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.

IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2431-2431
Author(s):  
Marta Coscia ◽  
Francesca Pantaleoni ◽  
Chiara Riganti ◽  
Candida Vitale ◽  
Micol Rigoni ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Board Of Directors ◽  
Stromal Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Spontaneous Apoptosis ◽  
Advisory Committees

Abstract Abstract 2431 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival. Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system. These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

scholarly journals Targeting BTN2A1 By a Unique Activating Mab Improves Vγ9Vδ2 T Cell Cytotoxicity Against Primary Acute Lymphoblastic Blasts

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2302-2302
Author(s):  
Anne-Charlotte Le Floch ◽  
Caroline Imbert ◽  
Aude De Gassart ◽  
Florence Orlanducci ◽  
Aude Le Roy ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemic Cells ◽  
Target Cells ◽  
Advisory Committees ◽  
Vγ9vδ2 T Cells ◽  
Privately Held

Abstract Introduction Vγ9Vδ2 T cells are new promising cytotoxic effectors in hematological malignancies. In acute myeloid leukemia and in non-Hodgkin lymphomas, Vγ9Vδ2 T cells-based immunotherapy has shown encouraging results both in preclinical models and in early phase clinical trials. Acute lymphoblastic leukemia (ALL) includes very heterogeneous clinico-biological entities, for which recent immunotherapy approaches are currently being developed. Nevertheless, global prognosis of ALL patients still be poor with a 5 years-overall survival of less than 40% and therefore, treatments need to be improved. Very few data are currently available on susceptibility of ALL blasts to Vγ9Vδ2 T cell cytotoxic activity. Vγ9Vδ2 T cells are activated by phosphoantigens bound to BTN3A1 on target cells. BTN3A molecules are targeted at clinical level, with the ICT01 agonist monoclonal antibody (mAb), that is currently tested in a multicentric phase ½ study (EVICTION study). Biology of Vγ9Vδ2 T cells has recently undergone a new paradigm with the identification of BTN2A1 as the direct ligand for Vγ9 chain of γδ TCR. BTN2A1 is mandatory for Vγ9Vδ2 T cell activation but its precise role in modulating functions of Vγ9Vδ2 T cells remains unknown. Here, we show that allogenic and autologous Vγ9Vδ2 T cells exert cytolytic functions against ALL cell lines and primary ALL blasts, and we report that Vγ9Vδ2 T cell cytotoxic activity is enhanced after treatment with a unique agonist mAb targeting BTN2A1. Material and methods 5 ALL cell lines (697, RS4;11, NALM-6, HPB-ALL, SUP-T1) and PBMC from 11 adults ALL patients at diagnosis (B-ALL, T-ALL and Ph+ ALL) were tested in functional assays. We evaluated apoptosis of ALL cell lines and of primary ALL blasts after coculture with allogenic Vγ9Vδ2 T cells. ALL samples were also tested for their expansion capacities and a degranulation assay was performed at D14. We assessed in parallel relative quantification of the level expression of BTN2A1 (ICT0302 and 7.48 epitopes), and BTN3A (20.1 and 108.5 epitopes) on surface of ALL blasts. DAUDI-BTN2AKO+2A1 and HEK293-BTN2AKO+2A1 cells were used in binding assays, and modulation of TCR binding was assessed using recombinant tetramerized Vγ9Vδ2 TCR. Results We showed that Vγ9Vδ2 T cells exert spontaneous cytotoxicity against ALL cell lines and primary ALL blasts with a heterogeneous susceptibility depending on the target. We demonstrated that anti-BTN2A1 ICT0302 agonist mAb significantly enhanced Vγ9Vδ2 T cells mediated apoptosis in comparison to control condition, even for the less spontaneously susceptible cells. We confirmed these observations with degranulation of autologous Vγ9Vδ2 T cells expanded from 5 ALL patients at diagnosis that was increased after treatment with anti-BTN2A1 ICT0302 agonist mAb. BTN3A and BTN2A1 were detected on surface of ALL blasts, and BTN3A 108.5 was the most expressed epitope. Interestingly, we observed that anti-BTN2A1 ICT0302 strongly increased binding of a recombinant Vγ9Vδ2 TCR to target cells using with HEK293 and DAUDI cells. Discussion Our results highlighted that Vγ9Vδ2 T cells exert cytolytic functions against ALL cells, both in allogenic and autologous setting and demonstrated that BTN2A1 targeting with our unique agonist mAb could potentiate effector activities of Vγ9Vδ2 T cells against ALL blasts. These results indicate that the sensitization of leukemic cells can be induced by activation BTN3A as well as BTN2A1 mAbs. These data bring novel understanding on the biology of BTN2A1 on leukemic cells and our ability to enhance both binding and function. These findings could be of great interest for the design of innovative Vγ9Vδ2 T cells-based immunotherapy strategies for treating ALL that could be extended to other cancer types. Disclosures De Gassart: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Cano: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Olive: Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Anti-BTN2A1 ICT0302 is a murine agonist monoclonal antibody targeting BTN2A1 whose aim is to increase Vgamma9Vdelta2 T cells functions.

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 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 The NLPHL Tumor Microenvironment Is Markedly Enriched in the Tigit and PD-1 Signalling Axes Compared to Classical Hodgkin Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3513-3513
Author(s):  
Jay Gunawardana ◽  
Muhammed B. Sabdia ◽  
Karolina Bednarska ◽  
Soi C. Law ◽  
Sandra Brosda ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Immune Checkpoint ◽  
Research Funding ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Immune Checkpoints ◽  
Advisory Committees ◽  
Tcr Repertoire

Abstract Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) comprises 5% of all Hodgkin lymphomas (HL). Its biology remains poorly characterized. Like classical HL (cHL), it contains minimal malignant cells embedded within a T cell rich intra-tumoral microenvironment (TME). Unlike cHL, it can transform to diffuse large B cell lymphoma (DLBCL). Immune-checkpoint blockade is effective in cHL but has minimal activity in DLBCL. No data is currently available regarding the potential to reactivate host anti-tumoral activity via immune-checkpoint blockade in NLPHL. Diagnostic FFPE samples from 49 NLPHL patients retrospectively collected from 4 Australian centres were interrogated. Inclusion criteria were sample availability and centrally confirmed histological NLPHL. Characteristics were in line with the literature: median age 45 years, range 13-82 years; F:M 1:3.5; stage I/II 55%, III/IV 35% (10% stage unknown) with the majority of cases were of immuno-architectural types A or C. RNA was digitally quantified using the NanoString 770-gene PanCancer Immune panel. Multi-spectral immunofluorescent (mIF) microscopy, plasma soluble PD-1 quantification, cell sorting, T cell receptor (TCR) repertoire analysis and functional immuno-assays were also performed. Results were compared with samples from 38 cHL and 35 DLBCL patients. We initially compared gene expression of NLPHL and cHL, looking for molecular similarities and differences. Ten non-lymphomatous nodes (NLN) were included as controls. Unsupervised clustering showed all but 3 NLPHL cases segregated from the cHL cluster. All NLN congregated in a discrete sub-cluster. As expected, RNA analysis showed significant enrichment for CD20 in NLPHL and CD30 in HL. Volcano plots (Fig. 1a), corrected for false-discovery showed marked variation in gene expression. For NLPHL (vs. cHL) there were 105 upregulated and 337 down regulated genes. Strikingly, the most significantly differentially over-expressed genes in NLPHL were all T cell related (CD247: CD3 zeta chain; CD3D: CD3 delta chain; GZMK: granzyme K; EOMES: marker of CD8 + T cell tolerance; and the immune checkpoints PDCD1: encodes for PD-1; and TIGIT). CD8B expression was increased in NLPHL. For cHL, the most over-expressed genes included macrophage-derived chemokines CCL17 and CCL22. Gene set enrichment analysis revealed activation of the PD-L1 expression and PD-1 checkpoint pathway and 9 of the top 10 Gene Ontology (GO) term enrichment scores involved lymphocyte signalling in NLPHL (Fig. 1b). To better appreciate the impact of the relevant immune checkpoints on their signalling axis, we compared gene ratios for PD-1 and TIGIT receptors with their ligands (PD-L1/L2 and PVR, respectively). NLPHL showed the highest enrichment ratios of these signalling pathways vs. cHL, DLBCL and NLN (Fig. 1c). Although it is known that CD4 +PD-1 +T cells form rosettes around NLPHL cells, the differential cellular localization of immune proteins has not been compared between HL entities. Using mIF, the proportion of intra-tumoral PD-1 + was markedly higher for CD4 + (~7-fold; p<0.0001) and CD8 + (~5-fold; p<0.001) T cells in NLPHL. However, the proportion of T cells expressing LAG3 was similar. Soluble PD-1 was elevated for both NLPHL and cHL, indicating circulating blood is influenced by the TME. For both HL entities over 80% of circulating CD4 + and CD8 + T cells expressed PD-1 alone or in combination with TIGIT. TCR repertoire analysis of sorted T cell subsets showed large intra-tumoral clonal T cell expansions were also detectable in circulating T cells. T cell clones were predominantly PD1 +CD4 + T cells in both HL types. Finally, we developed a functional assay using PD-L1/PD-L2 expressing NLPHL and cHL cell lines. These were co-cultured with genetically engineered PD-1 +CD4 + T cells that express a luciferase reporter. Similar levels of heightened T cell activation were seen with immune-checkpoint blockade for both HL entities, indicating that immune-checkpoint inhibition may also be of benefit in NLPHL. In conclusion, our multi-faceted analysis of the immunobiological features of the TME in NLPHL, provides a compelling rationale for early phase clinical studies that incorporate immune-checkpoint blockade in NLPHL. Figure 1 Figure 1. Disclosures Hawkes: Bristol Myers Squib/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Specialised Therapeutics: Consultancy; Merck KgA: Research Funding; Merck Sharpe Dohme: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Antigene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Speakers Bureau; Janssen: Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel and accommodation expenses, Research Funding, Speakers Bureau. Swain: Janssen: Other: Travel expenses paid; Novartis: Other: Travel expenses paid. Keane: BMS: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Karyopharm: Consultancy; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Talaulikar: Takeda: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Jansenn: Honoraria, Research Funding; Roche: Honoraria, Research Funding; EUSA Pharma: Honoraria, Research Funding. Gandhi: janssen: Research Funding; novartis: Honoraria.

scholarly journals Single-Cell Characterization of the Immune and Leukemic Cells Following Anti-TIM3 and Hypomethylating Agent Combination Therapy in Patients with AML or MDS

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 801-801
Author(s):  
Jani Huuhtanen ◽  
Oscar Brück ◽  
Karita Peltonen ◽  
Anna Kreutzman ◽  
Olli Dufva ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Immune Cells ◽  
Research Funding ◽  
Cell Types ◽  
Leukemic Cells ◽  
Healthy Controls ◽  
Advisory Committees ◽  
Ifn Γ

Abstract Background The success of allogeneic stem cell transplantation supports the notion that immunotherapy can have curative potential in AML, but immune checkpoint therapies (e.g., anti-PD1) have shown only modest clinical efficacy. TIM3 is an immune-checkpoint molecule expressed both on immune and leukemic cells but not on healthy hematopoietic stem cells (HSCs), making it a particularly interesting target in AML. In myeloid malignancies, the combination of anti-TIM3 therapy with hypomethylating agents (HMA), which may prime the tumor microenvironment for immune therapies, has shown promising initial response rates up to 50%-60% of patients, but their mechanism of action is not fully understood. Methods We analyzed the effects of anti-TIM3 (sabatolimab, MBG453) in combination with decitabine in 11 refractory/relapsed AML patients and 1 MDS patient recruited in a phase Ib trial (NCT03066648), with 5/12 responders (3 CR, 2 CRi). We studied paired bone-marrow (BM) and peripheral blood samples with scRNA+TCRαβ-seq enriched for CD45+ immune cells (90% of input) and blast cells (10%) and flow cytometry. Additionally, to explore the expression of TIM3 and other immune checkpoints in different cell populations, we combined scRNAseq data from 160 BM aspirate samples across 10 different hematological malignancies and healthy controls. Results Our pan-heme scRNA-seq data analysis of over 500'000 cells revealed that unlike PD1 and CTLA4, HAVCR2 (TIM3) was primarily expressed in NK and myeloid cells (including dendritic cells [DCs], macrophages, and monocytes). In healthy controls, the expression of HAVCR2 was low in T-cells, but in patients with heme-malignancies, expression was seen on activated T-cells. In HSC populations, AML patients had generally upregulated HAVCR2 expression compared with healthy subjects. ScRNAseq data of 20 samples (n=7 patients) treated with anti-TIM3+HMA revealed that at baseline, DCs were more highly represented in samples from the responding (n=4) than from the non-responding patients (n=3). Following anti-TIM3+HMA treatment, DCs expanded significantly, and upregulated pathways related to interleukin production (IL-1b, IL-18) in responders, suggestive of an activated inflammasome response. At baseline, the most expanded NK-phenotype expressed the highest amounts of HAVCR2, which varied between patients from CD56 bright to adaptive NK cells. Anti-TIM3+HMA therapy modulated NK cells especially in responders, in which NK cells downregulated HAVCR2 and upregulated the NF-κB pathway. Importantly, the NF-κB pathway was upregulated in other cell types in responding patients, but not in non-responding patients. In contrast, the IFN-γ response was downregulated in both responding and non-responding patients in multiple different cell types. The highest expression of HAVCR2 in T cells was seen in cells co-expressing NK-receptors and with the highest cytotoxicity. Analysis of the scTCRαβ-seq revealed that the combination treatment did not have a marked effect on T-cell clonality, but one patient with CR had a significantly expanded large granular lymphocyte (LGL) clone covering 4%-25% of the repertoire. In responding patients, HAVCR2+ regulatory T-cells were more numerous at baseline, contracted following therapy, and lost response to IFN-γ, a pattern not seen in non-responding patients. The analysis of predicted cell-cell interactions between leukemic and immune cells did not show significant interactions between inhibitory PD1 or CTLA4, and their ligands, but ubiquitous LGALS9 - HAVCR2 interactions were predicted in leukemic bone marrows. Responding patients had more these interactions, which decreased following therapy. Non-responding patients had multiple interactions between T/NK cells and blasts via PVR and its ligands which were not seen in responding patients, which represent a putative resistance mechanism for anti-TIM3+HMA therapy. Conclusions Unlike PD1 and CTLA4, TIM3 is expressed on leukemic, DC, myeloid, and NK cells, and consistent with this finding, the effects of TIM3 blockade in vivo were mainly observed in these cell types. In responding patients, NFκB pathway was activated in T/NK cells following anti-TIM3 and HMA treatment concomitant with a decrease in inhibitory interactions. Our results pinpoint the differential effects of TIM3-blockade on immune cells and may aid in developing predictive biomarkers for treatment outcome. Figure 1 Figure 1. Disclosures Kreutzman: Novartis: Current Employment. Kontro: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Orlando: Novartis: Current Employment. Cremasco: Novartis: Current Employment. Wagner: Novartis: Current Employment, Current holder of individual stocks in a privately-held company. Pelletier: Novartis: Current Employment. Sabatos-Peyton: Novartis: Current Employment. Rinne: Novartis: Current Employment; Qiagen: Consultancy. Mustjoki: Janpix: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; BMS: Research Funding.

scholarly journals Richter's Transformation after CD-19 Directed CAR-T Cells for Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1430-1430
Author(s):  
Amanda Blackmon ◽  
Alexey V. Danilov ◽  
Lili Wang ◽  
Raju Pillai ◽  
Hormoz Babaei Mirshkarlo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Abstract Introduction Approximately 5-10% of patients with chronic lymphocytic lymphoma (CLL) will develop transformation to a more aggressive lymphoma, usually diffuse large B-cell lymphoma (Richter's transformation, RT). The median overall survival after transformation is less than one year. It remains difficult to predict which patients will transform although there is a correlation with poor risk features of CLL, like del17p/TP53 mutation and Notch1 mutations among others. While data emerging from trials of CD19-directed CAR-T cells (CD19CART) in CLL are showing promising results in the relapsed/refractory setting, there appears to be an emergence of RT in some cases even when there is no measurable residual CLL. For instance, in the phase 1 portion of the TRANSCEND CLL 004 trial, in the monotherapy arm with lisocabtagene maraleucel (n=23), 5 RT cases emerged subsequently and 3 of these had no recurrent CLL or MRD conversion to positive [Siddiqi T, et al. ASH 2020]. Four of these RT events were in patients who had progressed on both ibrutinib and venetoclax. Here we describe patients who developed RT after receiving CD19CART for CLL at City of Hope. Methods A retrospective chart review was performed to identify RT emergence and to analyze key factors surrounding the development of RT after CD19CART for CLL at City of Hope. Patient characteristics were assessed including age, sex, prior number of treatments, CLL FISH panel, mutational analysis, time on BTK inhibitor therapy, response to CAR T cell therapy, time to RT after CD19CAR T cell therapy, and outcomes after RT. Pathology samples from RT were assessed for CD19 expression and will be assessed for PDL-1, MYC, SYK, ZAP70, AKT, ERK expression by IHC or flow cytometry. Results A total of 7 out of 27 patients have been identified who received CD19CART for CLL at City of Hope and subsequently relapsed with RT [Table 1]. The median age at the time of CD19CART was 66 years (range, 54-68) and median number of prior therapies was 5 (range 4-7). All patients had features associated with high risk CLL prior to CD19CART: 5/7 had del17p; 3/7 had TP53 mutations, 2/7 had NOTCH1 mutations, and 1/7 had SF3B1 mutations. Most patients, 6/7, achieved an objective response to CD19CART with 4/7 undetectable minimal residual disease to a level of <10 -4 cells (uMRD4) CRs on imaging and bone marrow examination, and 1 uMRD4 PR. The median time to transformation after administration of CD19CART was 9.5 months (range 3.5-40 months). All patients had received BTK inhibitor therapy prior to CAR T cells, with the median length of treatment being 1 year (5 months - 4 years) and 6/7 had received prior venetoclax as well. Biopsy material at the time of RT indicated 6/7 were positive for CD19 expression by immunohistochemistry or flow cytometry (1 was only weakly positive). PD-L1, MYC, SYK, ZAP70, AKT, ERK expression will be analyzed, and results presented at the meeting. Of these patients, 3/7 were unable to be treated for RT and died shortly after diagnosis of RT due to frailty, sepsis/respiratory failure/compartment syndrome, and CNS involvement/altered mental status/hypercalcemia/tumor lysis. Two patients achieved CR (one with R-CHOP, one with O-CHOP/pembrolizumab/acalabrutinib) and underwent allogeneic hematopoietic stem cell transplantation - one of which now has relapsed SLL 2.5 years later. Two patients are on clinical trials and are pending response evaluation. Conclusions Given the expression of CD19 in the RT pathology of most cases in this series, it appears that a different mechanism of escape or resistance is occurring in these cases. All 7 pts had poor risk features of their CLL before CD19CART like del17p/TP53 mutation, Notch1 mutation and SF3B1 mutation. We are investigating the RT pathology specimens further and will compare these RT cases with other CLL patients we have treated with CD19CART thus far and who have not relapsed/progressed with RT in order to examine the differences in treatment history, cytogenetic features, proliferative/accelerated nature of CLL at baseline, and PDL1 expression before and after CAR T cell therapy. Improved treatment combinations are needed in high risk, multiply relapsed CLL patients to prevent emergence of RT despite excellent responses of the CLL itself. Figure 1 Figure 1. Disclosures Danilov: Gilead Sciences: Research Funding; Pharmacyclics: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; TG Therapeutics: Consultancy, Research Funding; Takeda Oncology: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria. Siddiqi: Janssen: Speakers Bureau; Oncternal: Research Funding; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kite Pharma: 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; TG Therapeutics: Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: CD19 CAR T products used in clinical trials for relapsed/refractory chronic lymphocytic leukemia

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 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.

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