Manufacture and Immunological Characterization of GMP-Compliant Functional Sars-COV-2 Cytotoxic T Lymphocytes (CTLs) Utilizing the Clinimacs ® Cytokine Capture System

Abstract Background: The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pandemic that has taken millions of lives around the globe. Treatment of patients with moderate and severe COVID-19 disease has included dexamethasone, tocilizumab, Remdesivir, convalescent plasma, and targeted antibodies, however, currently, there are no FDA approved targeted cellular therapies in the treatment of mild or moderate SARS-CoV-2 disease. Virus-specific cytotoxic T cell lymphocytes (vCTLs) have shown therapeutic efficacy in immunocompromised patients with viral infections. We developed a multicenter and multidisciplinary Viral Cytotoxic T-Cell Consortium (VIRCTLC) to investigate the use of vCTLs manufactured by direct enrichment using the Cytokine Capture System (CCS) on the CliniMACS® Prodigy device. SARS-CoV-2 specific PepTivator Peptides consist of overlapping peptides that span the entire sequence of the protein (Protein N and M), or the length of its immunodominant domain (Protein S). The peptides can bind to either MHC class I or MHC class II molecules and are therefore able to target both CD4 and CD8 T cells. Objective: To screen, manufacture, and characterize SARS-CoV-2 vCTLs generated from convalescent COVID-19 donors using the CliniMACS® Cytokine Capture System on the CliniMACS® Prodigy device. Methods: Donor screening was done utilizing PBMNCs from 15 convalescent COVID-19 donors after informed consent. PBMNCs were stimulated with a mix of PepTivator peptides (Miltenyi Biotech®) contained in the S, M and N proteins. IFN-γ levels were examined in CD3, CD4, and CD8 T cells by flow cytometry analysis. After informed consent, PBMNCs from three convalescent COVID-19 donors who screened positively to the PepTivator® peptide pools of SARS-CoV-2 Proteins M, N and S were collected by apheresis using the SPECTRA Optia® apheresis instrument. PBMNCs were incubated with the PepTivator® peptide pools for 4 hours. After incubation, the SARS-CoV-2 vCTLs were enriched using the CliniMACS Cytokine Capture System as we have previously described (Flower/Cairo, et al, ASTCT, 2020). Samples were taken from the enriched vCTLs and tested in gram stains, sterility cultures, cell counts, viability and IFN-γ cytokine staining (CD3/CD4/CD8/IFN-γ marker panel) by flow cytometry. Amplification and sequencing of TCRβ CDR3 regions of pre-stimulated PBMNC, stimulated PBMNCs samples taken from the QC bag (QC samples) and the enriched SARS-CoV-2 vCTLs were performed on the ImmunoSEQ platform using ImmunoSEQ® TCRB Assay kit (Adaptive Biotechnologies, Seattle, WA, USA). Characterization of immune subsets was done by mass cytometry analysis with 41 Immunophenotypic markers. Transcriptome of the immune landscape of QC samples, and enriched vCTLs was compared with the pre samples using the human nCounter PanCancer Immune Profiling Panel on the nCounter system. Results: We demonstrate that 93.3% of convalescent donor blood samples passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that consisted of 79% + 21% (mean + SEM) IFNγ + T cells (Fig.1). TCRβ sequencing showed that convalescent COVID-19 donors have a highly diverse TCR repertoire and we identified TCRβ CDR3 clones that are known to be associated with SARS-CoV-2 T cell responses. Immunophenotyping analysis demonstrated more CD4 T cells than CD8 T cells in the SARS CoV-2 vCTLs, an increase in memory CD8 and CD4 cells, especially CD8 T EM, CD4 T cm and CD4 T EMRA cells (Fig.2) and an increase DC cells in the SARS CoV-2 vCTL products as compared to pre-stimulated PBMNCs. Expression of the exhaustion markers was not enhanced in the SARS CoV-2 vCTLs as compared to pre-stimulated PBMNCs. Transcriptome analysis showed increased gene expression in T-cell function, interleukin, pathogen defense, and TNF superfamily pathway genes in the SARS CoV-2 vCTLs as compared to pre-stimulated PBMNCs. Conclusion: Our study demonstrates that highly functional SARS-CoV-2 vCTLs can be rapidly generated by direct cytokine enrichment from convalescent donor peripheral blood mononuclear cells. These data serve as pre-clinical validation for an ongoing clinical trial utilizing related HLA-matched and haplo-identical SARS CoV-2 vCTLs for the treatment of patients with mild and moderate SARS-CoV-2 disease (IND #27260, NCT# 04896606). Figure 1 Figure 1. Disclosures Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company. Johnson: Miltenyi Biotec: Research Funding. Cairo: Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Sanofi: Speakers Bureau; Servier: Speakers Bureau; Sobi: Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees; Nektar: Membership on an entity's Board of Directors or advisory committees.

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Evaluation of T-Cell Compartment By Complex Multiparameter Flow Cytometry Reveals Distinct Patterns of T-Cell Exhaustion in DLBCL, FL and HL Patients

Blood ◽

10.1182/blood-2019-130647 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2806-2806 ◽

Cited By ~ 1

Author(s):

Alexis Vallée ◽

Mitra Shourian ◽

Nathalie Johnson ◽

Hélène Decaluwe

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

P Value ◽

Advisory Committees ◽

Cell Functions ◽

Pd1 Blockade

Background: Immune checkpoint (IC) blockade has revolutionized the treatment of chemo-refractory solid tumors and has demonstrated promising results for the treatment of resistant blood cancers, including lymphoma. However, clinical response to PD1 blockade depends on the subtype of lymphoma, with durable responses observed in 70 % of Hodgkin lymphoma (HL) patients versus only 36 to 40% of Diffuse Large B-Cell Lymphoma (DLBCL) and Follicular Lymphoma (FL) patients(1-3). The biological aspects that hinder the efficacy of immunotherapy in DLBCL and FL patient are not well-known. Primary tumor resistance to PD1 blockade could be driven by inherent genomic alterations or by ineffective anti-tumor T cell responses. ICs expressed at the surface of tumor-infiltrating-lymphocytes (TILs) use distinct suppressive mechanisms to enforce T-cell dysfunction. The collective expression of ICs defines multiple subsets of exhausted T cells during cancer(4). Since multiple of these ICs collaborate to repress anti-tumor T cell functions, we hypothesized that TILs from DLBCL and FL patients express high levels of ICs and present a more advanced exhausted phenotype compared to TILs from HL patients. Methods: We studied lymphoma samples collected at diagnosis and frozen as a viable cell suspension from 57 patients with HL, FL or DLBCL. We performed an extended immunophenotype of TILs through flow cytometry using maturation markers, exhaustion markers, homing and chemokine receptors. The results were analyzed with FlowJo. Boolean gating was used to assess IC co-expression. Statistical analyses were performed using Prism. Results: Our data indicated that CD8 TILs from DLBCL patients express higher levels of classical ICs such as PD1 (1059 vs 1015 and 648), Tim3 (1747 vs 868 and 918,) and Lag3 (2603 vs 1013 and 1031) compared to FL and HL patients (p-value < 0.01), while the expression of 2B4, BTLA and CD160 were similar. Boolean analysis revealed that DLBCL and FL had a higher frequency of CD8T cells that co-expressed 6-7 ICs, compared to HL, suggesting a more advanced state of exhaustion (19% and 26% vs 14%, p-value < 0.05). This correlated with an increased frequency of PD-1highCD39high T cells in DLBCL and FL patients compared to HL (32% and 24% vs 14%, p-value < 0.005), which correlated with increased frequency of EOMEShigh cells in both DLBCL and FL (84% and 76% vs 55%, p-value < 0.005). These markers identify populations of cells with phenotypic and transcriptomic profiles of terminal exhaustion with reduced cytokine production, proliferative capacity and persistence (5, 6). In contrast, stem-like exhausted CD8 T cells are defined by intermediate expression of PD-1 and expression of the chemokine receptor CXCR5, and are driven by the transcription factor TCF-1(7, 8). We observed that DLBCL exhibited significantly less progenitor Tim3-/CXCR5+ cells compared to FL and HL (27% vs 52% and 40%, p-value < 0.01). Further, the frequency and mean fluorescent intensity of TCF-1 was significantly reduced in DLBCL compared to FL and HL (43% / 495 ± 64 vs 70% / 752 ± 42 and 61% / 670 ± 74, p-value < 0.01). This data correlated with lower frequency of CD27+ CD8 T cells in DLBCL vs FL and HL (82% vs 96% and 96%, p-value < 0.01). Conclusion: Collectively, our data indicate that TILs from DLBCL and FL patients express a whole array of ICs that have non-redundant suppressive functions. Further, these patients present subsets of cells with increased functional exhaustion, subsets demonstrated to be unresponsive to PD-1 blockade(7). Further, DLBCL patients lack protective stem-like T-cell subsets which were recently demonstrated to correlate with clinical outcome(9). These results enlighten our knowledge of the cellular defects precluding potent anti-tumor T cell functions in DLBCL and FL and propose novel targets for immunotherapies in PD-1-unresponsive lymphomas. Research funding: This work is supported by the Canadian Institutes of Health Research (PJT-155996), Canadian Cancer Society and Cole Foundation (grant #705478). References: 1. S. M. Ansell et al., in N Engl J Med 372, 311 (2015). 2. R. Chen et al., J Clin Oncol35, 2125 (2017). 3. A. M. Lesokhin et al., J Clin Oncol34, 2698 (2016). 4. B. Bengsch et al., Immunity48, 1029 (2018). 5. M. A. Paley et al., Science338, 1220 (2012). 6. P. K. Gupta et al., PLoS pathogens11, e1005177 (2015). 7. S. J. Im et al., Nature537, 417 (2016). 8. D. T. Utzschneider et al., Immunity45, 415 (2016). 9. M. Sade-Feldman et al., Cell176, 404 (2019). Disclosures Johnson: Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria; BMS: Consultancy, Honoraria; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Seattle Genetics: Honoraria; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding.

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CD4+ T Cell Fate in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117674 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4494-4494

Author(s):

Rachel Elizabeth Cooke ◽

Jessica Chung ◽

Sarah Gabriel ◽

Hang Quach ◽

Simon J. Harrison ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Flow Cytometric Analysis ◽

Advisory Committees ◽

Mitochondrial Mass ◽

Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Phase 1/2 Study of Nexi-001 Donor-Derived Multi-Antigen Specific CD8+ T Cells for the Treatment of Relapsed Acute Myeloid Leukemia (AML) after Allogeneic Hematopoietic Transplantation

Blood ◽

10.1182/blood-2021-152419 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4819-4819

Author(s):

Monzr M. Al Malki ◽

Sumithira Vasu ◽

Dipenkumar Modi ◽

Miguel-Angel Perales ◽

Lucy Y Ghoda ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Phase 1 ◽

Clinical Activity ◽

Advisory Committees ◽

Over Time

Abstract Patients who relapse after allogeneic HCT have a poor prognosis and few effective treatment options. Responses to salvage therapy with donor lymphocyte infusions (DLI) are driven by a graft versus leukemia (GvL) effect. However, relapses and moderate to severe graft versus host disease (GVHD) are common. Therapies that increase the GvL effect without inducing GVHD are needed. The NEXI-001 study is a prospective, multicenter, open-label phase 1/2 trial designed to characterize the safety, immunogenic, and antitumor activity of the NEXI-001 antigen specific T-cell product. This product is a donor-derived non-genetically engineered therapy that consists of populations of CD8+ T cells that recognize HLA 02.01-restricted peptides from the WT1, PRAME, and Cyclin A1 antigens. These T cells consist of populations with key memory phenotypes, including stem-like memory, central memory, and effector memory cells, with a low proportion (<5%) of potentially allogeneic-reactive T-naïve cells. Patients enrolled into the first cohort of the dose escalation phase received a single infusion of 50 million (M) to 100M cells of the NEXI-001 product. Bridging anti-AML treatment was permitted during the manufacture of the cellular product with a wash-out period of at least 14 days prior to lymphodepletion (LD) chemotherapy (intravenous fludarabine 30 mg/m 2 and cyclophosphamide 300 mg/m 2) that was administered on Days -5, -4, and -3 prior to the infusion of the NEXI-001 product up to 72 hours later (Day1). Lymphocyte recovery to baseline levels occurred as early as three days after the NEXI-001 product infusion with robust CD4 and CD8 T cell reconstitution after LD chemotherapy. NEXI-001 antigen specific T cells were detectable in peripheral blood (PB) by multimer staining and were found to proliferate over time and to traffic to bone marrow. The phenotype composition of detectable antigen specific T cells at both sites was that of the infused product. T-cell receptor (TCR) sequencing assays revealed T cell clones in the NEXI-001 product that were not detected in PB of patients tested at baseline. These unique clones subsequently expanded in PB and bone marrow (BM) and persisted over time. Neutrophil recovery, decreased transfusion burden of platelets and red blood cells, and increased donor chimerism were observed. Decreases in myeloblasts and reduction in the size of an extramedullary myeloid sarcoma were suggestive of clinical activity. One patient, a 23-year- old with MRD+ disease at baseline, received two doses of 200M NEXI-001 cells separated by approximately 2 months. Following the first infusion, antigen specific CD8+ T cells increased gradually in PB to 9% of the total CD3+ T cell population just prior to the second infusion and were found to have trafficked to bone marrow. By Day 2 following the second infusion, which was not preceded by LD chemotherapy, the antigen specific CD8+ T cells again increased to 9% of the total CD3+ T cell population in PB and remained at ≥5% until the end of study visit a month later. The absolute lymphocyte count increased by 50% highlighting continued expansion of the NEXI-001 T cells. These cells also maintained significant Tscm populations. Treatment related adverse events, including infusion reactions, GVHD, CRS, and neurotoxicity (ICANS), have not developed in these patients who have received 50M to 200M T cells of the NEXI-001 product either as single or repeat infusions. In conclusion, these results show that infusion of the NEXI-001 product is safe and capable of generating a cell-mediated immune response with early signs of clinical activity. A second infusion is associated with increasing the level of antigen specific CD8+ T cells and their persistence in PB and BM. TCR sequencing and RNA Seq transcriptional profiling of the CD8+ T cells are planned, and these data will be available for presentation during the ASH conference. At least two cycles of 200M NEXI-001 cells weekly x 3 weeks of a 4-week cycle is planned for the next dose-escalation cohort. Early data suggest that the NEXI-001 product has the potential to enhance a GvL effect with minimal GVHD-associated toxicities. Disclosures Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Neximmune: Consultancy; Hansa Biopharma: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy. Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: travel support; Kiadis, Inc.: Research Funding; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Modi: MorphoSys: Membership on an entity's Board of Directors or advisory committees; Seagen: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding. Perales: Sellas Life Sciences: Honoraria; Novartis: Honoraria, Other; Omeros: Honoraria; Merck: Honoraria; Takeda: Honoraria; Karyopharm: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; MorphoSys: Honoraria; Kite/Gilead: Honoraria, Other; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Medigene: Honoraria; NexImmune: Honoraria; Cidara: Honoraria; Nektar Therapeutics: Honoraria, Other; Servier: Honoraria; Miltenyi Biotec: Honoraria, Other. Edavana: Neximmune, Inc: Current Employment. Lu: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Suarez: Neximmune, Inc: Current Employment. Oelke: Neximmune, Inc: Current Employment. Bednarik: Neximmune, Inc: Current Employment. Knight: Neximmune, Inc: Current Employment. Varela: Kite: Speakers Bureau; Nexlmmune: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-130197 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3202-3202

Author(s):

Cameron S. Bader ◽

Henry Barreras ◽

Casey O. Lightbourn ◽

Sabrina N. Copsel ◽

Dietlinde Wolf ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Activation ◽

Cd8 T Cell ◽

Advisory Committees ◽

Hematopoietic Stem

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells, leading to damage of particular host tissues characteristic of GVHD. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to pro-inflammatory cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) aHSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of MUD C3H.SW bone marrow (BM) + T cells into irradiated B6 wildtype (WT) or STING-/- recipients. Colon tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs WT. MUD STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 wks post-HSCT vs WT. Double chimerism studies showed that the absence of STING in non-hematopoietic cells was responsible for GVHD amelioration. Conversely, a single dose of the highly specific STING agonist DMXAA given in vivo increased IFNβ, TNFα and IL-6 mRNA expression in WT, but not STING-/-, colon tissue 48 hrs after transplant and increased GVHD scores and lethality post-HSCT. Post-transplant cytoxan treatment abolished the ability of DMXAA to augment GVHD, supporting the notion that STING signaling increases donor T cell activation during aHSCT. To evaluate the potential impact of STING in the clinical setting, we transplanted C3H.SW BM + T cells into mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) and found that these mice also exhibited diminished GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD aHSCT contrasts to reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) aHSCT (Fischer J, et al, Sci. Transl. Med. 2017). Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we hypothesized that STING's effect on the predominant T cell subset in each model may explain these seemingly paradoxical results in STING-/- vs WT recipients. Therefore, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING-/- mice and found that - in contrast to MMUD recipients of combined CD4+ and CD8+ T cells - STING-/- recipients developed lower GVHD clinical scores, reduced skin pathology and had lower frequencies of activated T cells 8 wks post-HSCT vs WT, supporting a role for STING in the promotion of CD8+ T cell-mediated GVHD. Next, we investigated if recipient APCs played a role in STING's enhancement of CD8+ T cell-mediatedGVHD. We found that STING-/- mice had greater frequencies and numbers of recipient splenic CD11b+CD11c+ APCs 1 day after MMUD B6 into BALB/c aHSCT (Fig. A). BALB/c-STING-/- APCs also expressed reduced MHC class I protein levels (Fig. B). Moreover, STING-/- recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C). These data support the hypothesis that STING contributes to early activation of donor CD8+ T cells and elimination of recipient APCs. Next, to identify if the loss of host MHC II+ APCs affected subsequent donor CD4+ T cell activation, B6-Nur77GFP transgenic donor T cells were used to explicitly monitor T cell receptor signaling. Consistent with increased numbers of host MHC II+ APCs in the spleens of STING-/- recipients 1 day post-aHSCT, we found greater frequencies and numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ in STING-/- spleens 6 days after transplant (Fig. D). In summary, our studies demonstrate that STING plays an important role in regulating aHSCT and provide one potential mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Overall, our studies suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Blazar: BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: 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; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding. Levy:Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

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Early Mycosis Fungoides Lesions with Increased CD3-CD4+ Histiocytes Are Associated with Dyslipidemia and Use of Statins

Blood ◽

10.1182/blood.v126.23.4609.4609 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4609-4609

Author(s):

Chee Won Oh ◽

Carlos Torres-Cabala ◽

Mikyoung Chang ◽

Madeleine Duvic

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Skin Lesions ◽

Medication History ◽

Advisory Committees ◽

History Of ◽

Skin Biopsies

Abstract Background The term "histiocyte" includes cells of the monocyte/macrophage series as antigen processing cells and the Langerhans cell/DC series as antigen-presenting cells. At least three DC subsets exist in skin: two expressing either CD1a or CD14 are dermal and Langerhans cells expressing CD1a are epidermal. Since the phenotype of histiocytic cells is typically CD3-CD4+, an estimation of the CD4+ histiocytic population can be made by comparing the numbers of CD3+ T cells with CD4+ cells. Programmed Cell Death 1 (PD-1) is an inhibitory receptor expressed on T cells, B cells, and some myeloid cells. During chronic antigen exposure, expression of PD-1 is sustained. Statins, inhibitors of cholesterol biosynthesis, are immunomodulatory agents acting on T cells and DCs, but their effects on skin immunology are unknown. Objectives To investigate whether infiltrates of CD3-CD4+histiocytes in early mycosis fungoides (MF) lesional skin biopsies are associated with any other factors, including history of medication and to reveal their histopathological pattern. Methods From Jan to Dec 2014, we identified cases of early MF from the clinic in which CD4+ cells exceeded CD3+ cells with biopsies to identify increased histiocytic population. Exclusion criteria included Sézary syndrome, granulomatous MF, T cell receptor beta monoclonality, abnormal T cell populations by flow cytometry, retinoid treatment, and progression of disease after treatment (n=12). Clinical and laboratory findings were retrospectively reviewed. Skin biopsies stained for H&E, CD3, CD4, CD7, and CD8 were reviewed. In 3 cases with paraffin blocks available, immunohistochemical stains for CD68, CD1a, CD163, PD-1, and PD-1 ligand PD-L1 were done. Results Clinical manifestations of early MF were pink scaly patches (9/12), capillaritis (2/12), and annular erythema - like patches (1/12). Eleven also had an increased monocytes in peripheral blood. All cases had a medication history of taking statins (atorvastatin 5/12; simvastatin 2/12; rosuvastatin 1/12) for dyslipidemia (hypercholesterolemia 7/12; both hypercholesterolemia and hypertriglyceridemia 3/12). In 9/12, symptoms persisted after MF treatment. A lichenoid or superficial perivascular lymphohistiocytic infiltration was observed in skin lesions. Focal basal vacuolization was found in all 12 patients. Upper dermal perivascular extravasation of RBCs suggesting vasculopathy was also found in 12/12 cases. All twelve cases showed predominant CD4+ T cells compared to CD8+ T cells in dermis and the CD4+ T cells were more prominent in dermis rather than in epidermis. CD7+ T cells were preserved (3/12) or partially lost (9/12). In all 3 cases, macrophage markers CD68 and CD163 were positive in dermal infiltrates. CD1a+ DCs were increased in both epidermis and dermis in all 3/3. Only one case of three showed PD1/PD-L1+ T cells in dermis. Discussion and Conclusion All our cases had a medication history of statins for dyslipidemia. Of interest, skin biopsies showed a vasculopathy previously reported during high-dose atorvastatin treatment (Tehrani et al, 2013) and infiltration of CD4/CD8+ T cells, CD1a+DCs and CD163/CD68+ macrophages. We hypothesize that statins or dyslipidemia in early MF were associated with cutaneous T cell immune reaction. In support of our hypothesis that dyslipidemia is associated with histiocytosis, we found a report of nine cases of granulomatous pigmented purpuric dermatosis with concurrent hyperlipidemia (Battle et al, 2015). Cholesterol induces monocytosis and M1 macrophages in mice. One study showed that predominant migration of mature CD1a+ DC is associated with release of IL-12p70 and efficient expansion of Th 1 cells and functional CD8+ T cells. On the contrary, IL-10 up-regulates migration of immature CD14+ DC, expression of the M2 macrophage marker CD163, poor expansion of CD4+ and CD8+ T cells, and skewing of Th responses conducive to expression of PD-L1. We cannot know whether skin lesions are secondary to hyperlipidemia or to treatment with statins. Although M1 and M2 macrophages can be distinguished by diverse markers, none of these antigens are suitable for single-marker identification by immunohistochemistry in paraffin embedded tissue blocks. Further study of the cutaneous effect and immunologic mechanisms leading to increased expression of DCs and T cell dysfunction after statin medication is necessary. Disclosures Duvic: Oncoceutics: Research Funding; Therakos: Research Funding, Speakers Bureau; Huya Bioscience Int'l: Consultancy; Tetralogics SHAPE: Research Funding; Innate Pharma: Research Funding; Cell Medica Ltd: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; MiRagen Therapeutics: Consultancy; Soligenics: Research Funding; Allos (spectrum): Research Funding; Array Biopharma: Consultancy; Spatz Foundation: Research Funding; Rhizen Pharma: Research Funding; Eisai: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin, Co: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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NME-2 Protein Functions as a Tumour Associated Antigen in HLA-A2+ Cells and Is Over Expressed in CML Via a Bcr/Abl Dependent Post Transcriptional Mechanism.

Blood ◽

10.1182/blood.v114.22.3266.3266 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3266-3266

Author(s):

Sabine Tschiedel ◽

Melanie Adler ◽

Karoline Schubert ◽

Annette Jilo ◽

Enrica Mueller ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cell Response ◽

Research Funding ◽

Kinase Activity ◽

T Cell Response ◽

Advisory Committees

Abstract Abstract 3266 Poster Board III-1 Introduction: NmE2 (Nm23-H2, NDP kinase B) is one of a family of proteins that catalyze the transfer of gamma-phosphate between nucleoside-triphosphates and diphosphates. The two major family members, NmE1 and NmE2 are strongly implicated in the control of differentiation, proliferation, migration and apoptosis via interactions which are often independent of their kinase activity, NmE2 being a transcriptional activator of the c-myc gene. We recently identified NmE2 as a tumour associated, HLA-A32+ restricted, antigen in a patient with CML and found the protein (but not the mRNA) to be generally over expressed in CML but not in other haematological malignancies. We also detected a specific T-cell response in peripheral blood cells of a patient 5 years after transplantation. This identifies NmE2 as a potential target for both molecular and immunotherapy of CML. However, the development of immunotherapeutic approaches will depend on the ability of NmE2 to function as a tumour antigen in common HLA backgrounds. The aims of this study were firstly to investigate the antigenicity of NmE2 in the HLA-A2 background (which accounts for more than 50% of the Caucasian population), and secondly to characterise the regulatory relationship between Bcr/Abl and NmE2 using a cell line model of CML. Materials and Methods: 5 nonameric NmE2 peptides with predicted anchor amino acids for HLA-A2 were loaded at concentrations of 10μM separately onto HLA-A2 expressing antigen presenting cells. Elispot Assays were carried out with CD8+ MLLCs (for the identification of antigenic peptides) or CD8+ cells isolated directly from a CML patient at different time points after HCT. Ba/F3 cells stably expressing wild type and mutant forms of Bcr/Abl were treated with imatinib and nilotinib (0 – 10 μM) for 48h. Bcr/Abl activity was assessed by FACS using antibodies specific for the phosphorylated forms of CrkL and Stat5. NmE2 and c-Myc protein were detected by immunocytochemistry and Western blotting with specific antibodies [Santa Cruz, clones L-16 and 9E10 respectively]. Levels of nme2 and c-myc mRNA were determined by quantitative real time PCR. Results: Full length NmE2 protein and 2 of 5 HLA-A2 anchor-containing peptides tested (NmE2132–140 and NmE2112–120) were specifically recognized by the HLA-A2+ CD8+ MLLC, demonstrating the antigenicity of NmE2 in the HLA-A2 background in vitro. Furthermore, while CD8+ T-cells from a transplanted HLA-A2+ CML patient showed little or no specific reactivity in the first 10 months after HCT, a distinct reactivity (up to 0.6 % NmE2 reactive CD8+ T cells) became apparent at later stages, consistent with the development of an immune response against NmE2-expressing cells in vivo. The patient remained negative for bcr/abl transcripts throughout this period. BA/F3 Bcr/Abl cells expressed increased levels of NmE2 protein (but not mRNA) compared to the parent BA/F3 line. Interestingly, treatment with imatinib or nilotinib reduced NmE2 protein expression in BA/F3 Bcr/Abl, but not in cells expressing Bcr/Abl mutants resistant to the respective inhibitors. Treatment of BA/F3 Bcr/Abl cells with the PI3K inhibitor Ly294002 resulted in reduced Bcr/Abl activity and a corresponding reduction in both c-Myc and NmE2 protein levels, without affecting mRNA levels. Conclusion: The over expression of NmE2 is closely linked to Bcr/Abl kinase activity, the predominant level of regulation being post-transcriptional and dependent on PI-3K activity. The NmE2 protein is restricted by HLA-A2 as well as by HLA-A32. The development of an NmE2-specific T-cell response in a CML patient after stem cell transplantation suggests that NmE2 functions as a tumour antigen in HLA-A2+ patients in vivo and may be relevant to the long term immune control of CML. NmE2 is therefore a promising candidate for the development of new immunotherapeutic strategies for the treatment of CML. Disclosures: Lange: BMS: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Niederwieser:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding.

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Single-Cell Analysis of the Classical Hodgkin Lymphoma Immune Environment Reveals a Clonally-Expanded CD8+ T Cell Population with a Cytotoxic Phenotype

Blood ◽

10.1182/blood-2020-137171 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 40-41

Author(s):

Jovian Yu ◽

Xiufen Chen ◽

James Godfrey ◽

Girish Venkataraman ◽

Sonali M. Smith ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Immune Cell ◽

Clonal Expansion ◽

Advisory Committees ◽

Cell Clusters

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by a robust and complex immune cell infiltrate and the rare presence of malignant Hodgkin-Reed-Sternberg (HRS) cells. At the genetic level, HRS cells recurrently acquire alterations that lead to defective antigen presentation (β2 microglobulin mutations) and mediate T cell dysfunction (PD-L1 copy gains/amplifications) in order to subvert host immune surveillance. The clinical relevance of PD-L1 protein over-expression in cHL is clear, as response rates to PD-1 blockade therapy are extremely high among patients with relapsed/refractory (r/r) disease. Despite its remarkable efficacy, the cells that mediate response to anti-PD-1 therapy in cHL remain undefined. Recent analyses have highlighted a possible role for CD4+ T cells in mediating the clinical activity of anti-PD-1 therapy in cHL. CD4+ T cells significantly outnumber CD8+ T cells in cHL lesions and are more frequently juxtaposed to HRS cells in situ. Furthermore, HLA class II expression on HRS cells predicted higher complete response rates to PD-1 blockade therapy in r/r cHL patients. However, a candidate T cell population capable of specific reactivity to antigens expressed by HRS cells has yet to be identified. This information is critical as such T cells might be functionally reinvigorated to mediate HRS cell elimination following PD-1 blockade therapy. In order to address this key knowledge gap, we analyzed data at single cell (sc) resolution using paired RNA and T cell receptor (TCR) sequencing in 9 diagnostic cHL and 5 reactive lymph node (RLN) specimens. Methods: Sequencing was performed using the 10x Genomics Chromium Single Cell 5' Gene Expression and V(D)J workflows. B-cell depletion of each sample was achieved using CD19 microbeads and negative selection to enrich T cell populations. Reads were analyzed and aligned with CellRanger (v3.1.0) and Seurat (v3.2.0) was used to conduct clustering by a shared nearest neighbor (SNN) graph on scRNA data. TCR sequencing data was integrated using scRepertoire (v1.0.0). Results: A detailed map of the immune cell states in cHL was created using scRNA-seq (10X) data on 79,085 cells from 9 cHL (52,602 cells) and 5 RLN samples (26,484 cells) expressing a total of 21,421 genes (mean 5649 cells/sample; mean 2849 mRNA reads/cell). Dimensionality reduction and unsupervised graph-based clustering revealed 21 distinct cell type and activation state clusters, including T cells, NK cells, macrophages, and dendritic cells (Fig 1A-B). A cluster identifying HRS cells was not observed, consistent with a recently published report. Ten T cell clusters were identified (47,573 cells), including naive- and memory-like T cells, effector/cytotoxic CD8+ T cells, regulatory T cells, and T follicular helper cells. Unexpectedly, a putative exhausted T cell cluster was not clearly observed. The relative contributions of cHL and RLNs cases to these clusters are shown in Fig 1C. Paired TCR sequencing was available for 23,943 cells. Overall TCR diversity was lower among cHL samples compared to RLN specimens (Fig 1D). In cHL samples, modest clonal expansion within regulatory T cell and memory CD4+ T cell clusters was observed, but the most striking clonal expansion occurred among cells assigned to effector/cytotoxic CD8+ T cell clusters - a finding not observed in most RLN specimens (Fig 1E). Clonally-expanded effector/cytotoxic CD8+ T cells displayed high expression of granzymes (GZMA, GZMH, GZMK), cytokines (TNF, IFNG) and chemokines (CCL4/CCL5), and modest expression of exhaustion markers (PDCD1, ENTPD1, HAVCR2, ITGAE), contrasting with data from single-cell analyses of solid tumors. Clonal expansion of effector/cytotoxic CD8+ T cells was particularly robust in EBV-positive cHLs, likely due to recognition of viral-derived epitopes displayed on HRS cells (Fig 1F). Phenotypic and functional validation of key immune cell clusters in cHL specimens using spectral cytometry is underway and will be reported at the meeting. Conclusions: For the first time, our data have unveiled the nature of the T cell repertoire in cHL at single cell resolution. Our results reveal a recurrent pattern of clonal expansion within effector CD8+ cells, which may be the HRS antigen-specific T cells that mediate response to PD-1 blockade. This hypothesis requires confirmation through similar analyses of pre- and on-treatment biopsies of cHL patients receiving anti-PD-1 therapy. Disclosures Godfrey: Gilead: Research Funding; Merck: Research Funding; Verastem: Research Funding. Venkataraman:EUSA Pharma: Speakers Bureau. Smith:Janssen: Consultancy; BMS: Consultancy; TG Therapeutics: Consultancy, Research Funding; Genentech/Roche: Consultancy, Other: Support of parent study and funding of editorial support, Research Funding; Karyopharm: Consultancy, Research Funding; FortySeven: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; Celgene: Consultancy, Research Funding. Kline:Kite/Gilead: Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Venetoclax Enhances Anti-Leukemia Activity of CD123-Specific BiTE-Secreting T-Cells in AML

Blood ◽

10.1182/blood-2020-139625 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-13

Author(s):

Hong Mu-Mosley ◽

Lauren B Ostermann ◽

Ran Zhao ◽

Challice L. Bonifant ◽

Stephen Gottschalk ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Vehicle Control ◽

Advisory Committees ◽

Cellular Therapies

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p<0.001). In the Molm13 luciferase xenograft model, NSGS mice were randomized into 5 groups after AML engraftment was confirmed: 1) vehicle control, 2) Venetoclax (Ven) only, 3) CD123-ENG T-cells only, 4) Ven+CD123-ENG T-cells, 5) Ven+CD123-ENG T-cells/2-day-off Ven post T-cell infusion (Ven[2-day-off]+CD123-ENG). Venetoclax treatment (100 µg/kg daily via oral gavage) was started on day 4 post Molm13 injection, and on day 7, mice received one i.v. dose of CD123-ENG T-cells (5x106 cells/mouse). Venetoclax or CD123-ENG T-cell monotherapy reduced leukemia burden compared to the control group, and combinational treatments further inhibited leukemia progression as judged by BLI and circulating AML cells (%GFP+mCD45-/total live cells) by flow cytometry on day 15 post MOLM-13 injection: vehicle control: 19.6%; Ven+: 3.4%; CD123-ENG T-cells:1.2 %; Ven+CD123-ENG T-cells: 0.3%; Ven[2-day-off]+CD123-ENG T-cells (p<0.01 Ven+ or CD123-ENG T-cells versus control; p<0.001 Ven+CD123-ENG or Ven[2-day-off]+CD123-ENG T cells versus CD123-ENG T cells, n=5). The enhanced anti-AML activity of combining venetoclax and CD123-ENG T-cells translated into a significant survival benefit in comparison to single treatment alone (Fig. 2). However, while Ven+CD123-ENG and Ven[2-day-off]+CD123-ENG T-cell treated mice had a survival advantage, they had reduced circulating numbers of human CD3+ T cells on day 8 post T-cells infusion compared to mice that received CD123-ENG T-cells, indicative of potential adverse effect of venetoclax on T-cell survival in vivo. Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

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CD3xCD19 Dart Treatment Is Efficient in Venetoclax Resistant CLL and Reverses T Cell Dysfunction

Blood ◽

10.1182/blood-2019-123350 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3043-3043

Author(s):

Anne W. J. Martens ◽

Susanne R. Janssen ◽

Ingrid A.M. Derks ◽

Sanne Tonino ◽

Eric Eldering ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Killing ◽

Advisory Committees ◽

Cell Dysfunction ◽

T Cell Dysfunction

Intro - Agents targeting the apoptosis pathway, like the Bcl-2 inhibitor venetoclax, are highly effective in chronic lymphocytic leukemia (CLL). However, not all patients experience deep responses and acquired resistance has already been described. T cell mediated lysis is another tool currently exploited in hematologic malignancies. In contrast to acute lymphoblastic leukemia (ALL) however, efficacy of autologous based T cell therapy, such as CAR T cells, in CLL has been low. This is linked to a CLL mediated acquired T cell dysfunction. Bispecific T cell engagers targeting CD19 are successfully applied in ALL, but whether it overcomes the acquired T cell dysfunction in CLL is unknown. We therefore tested efficacy of a CD3xCD19 Dual Affinity Re-Targeting molecule (DART) in CLL. Since it has been observed that bispecific antibodies can overcome deficient synapse formation in CLL (Robinson et al, 2018) and based on our assumption that T cell mediated lysis differs from venetoclax-mediated killing, we hypothesized that usage of a CD3xCD19 DART in CLL overcomes T cell dysfunction and will be effective against venetoclax resistant CLL. Methods - Co-culture of CLL derived or aged-matched healthy donor (HD) CD4+ and/or CD8+ T cells with (CD40 activated) primary CLL or CD19+ cell lines JeKo-1 or Ramos in presence of CD3xCD19 (JNJ-64052781), CD3xFITC, anti-CD3/28 antibodies was performed. R esults - JeKo-1 cells were highly sensitive to CD3xCD19 mediated HD T cell killing with close to 70% of lysis in a concentration of 10ng/mL using an E:T ratio of 4:1. In the same conditions, primary CLL cells proved sensitive for CD3xCD19 mediated HD T cell killing with 50% of lysis. Killing was observed irrespective of IGHV mutation or chemorefractory status. We next compared HD with CLL-derived T cells by measuring activation levels between direct TCR (anti-CD3/CD28) and CD3xCD19 stimulation. As described, TCR stimulation resulted in impaired CLL CD4+ and CD8+ T cell activation and proliferation when compared to HD. In contrast, treatment of CLL derived PBMCs with CD3xCD19 did not resulted in dysfunctional CLL-derived T cell responses (Fig 1A-C). Consistently, co-culture of CLL derived CD4+, CD8+ or a combination with either JeKo-1 or allogeneic CLL cells in the presence of CD3xCD19 resulted in significant cytotoxicity (Fig. 1D). In the allogeneic setting, cytotoxic capacity of CD4+ T cells was similar to their CD8+ counterparts. When targeting autologous CLL, a benefit was observed when both CD4+ and CD8+ T cells were present (Fig. 1D). We then studied whether venetoclax resistant CLL cells could be targeted by CD3xCD19 mediated T cell killing. Bcl-2 overexpressing Ramos were equally lysed in presence of the CD3xCD19 DART as their wildtype counterpart, indicating that Bcl-2 expression does not inhibit CD3xCD19 mediated cell death. Following CLL cell stimulation by CD40 ligation, anti-apoptotic Bcl-XL, Bfl-1 and Mcl-1 are highly induced (Thijssen et al., 2015) resulting in venetoclax resistance (Fig 1E). Nevertheless, CD40L stimulated CLL cells were as efficiently lysed upon CD3xCD19 treatment as unstimulated CLL. (Fig 1F). This indicates that an augmented apoptotic threshold does not impact efficacy of CD3xCD19. Further examination of the mechanism of CD3xCD19 mediated killing showed that lysis depended on granzymes, as blocking granule exocytosis prevented cell death. Independence of the mitochondrial apoptotic pathway was shown by equal sensitivity to CD3xCD19 mediated T cell lysis comparing BAX/BAK knockout Jeko-1 cells to the parental cell line. Also, caspase blockage did not inhibit cell death, pointing to apoptosis independent killing. In concordance, PARP cleavage could only be detected when caspase activity was not blocked. Conclusion - This is the first report describing reversal of CLL mediated T cell dysfunction by applying a CD3xCD19 DART. Furthermore, it shows that venetoclax resistant CLL can still be efficiently targeted by T cells, in a non-apoptotic fashion. These results imply that T cell mediated therapy could be used alongside venetoclax. Figure 1 Disclosures Eldering: Celgene: Research Funding; Roche: Research Funding; Janssen Pharmaceutical Companies: Research Funding. van der Windt:Genmab: Employment. Kater:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding.

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More Than a BTK Inhibitor; Ibrutinib Profoundly Impacts the Function of Cell Mediated Immunity

Blood ◽

10.1182/blood-2018-99-117618 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4417-4417

Author(s):

Chia Sharpe ◽

Joanne Davis ◽

Kylie D. Mason ◽

Dale I Godfrey ◽

Adam Uldrich ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Function ◽

T Cell Proliferation ◽

Advisory Committees ◽

The Impact ◽

Btk Inhibitors

Abstract Background: Chronic lymphocytic leukaemia (CLL) is associated with profound immune dysfunction, which is often exacerbated by CLL therapies. This study aimed to understand the impact of multiple BTK inhibitors on the function of cytotoxic T cells. BTK, along with ITK, TXK, BMX and TEC is a member of the Tec family of nonreceptor tyrosine kinases which are integral to the downstream signalling of many immune receptors. Critically, BTK and ITK are downstream of the B cell and T cell receptors. Whilst identified as a selective BTK inhibitor, ibrutinib inhibits all five members of the Tec family at clinically meaningful doses (Long et al, JCI, 2017). More selective BTK inhibitors have been developed, including acalabrutinib and zanubrutinib, which have less affinity for ITK but variable inhibition for other Tec family members. Functional impairment of NK cells by ibrutinib is well established (Kohrt et al, Blood 2014), however the effects on T cell function remains unclear. It has been suggested that inhibiting ITK results in skewing of CD4 T cells toward a T helper 1 phenotype whilst CD8 T cell function is preserved due to redundant kinase signalling (Dubovsky et al, Blood, 2013). This is yet to be demonstrated in patients and little is known about the impact of BTK inhibitors on cytotoxic T cell populations. Methods: Peripheral blood mononuclear cells were isolated from CLL patients receiving either ibrutinib or zanubrutinib and from treatment naive CLL patients or age matched healthy donors. T cell proliferation was measured using CellTrace Violet, cultures of whole PBMC were stimulated with CD3/CD28 beads and 20 IU/mL IL-2 and treated with 1uM ibrutinib or zanubrutinib or acalabrutinib or vehicle control for 7 days, n=7. CD8 T cell and NKT cell degranulation in response to CD3/CD28 stimulation was assessed using CD107a mobilisation and IFN𝛾 production over 4 hours, n=6 and n=7. Whole PBMC from patients treated with either ibrutinib or zanubrutinib were stimulated for 24 hours using CD3/CD28 beads, supernatants were analysed using a BD™ Cytometric Bead Array. Results: In vitro treatment with ibrutinib significantly impairs the function of cytotoxic T cells. Both CD4 and CD8 T cells cultured in the presence of ibrutinib had significantly decreased proliferation, whilst zanubrutinib and acalabrutinib did not significantly impact T cell proliferation (Figure 1, p=0.0002 and p<0.0001). Furthermore, CD8 T cells from CLL patients and healthy donors had significant abrogation of degranulation and cytokine production when treated with Ibrutinib but not the more selective BTK inhibitors. Similarly, ibrutinib treated healthy donor NKT cells showed significantly diminished degranulation and IFN𝛾 production (Figure 2, p =0.0004 and p=0.0003). Finally, PBMC isolated from patients after treatment with ibrutinib had muted cytokine production including IL-2 (p= 0.003), IL-17A (p=0.023), TNF (p=0.031) and IL-10 (p=0.016) as compared to PBMC isolated before ibrutinib treatment. However there was no significant change in Th1 or Th2 cytokines. Discussion: Together these results highlight the impact of Ibrutinib on cell mediated cytotoxicity. In both in vitro and ex vivo functional assays ibrutinib, but not more selective BTK inhibitors perturbed proper T cell function. Whilst CD4 T cell proliferation was suppressed Th1 skewing was not observed in ibrutinib treated patients. Furthermore. CD8 T cells had profoundly impaired responses to TCR stimulation. Understanding how BTK inhibitors alter the function of cytotoxic cells is essential for the combination of these therapies with immunotherapies and may inform the use of these therapies in the context of adoptive cellular therapies and transplantation. Disclosures Tam: Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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