Blazar: Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Davila:Atara: Research Funding; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Anixa: Consultancy; Novartis: Research Funding.

scholarly journals Pre-Existing T Cell Subsets Determine Anti-PD1 Blockade Response in Richter's Transformation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 42-43
Author(s):  
Prajish Iyer ◽  
Lu Yang ◽  
Zhi-Zhang Yang ◽  
Charla R. Secreto ◽  
Sutapa Sinha ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Gene Signature ◽  
T Cell Subsets ◽  
Rna Seq ◽  
Advisory Committees

Despite recent developments in the therapy of chronic lymphocytic leukemia (CLL), Richter's transformation (RT), an aggressive lymphoma, remains a clinical challenge. Immune checkpoint inhibitor (ICI) therapy has shown promise in selective lymphoma types, however, only 30-40% RT patients respond to anti-PD1 pembrolizumab; while the underlying CLL failed to respond and 10% CLL patients progress rapidly within 2 months of treatment. Studies indicate pre-existing T cells in tumor biopsies are associated with a greater anti-PD1 response, hence we hypothesized that pre-existing T cell subset characteristics and regulation in anti-PD1 responders differed from those who progressed in CLL. We used mass cytometry (CyTOF) to analyze T cell subsets isolated from peripheral blood mononuclear cells (PBMCs) from 19 patients with who received pembrolizumab as a single agent. PBMCs were obtained baseline(pre-therapy) and within 3 months of therapy initiation. Among this cohort, 3 patients had complete or partial response (responders), 2 patients had rapid disease progression (progressors) (Fig. A), and 14 had stable disease (non-responders) within the first 3 months of therapy. CyTOF analysis revealed that Treg subsets in responders as compared with progressors or non-responders (MFI -55 vs.30, p=0.001) at both baseline and post-therapy were increased (Fig. B). This quantitative analysis indicated an existing difference in Tregs and distinct molecular dynamic changes in response to pembrolizumab between responders and progressors. To delineate the T cell characteristics in progressors and responders, we performed single-cell RNA-seq (SC-RNA-seq; 10X Genomics platform) using T (CD3+) cells enriched from PBMCs derived from three patients (1 responder: RS2; 2 progressors: CLL14, CLL17) before and after treatment. A total of ~10000 cells were captured and an average of 1215 genes was detected per cell. Using a clustering approach (Seurat V3.1.5), we identified 7 T cell clusters based on transcriptional signature (Fig.C). Responders had a larger fraction of Tregs (Cluster 5) as compared with progressors (p=0.03, Fig. D), and these Tregs showed an IFN-related gene signature (Fig. E). To determine any changes in the cellular circuitry in Tregs between responders and progressors, we used FOXP3, CD25, and CD127 as markers for Tregs in our SC-RNA-seq data. We saw a greater expression of FOXP3, CD25, CD127, in RS2 in comparison to CLL17 and CLL14. Gene set enrichment analysis (GSEA) revealed the upregulation of genes involved in lymphocyte activation and FOXP3-regulated Treg development-related pathways in the responder's Tregs (Fig.F). Together, the greater expression of genes involved in Treg activation may reduce the suppressive functions of Tregs, which led to the response to anti-PD1 treatment seen in RS2 consistent with Tregs in melanoma. To delineate any state changes in T cells between progressors and responder, we performed trajectory analysis using Monocle (R package tool) and identified enrichment of MYC/TNF/IFNG gene signature in state 1 and an effector T signature in state 3 For RS2 after treatment (p=0.003), indicating pembrolizumab induced proliferative and functional T cell signatures in the responder only. Further, our single-cell results were supported by the T cell receptor (TCR beta) repertoire analysis (Adaptive Biotechnology). As an inverse measure of TCR diversity, productive TCR clonality in CLL14 and CLL17 samples was 0.638 and 0.408 at baseline, respectively. Fifty percent of all peripheral blood T cells were represented by one large TCR clone in CLL14(progressor) suggesting tumor related T-cell clone expansion. In contrast, RS2(responder) contained a profile of diverse T cell clones with a clonality of 0.027 (Fig. H). Pembrolizumab therapy did not change the clonality of the three patients during the treatment course (data not shown). In summary, we identified enriched Treg signatures delineating responders from progressors on pembrolizumab treatment, paradoxical to the current understanding of T cell subsets in solid tumors. However, these data are consistent with the recent observation that the presence of Tregs suggests a better prognosis in Hodgkin lymphoma, Follicular lymphoma, and other hematological malignancies. Figure 1 Disclosures Kay: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Sunesis: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; MEI Pharma: Research Funding. Ansell:AI Therapeutics: Research Funding; Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding. Ding:Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: pembrolizumab

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