Ibrutinib Treatment in CLL Patients Improves T Cell Function and Blinatumomab Redirected Cytotoxicity

Introduction:T cell engaging bispecific antibodies (T-BsAb) redirect cytotoxic T cells to tumor associated antigen (TAA)-positive target cells, leading to T-cell activation and lysis of target cells. Blinatumomab is the only T-BsAb approved by FDA. Numerous T-BsAb are undergoing clinical trials, targeting both hematological malignancies (e.g. BCMA, CD123 and CD33) and solid tumors (e.g. CEA, EpCAM, and GPC3). While these T-BsAbs represent a promising novel therapeutic approach, their efficacy remains modest. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and induces durable remissions in chronic lymphocytic leukemia (CLL). Our group has reported that ibrutinib has favorable immunomodulatory effects due to inhibition of interleukin-2 inducible T-cell kinase (ITK). Ibrutinib decreased key immunosuppressive checkpoint molecules and the frequency of regulatory T cells (Treg)(Long, Beckwith et al. 2017). Moreover, Ibrutinib significantly increased the number of the activated T cells by rescuing them from activation induced cell death (AICD). However, the function of ibrutinib 'rescued' T cells and their ability to mediate T-BsAb redirected cytotoxicity is unknown. Therefore we proceeded to study how ibrutinib treatment affects the functional competency of T cells by measuring their capability to mediate Blinatumomab redirected cytotoxicity. To examine T cell function post ibrutinib treatment, T cells were isolated from pre and post treatment PBMC samples by fluorescence activated cell sorting (FACS), and used as effector cells with pre-treatment CLL B-cells as target cells. Blinatumomab redirected T-cell cytotoxicity against tumor cells and T cell survival were assayed by flow cytometry based methods. Effector cells (T cells) were labeled with CFSE. Target (CLL) cells were labeled with CellTrace Violet. They were co-cultured at an E:T ratio of 4:1 with Blinatumomab for 24 hours. Effector cells and target cells can be distinguished by different fluorescence labels. Apoptosis and cell death were evaluated by Annexin-V and PI staining. The number of viable target cells and effector cells were calculated by Countbright counting bead. Results: We observed that post-ibrutinib T cells demonstrated significantly superior cytotoxicity against autologous CLL cells in the presence of blinatumomab in three independent patient samples. Numbers of viable CLL cells co-cultured with post-ibrutinib T cells were lower when compared to those co-cultured with pre-ibrutinib T cells (33.6%, 68% and 55.1% reduction in viable CLL cells for the three patient samples, respectively). We also found that Treg depletion (by depleting CD25high/CD127low CD4+ T cells) further enhanced cytotoxicity for both pre and post-ibrutinib treatment T cells, with the numbers of viable CLL cells reduced another 30-40%. Moreover, compared to pre-ibrutinib T cells, post-ibrutinib treatment T cells again demonstrated superior cytotoxicity when Treg cells were depleted, with decreased numbers of viable CLL cells at the end of co-culture (43%, 69% and 49.4% reduction, respectively). We also observed that the numbers of viable T cells from pre-ibrutinib samples were much lower than post-ibrutinib samples (52.5%, 77.9% and 74% reduction in viable T cells, respectively). Conclusions: Our findings suggest that ibrutinib-rescued T cells are functionally competent. CLL patients' T cells post ibrutinib treatment demonstrated superior cytotoxicity against autologous leukemia cells compared to T cells from treatment baseline. Moreover, we show that increased T cell activity is not solely due to ibrutinib-related Treg depletion. Treg depletion in our experiments further enhanced the cytotoxicity of both post and pre-ibrutinib treatment T cells while post-ibrutinib T cells still demonstrated superior cytotoxicity (Fig 1). Lastly, T cells isolated from post-ibrutinib samples demonstrated significantly improved viability after in-vitro stimulation with blinatumomab, which is likely a result of reduced AICD. Disclosures Bhat: Pharmacyclics: Consultancy; Janssen: Consultancy. Rogers:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Acerta Pharma: Consultancy. Woyach:Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau.

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Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Blood ◽

10.1182/blood-2021-150351 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1172-1172

Author(s):

Maryam Kazerani Pasikhani ◽

Anetta Marcinek ◽

Bettina Brauchle ◽

Jonathan Jonas Taylor ◽

Helena Domínguez Moreno ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Research Funding ◽

Cell Function ◽

Target Cells ◽

Cell Cytotoxicity ◽

T Cell Cytotoxicity ◽

Support Research

Abstract Novel immunotherapeutic strategies like BiTE ® (bispecific T cell engager) constructs aim to eradicate neoplastic cells by TCR-independent T-cell activation, and therefore rely on the function of autologous T cells. Currently, their efficacy is also evaluated in heavily pre-treated patients with relapsed/refractory acute myeloid leukemia (AML). Previous data demonstrated dysfunction in CD8 + T cells of AML patients (Knaus et al 2018). Thus, we aimed to characterize the progressive modulation of T-cell activity over the course of AML progression to improve the optimal application of T-cell based immunotherapeutic approaches. Bone marrow mononuclear cells (BMMCs) from AML patients at time of initial diagnosis (ID), complete remission (CR), relapse (RL), as well as of age-matched healthy donors (HD) were analyzed for T-cell subset distribution and expression of exhaustion markers by flow cytometry. Additionally, T-cell function was assessed after stimulation with 1) CD3/CD28 beads; 2) AMG 330, a CD33/CD3 specific BiTE ® construct, after incubation with OCI-AML3 target cells; or 3) AMG 330 in an autologous ex vivo long-term culture system after incubation with primary AML cells (pAML). After 6 days, T cell proliferation, expression of effector molecules and cytokines, and AMG 330-mediated T-cell cytotoxicity were assessed by flow cytometry. Lastly, we performed longitudinal bulk RNA-sequencing on 5000 sorted T cells from 7 matched ID-RL primary AML samples. Immunophenotypic analysis of BM T-cell subsets revealed a shift from T NAIVE toward central/effector memory subsets during AML progression. We observed lower percentages of T NAIVE in RL (n=3) compared to CR (n=3) CD8 + T cells(11.8 vs. 45.2%, p=0.07; RL vs. CR). Conversely, RL patients showed increased percentages of CD8 + memory T cells (T CM: 23.4 vs. 6.7%; T EM: 29.4 vs. 20.2%; T EMRA: 35.3 vs. 27.8%; RL vs. CR). Further characterization of exhaustion markers exhibited a significantly higher percentage of both CD4 + and CD8 + T cells expressing 2B4 (CD244) in ID (n=19) and RL (n=13) compared to HD (n=10, both p < 0.001). A higher percentage of PD-1 + CD8 + and TIM-3 + CD4 + T cells was detected in both ID and RL relative to HD (all p < 0.05). However, a significantly increased percentage of CD8 + T cells expressing TIM-3 and CD160 was detected in ID relative to HD (p < 0.05). Intriguingly, RL CD4 + T cells demonstrated a significantly higher level of LAG3 compared to ID (p < 0.01). In line with phenotypic exhaustion features, ID (n=4) and RL (n=5) CD8 + T cells showed reduced proliferation compared to HD (n=4) CD8 + T cells after CD3/CD28 bead stimulation (both p < 0.01). Correspondingly, we observed a marked reduction in the expression of Granzyme B (GZMB) by CD8 + T cells (both p < 0.05). Interestingly, when compared to ID, RL CD4 + T cells showed decreased TNF-α secretion (p < 0.05). In contrast to these findings, AMG 330-mediated T cell cytotoxicity against OCI-AML3 target cells was superior with RL T cells compared to ID T cells (p < 0.001). The percentage of GZMB + CD8 + T cells strikingly enhanced in RL relative to ID (p < 0.01). In an autologous setting with pAML samples, T cells from RL patients (n=6) showed higher AMG 330-mediated cytotoxicity compared to ID (n=9) T cells (67.7 vs. 35.2; RL vs. ID). In our longitudinal RNA-sequencing, differentially expressed genes analysis detected 61 up- and 30 downregulated genes (log2 FC > 1 or < -1; p < 0.01) in RL T cells compared to their matched ID counterparts. Among the significantly upregulated genes in RL, we identified genes associated with memory T cell function (TP53INP2, DUSP4) and exhaustion (NR4A1, TOX2). Moreover, Gene set enrichment analysis showed significant enrichment of gene signatures associated to memory and exhausted T cells (normalized enrichment score (NES)=1.2 and 1.3; p-value= 0.026 and 0.008, respectively), depletion of oxidative phosphorylation (NES=-2.05; p adj < 0.0001) and protein secretion (NES=-1.49; p adj < 0.05) gene signatures in RL vs. ID T cells. Taken together, our data show that patient T cells acquire an activated/exhausted phenotype upon AML progression. However, this is not reflected in the T-cell effector functions upon AMG 330 stimulation, in contrast to bead stimulation. These observations may highlight the significant role of the AML target cells in shaping a T-cell response. To this end, we will further analyze the longitudinal communication between T cells and their corresponding AML blasts. Disclosures Brauchle: Adivo: Current Employment. Kischel: Amgen GmbH Munich: Current Employment. Buecklein: BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Roche: Research Funding; Miltenyi: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment.

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Preservation of CD20-Specific Chimeric Antigen Receptor T Cell Function in the Presence of Residual Rituximab

Blood ◽

10.1182/blood.v126.23.4428.4428 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4428-4428

Author(s):

Gregory A. Rufener ◽

Philip Olsen ◽

Sang Yun Lee ◽

Michael C Jensen ◽

Ajay K. Gopal ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Binding Sites ◽

Research Funding ◽

Cell Function ◽

Target Cells ◽

T Cell Function ◽

Car T ◽

Anti Cd20

Abstract BACKGROUND: The CD20 antigen is an attractive immunotherapy target for B cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation of this approach is that residual serum rituximab from prior chemoimmunotherapy regimens might block CAR binding to CD20 and prevent T cell mediated anti-lymphoma responses. However, previous data from our group and others have suggested that CD20 CAR+ T cell function is only partially blocked by anti-CD20 antibody (Ab), and T cell function in the setting of anti-CD3 x anti-CD20 bispecific Ab is not blocked by rituximab levels of up to 100 μg/ml. We have further tested the impact of various concentrations of rituximab on CD20-CAR T cell activity in vitro and in vivo. METHODS: CD3+ T cells (proliferation and cytokine assays) or CD8+ selected T cells (cytotoxicity assays) were isolated from healthy donors, activated with anti-CD3/CD28 beads, and transduced with epHIV7 lentiviral vectors encoding 2nd or 3rd generation anti-CD20 CAR constructs (Leu16-28-ζ, Leu16-28-BB-ζ, or fully human 1-5-3-NQ-28-BB-ζ). Functional assays, performed using target cells pre-incubated for 30 min. with varying concentrations of rituximab, included a CFSE assay to assess CAR T cell proliferation, Luminex assays for cytokine secretion, and a 5-hour standard 51 chromium release assay for cytotoxicity. Target cells included K562 cells transduced to express CD80 with or without CD20 (denoted "K80" and "K80-20"), Raji, Daudi, Granta, Rec-1, and FL-18 lymphoma cells. K80-20 cell lines expressing low, medium, and high CD20 were established by limiting dilution cloning. For in vivo experiments, NOD/SCID/γ-/- (NSG) mice were inoculated i.v. with rituximab-resistant Raji-ffLuc lymphoma cells. After 5 days, rituximab was administered i.p. at 25 μg/ml or 200 μg/ml, and then at 24 hours after rituximab administration CAR+ central memory T cells expressing the 1-5-3-NQ-28-BB-ζ vector were injected i.v. Tumor growth was measured with bioluminescence imaging twice weekly and mice were followed for survival. RESULTS: The availability of CD20 binding sites on Ramos lymphoma cells pre-incubated with various concentrations of rituximab was assessed with flow cytometry, and as expected, we found a dose-dependent blockade of CD20, with complete blockade at 50 μg/ml rituximab at 4°C. However, when anti-CD20-PE was incubated at 37°C, low-level CD20 binding could occur even at 200 μg/ml of rituximab. Despite the low number of available CD20 binding sites after rituximab, proliferation of CFSE-labeled CAR+ T cells was largely unimpaired in rituximab concentrations up to 200 μg/ml. In contrast, cytokine secretion was impaired in a dose-dependent manner, although even at 100 μg/ml of rituximab, interferon-γ, interleukin-2, and tumor necrosis factor a were still produced at 34-51%, 70-92%, and 79-108% of baseline levels, respectively. Cytotoxicity also decreased with increasing rituximab concentration but >75% of baseline cytolytic activity was retained at 100 μg/ml. We hypothesized that the level of CD20 expression on target cell lines might impact sensitivity to rituximab blockade. Using K80-20 cells with low, medium, or high CD20 expression we found that cytokine secretion and cytotoxicity (but not proliferation) were highly impaired upon stimulation with CD20low target cells, whereas T cell function remained completely intact when CD20high cells were used as targets. In vivo, mice bearing rituximab-refractory Raji-ffLuc tumors experienced only slight delay of tumor growth when treated with either low or high doses of rituximab, and mice treated with T cells alone had significant clearance of tumor. In mice that received low or high-dose rituximab prior to T cell infusions, tumor rejection and survival prolongation were equivalent to or better than that observed with mice receiving T cells alone (see figure). CONCLUSION: We have shown that the in vitro and in vivo activity of CD20-targeted CAR T cells is minimally impacted after rituximab, despite a low number of available CD20 binding sites. These data suggest that residual serum rituximab levels will not present a significant impediment to CD20-targeted CAR therapy in patients who have received rituximab-containing chemotherapy regimens. Figure 1. Figure 1. Disclosures Jensen: Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Gopal:Merck: Research Funding; BioMarin: Research Funding; Seattle Genetics: Consultancy, Honoraria; Gilead: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Piramal: Research Funding; Millenium: Honoraria, Research Funding; BMS: Research Funding; Janssen: Consultancy; Emergent/Abbott: Research Funding; Sanofi-Aventis: Honoraria. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy. Till:Pfizer: Research Funding; Roche/Genentech: Research Funding.

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Potent HIV-specific responses are enriched in a unique subset of CD8+ T cells that coexpresses CD4 on its surface

Blood ◽

10.1182/blood-2009-02-202481 ◽

2009 ◽

Vol 114 (18) ◽

pp. 3841-3853 ◽

Cited By ~ 26

Author(s):

Andrew Zloza ◽

Jason M. Schenkel ◽

Allan R. Tenorio ◽

Jeffrey A. Martinson ◽

Paul M. Jeziorczak ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cell Function ◽

Target Cells ◽

Effector Cells ◽

Multiple Parameters ◽

Mhc Ii ◽

Class Ii Mhc ◽

Anti Hiv

AbstractIn humans, approximately 3% of peripheral CD8+ T cells coexpress CD4 dimly on their surface and hence are designated as CD4dimCD8bright T cells. We evaluated the contribution of this CD4dimCD8bright T-cell population to anti-HIV immunity. We demonstrate that CD4dimCD8bright T cells generate greater than 55% of CD8+ T-cell antigen recognition and effector response to HIV, as evaluated by multiple parameters for assessing T-cell antiviral immunity, including HIV tetramer recognition, cytokine production, and cytolytic potential. Inhibition of major histocompatibility class II (MHC-II) on target cells or CD4 on CD4dimCD8bright T cells diminishes their anti-HIV responses, suggesting that CD4 on effector cells and MHC-II on target cells provides an additional arm of contact between effector and target cells which is critical to CD4dimCD8bright T-cell function. CD4dimCD8bright T cells also exhibit features that are indicative of central memory T cells. Finally, CD4dimCD8bright T cells are elevated in blood of HIV+ long-term nonprogressors in comparison to HIV− donors. Collectively, our findings show that CD4dimCD8bright T cells designate an enriched antiviral subpopulation of CD8+ T cells that should be targeted for therapeutic intervention or evaluation of vaccine efficacy.

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The Protein Kinase C Inhibitor MS-553 for the Treatment of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-128871 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2077-2077

Author(s):

Elizabeth M. Muhowski ◽

Amy M. Lehman ◽

Sean D. Reiff ◽

Janani Ravikrishnan ◽

Rose Mantel ◽

...

Keyword(s):

Research Funding ◽

Ohio State University ◽

Lymphocytic Leukemia ◽

State University ◽

Advisory Committees ◽

University Patents ◽

Kinase C ◽

Hla Dr ◽

Bcr Signaling ◽

Equity Ownership

Introduction: Treatment of chronic lymphocytic leukemia (CLL) has been transformed by small molecule inhibitors targeting the B-cell receptor (BCR) signaling cascade. The first-in-class small molecule inhibitor of Bruton's Tyrosine Kinase (BTK), ibrutinib, is FDA approved as a frontline therapy for CLL. However, resistance to BTK inhibition has emerged in patients through acquisition of mutations in BTK or its immediate downstream target, PLCG2, emphasizing the need for alternative targets and therapies. BCR signaling remains intact in the presence of these mutations, making targeted inhibition of proteins downstream of BTK an attractive therapeutic strategy. Protein kinase C-β (PKCβ) is a downstream member of the BCR signaling pathway that we have previously demonstrated as an effective therapeutic target in CLL. MS-553 is a potent, ATP-competitive, reversible inhibitor of several PKC isoforms including PKCβ. Therefore, we evaluated the effects of MS-553 in primary CLL cells. Methods: Primary CLL cells were isolated by negative selection and treated with increasing concentrations of MS-553 to a maximum dose of 10 µM. BCR signaling changes were interrogated by change in target protein phosphorylation by immunoblot following a 24 hour drug incubation with and without phorbol ester stimulation (90 minutes) in CLL samples. Inhibition of CpG-mediated activation of CLL cells was measured using flow cytometry (CD86 and HLA-DR) in ibrutinib refractory patient samples at baseline and post-relapse due to the emergence of the p.C481S BTK mutation. CCL3 and CCL4 expression was measured by ELISA after 24 hours in primary CLL cells in the presence or absence of anti-IgM ligation. TNFα expression was also measured by ELISA in negatively selected, healthy donor T cells treated with MS-553 for 24 hours with or without anti-CD3 and anti-CD28 stimulation. Results: At 24 hours, 5 µM MS-553 inhibited downstream BCR signaling in primary CLL cells, demonstrated by 31% reduced phosphorylation of PKCβ (p=0.08, n=5) and several of its downstream targets including GSK3β (40%, p<.01, n=5) , ERK (46%, p=0.02, n=4) , and IκBα (56%, p=0.04, n=5) compared to vehicle treated, stimulated samples. CpG-mediated TLR9 stimulation increases expression of CD86 and HLA-DR in primary CLL cells. In baseline samples from ibrutinib treated patients, 10 µM MS-553 decreased expression of CD86 by 34% and HLA-DR by 91%. In matched patient samples post-relapse due to ibrutinib resistance, MS-553 (10 µM) maintained the ability to decrease expression of CD86 (49%) and HLA-DR (84%). Pro-inflammatory cytokine expression by primary CLL cells stimulated with anti-IgM decreased in the presence of 5 µM MS-553, with CCL3 decreasing by 36% (p=0.06, n=5) and CCL4 decreasing by 79% (p<.01, n=4) compared to vehicle treated, stimulated controls. TNFα expression by healthy T cells increased with anti-CD3 and anti-CD28 stimulation; 1 µM MS-553 reduced TNFα expression by 97% compared to vehicle treated, stimulated controls (p<.01, n=9). Conclusions: MS-553 is a novel and potent inhibitor of PKC demonstrating in vitro efficacy in CLL. MS-553 is able to inhibit BCR signaling by blocking phosphorylation of PKCβ and its downstream targets. CpG-mediated activation is reduced with MS-553 treatment in ibrutinib refractory patient samples both at baseline and post-relapse. Inflammatory signaling by primary CLL cells is further abrogated by MS-553 in its ability to decrease CCL3 and CCL4 cytokine expression. In an ongoing phase I clinical trial of MS-553, patient samples show a potent and dose dependent decrease in PKCβ activity as measured by a clinical biomarker assay. Together, our results suggest that MS-553 targets PKCβ in primary CLL to inhibit signaling and survival, establishing MS-553 as a potential therapeutic for treating CLL. These data justify continued preclinical and clinical work in the development of MS-553 for the treatment of CLL. Disclosures Niesman: MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zhang:MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Byrd:BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding. Woyach:Verastem: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding.

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Phase 2 Study of Combination of Cytarabine, Idarubicin, and Nivolumab for Initial Therapy of Patients with Newly Diagnosed Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.815.815 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 815-815

Author(s):

Farhad Ravandi ◽

Naval Daver ◽

Guillermo Garcia-Manero ◽

Christopher B Benton ◽

Philip A Thompson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Myeloid Leukemia ◽

Research Funding ◽

T Cell Subsets ◽

Newly Diagnosed ◽

Effector Cells ◽

T Effector Cells ◽

Grade 3

Abstract Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses by enabling T-cells in murine models of AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, AACR 2016). PD1 inhibition has shown activity in AML (Berger et al, Clin Cancer Res 2008). We hypothesized that addition of nivolumab to an induction regimen of ara-C and idarubicin may prolong relapse-free survival (RFS) and overall survival (OS); this study was designed to determine the feasibility of this combination. Methods: Pts with newly diagnosed acute myeloid leukemia (by WHO criteria; ≥20% blasts) and high risk MDS (≥10% blasts) were eligible to participate if they were 18-65 yrs of age and had adequate performance status (ECOG ≤3) and organ function (LVEF ≥ 50%; creatinine ≤ 1.5 g mg/dL, bilirubin ≤ 1.5 mg/dL and transaminases ≤ 2.5 times upper limit of normal). Treatment included 1 or 2 induction cycles of ara-C 1.5 g/m2 over 24 hours (days 1-4) and Idarubicin 12 mg/m2 (days 1-3). Nivolumab 3 mg/kg was started on day 24 ± 2 days and was continued every 2 weeks for up to a year. For pts achieving complete response (CR) or CR with incomplete count recovery (CRi) up to 5 consolidation cycles of attenuated dose ara-C and idarubicin was administered at approximately monthly intervals. Eligible pts received an allogeneic stem cell transplant (alloSCT) at any time during the consolidation or thereafter. Results: 3 pts with relapsed AML were treated at a run-in phase with a dose of nivolumab 1 mg/kg without specific drug-related toxicity. Subsequently, 32 pts (median age 53 yrs; range, 26-65) were treated as above including 30 with AML (24 de novo AML, 2 therapy-related AML, 3 secondary AML and 1 therapy-related secondary AML) and 2 high risk MDS. Pre-treatment genetic risk by ELN criteria was 11 adverse, 16 intermediate, and 5 favorable, including 2 FLT3 -ITD mutated, 5 NPM1 mutated, and 7 TP53 mutated. All 32 pts were evaluable for response and 23 (72%) achieved CR/CRi (19 CR, 4 CRi). The 4-week and 8 week mortality was 6% and 6%. The median number of doses of nivolumab received was 6 (range, 0-13); one pt did not receive nivolumab due to insurance issues. 9 pts underwent an alloSCT. After a median follow-up of 8.3 mths (range, 1.5-17.0) the median RFS among the responding pts has not been reached (range, 0.1 - 15.8 mths) and the median OS has not been reached (range 0.5-17.0 mths). Grade 3/4 immune mediated toxicities have been observed in 5 pts and include rash, pancreatitis, and colitis. Other grade 3/4 toxicities thought to be potentially related to nivolumab include cholecystitis in one pt. 9 pts proceeded to an alloSCT. Donor source was matched related in 2, matched unrelated in 6 and haplo-identical in 1 pt. Conditioning regimen was Fludarabine plus busulfan-based in 8, and fludarabine plus melphalan in 1 pt. 4 pts developed graft versus host disease (GVHD)(grade I/II in 3, grade III/IV in 1), which responded to treatment in 3. Multicolor flow-cytometry studies are conducted by the Immunotherapy Platform on baseline (prior to first dose of nivolumab) and on-treatment BM aspirate and peripheral blood to assess the T-cell repertoire and expression of co-stimulatory receptors and ligands on T-cell subsets and leukemic blasts, respectively. The baseline BM was evaluated on 23 of the 32 evaluable pts, including 18 responders and 5 non-responders. Pts who achieved a CR/CRi had a trend of higher frequency of live CD3+ total T cell infiltrate as compared to non-responders in the baseline BM aspirates (Fig 1A). We evaluated expression of immune markers on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. At baseline, BM of non-responders had significantly higher percentage of CD4 T effector cells co-expressing the inhibitory markers PD1 and TIM3 (p<0.05) and a trend towards higher percentage of CD4 T effector cells co-expressing PD1 and LAG3 compared to responders (Fig 1B). Co-expression of TIM3 or LAG3 on PD1+ T cells have been shown to be associated with an exhausted immune phenotype in AML (Zhou et al., Blood 2011). Conclusion: Addition of nivolumab to ara-C and anthracycline induction chemotherapy is feasible and safe in younger pts with AML. Among the pts proceeding to alloSCT the risk of GVHD is not significantly increased. Figure 1 Figure 1. Disclosures Daver: Pfizer Inc.: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Kiromic: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Incyte Corporation: Honoraria, Research Funding. Thompson: Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour: Bristol-Myers Squibb: Consultancy. Takahashi: Symbio Pharmaceuticals: Consultancy. DiNardo: Novartis: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Sharma: Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Astellas: Consultancy; EMD Serono: Consultancy; Amgen: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy; Consetellation: Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Kite Pharma: Consultancy, Other: stock; BMS: Consultancy. Cortes: BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding. Kantarjian: Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding.

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B-CLL Mediated Resistance to CAR T Cell Therapy Via Insufficient Activation Is CAR-Independent

Blood ◽

10.1182/blood-2020-137638 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 44-44

Author(s):

McKensie Collins ◽

Weimin Kong ◽

Inyoung Jung ◽

Stefan M Lundh ◽

J. Joseph Melenhorst

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Cytokine Production ◽

Cell Function ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T

Chronic Lymphocytic Leukemia (CLL) is a B cell malignancy that accounts for nearly 1/3rd of adult leukemia diagnoses in the Western world. Conventional chemo-immunotherapies initially control progression, but in the absence of curative options patients ultimately succumb to their disease. Chimeric Antigen Receptor (CAR) T cell therapy is potentially curative, but only 26% of CLL patients have a complete response. CLL-stimulated T cells have reduced effector functions and B-CLL cells themselves are believed to be immunosuppressive. Our work demonstrates that insufficient activation of CAR T cells by CLL cells mediates some of these effects and that the results are conserved between ROR1- and CD19-targeting CARs. Results: In this study we used an in vitro system to model the in vivo anti-tumor response in which CAR T cells serially engage with CLL cells. Multiple stimulations of CD19 or ROR1-targeting CAR T cells with primary CLL cells recapitulated many aspects of known T cell dysfunction including reduced proliferation, cytokine production, and activation. While the initial stimulation induced low level proliferation, subsequent stimulations failed to elicit additional effector functions. We further found that these functional defects were not permanent, and that CAR T cell function could be restored by switching to a stimulus with an aAPC (artificial Antigen Presenting Cell) control cell line. The aAPCs are well-characterized as potent stimulators of CAR T cell effector responses. Flow cytometry revealed that CLL-stimulated CAR T cells retained a non-activated, baseline differentiation profile, suggesting that CLL cells fail to stimulate CAR T cells rather than rendering them non-functional. One mechanism that could dampen activation is immune suppression. We assessed this at a high level by stimulating CAR T cells with CLL cells and aAPCs mixed at known ratios. However, even cultures containing 75% CLL cells stimulated proliferation and cytokine production. Extensive immune-phenotyping revealed high level expression of the IL-2 Receptor on 90% (18/20) of the B-CLL cells tested. Since cytokine sinking via IL-2 receptor expression is a well-known mechanism of regulatory T cell suppression, we hypothesized that CLL cells similarly sink IL-2, blunting T cell activation. To test this, we supplemented IL-2 into CLL/CAR T cell co-cultures and showed that this rescued proliferation but only partially restored cytokine production. In contrast to our hypothesis, analysis of cytokine production by flow cytometry showed that CLL-stimulated CAR T cells did not produce IL-2 following a 6- or 12-hour stimulus, but TNFα was expressed after 12-hours. Similarly, CAR T cell degranulation, a prerequisite for target cell lysis was triggered after CLL recognition. These data again suggested that CLL cells insufficiently stimulate CAR T cell cytokine production, but also showed that cytolytic activity against CLL cells is intact. We further proposed that CLL cells express insufficient levels of co-stimulatory and adhesion molecules to activate CAR T cells. Flow cytometry showed that most CLL cells expressed co-stimulatory and adhesion molecules at low levels; we hypothesized that up-regulating these molecules would enhance CAR T cell targeting of CLL cells. CLL cells were activated with CD40L and IL-4, which increased expression of CD54, CD58, CD80, and CD86. Stimulating CAR T cells with activated CLL cells enhanced CAR T cell proliferation and induced cell conjugate formation, indicating cell activation. Therefore, improving CLL stimulatory capacity can rescue T cell dysfunctions. To assess whether IL-2 addition and CD40 ligation were synergistic, we combined the two assays; however, we saw no additional improvement over IL-2 addition alone, suggesting that the two interventions may act upon the same pathway. Importantly, we also showed that rescue of CAR T cell function via IL-2 addition or CD40 ligation was not CAR-specific, as we observed the functional defects and subsequent rescue with both a ROR1-targeting CAR and the gold standard CD19-targeting CAR. Conclusions: Together, these data show that CAR T cell "defects" in CLL are actually insufficient activation, and improving the stimulatory capacity of CLL cells may enable better clinical responses. Further, this effect is not CAR-specific and these results may therefore be broadly applicable to multiple therapies for this disease. Disclosures Melenhorst: IASO Biotherapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Novartis: Other: Speaker, Research Funding; Johnson & Johnson: Consultancy, Other: Speaker; Simcere of America: Consultancy; Poseida Therapeutics: Consultancy.

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Treatment with Acalibrutinib, Ibrutinib and CD19 CAR T Cells Restore the Number of Granulocytic Myeloid Derived Supressor Cells in CLL-Bearing Mice

Blood ◽

10.1182/blood-2019-126697 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3032-3032

Author(s):

Arantxa Romero-Toledo ◽

Robin Sanderson ◽

John G. Gribben

Keyword(s):

T Cells ◽

T Cell ◽

Cell Population ◽

Research Funding ◽

Cell Function ◽

Car T Cells ◽

Car T Cell ◽

T Cell Function ◽

Car T ◽

Secondary Lymphoid Organs

The complex crosstalk between malignant chronic lymphocytic leukemia (CLL) cells and the tumor microenvironment (TME) is not fully understood. CLL is associated with an inflammatory TME and T cells exhibit exhaustion and multiple functional defects, fully recapitulated in Eµ-TCL1 (TCL1) mice and induced in healthy mice by adoptive transfer (AT) of murine CLL cells, making it an ideal model to test novel immunotherapies for this disease. Myeloid-derived suppressor cells (MDSCs), a non-leukemic cell type within the TME, are immature myeloid cells with the ability to suppress T cell function and promote Treg expansion. In humans, CLL cells can induce conversion of monocytes to MDSCs provoking their accumulation in peripheral blood (PB). MDSCs include two major subsets granulocytic (Gr) and monocytic (M)-MDSC. In mice, Gr-MDSCs are defined as CD11b+Ly6G+Ly6Clo and M-MDSC as CD11b+Ly6G-Ly6Chi. Both murine and human MDSCs express BTK. We observed that in CLL-bearing mice, MDSCs cells are lost in PB as disease progresses. Treatment with both BTK inhibitors (BTKi), ibrutinib (Ibr) and acalabrutinib (Acala), result in shift of T cell function from Th2 towards Th1 polarity and increase MDSC populations in vivo. We aimed to determine whether combination treatment with BTKi and chimeric antigen receptor (CAR) T cells renders recovery of the MDSC population in CLL-bearing mice. To address this question we designed a two-part experiment, aiming to mimic the clinically relevant scenario of pre-treatment of CLL with BTKi to improve CAR T cell function. Part 1 of our experiment consisted of 4 groups (n=12) of 2.5 month old C57/Bl6 mice. Three groups had AT with 30x106 TCL1 splenocytes. A fourth group of WT mice remained CLL-free as a positive control and donors for WT T cells. When PB CLL load reached >10% (day 14) animals were randomized to either Ibr or Acala at 0.15 mg/l in 2% HPBC or no treatment for 21 days. All animals from part 1 were culled at day 35 post-AT and splenic cells were isolated, analyzed and used to manufacture CAR T cells. WT, CLL, Ibr and Acala treated T cells were activated and transduced with a CD19-CD28 CAR to treat mice in part 2. Here, 50 WT mice were given AT with 20x106 TCL1 splenocytes for CLL engraftment. All mice were injected with lymphodepleting cyclophosphamide (100mg/kg IP) one day prior to IV CAR injection. At day 21 post-AT, mice were treated with WT CAR, CLL CAR, IbrCAR, AcalaCAR or untransduced T cells. MDSC sub-populations were monitored weekly in PB and SP were analysed by flow cytometry. As malignant CD19+CD5+ cells expands in PB, the overall myeloid (CD19-CD11b+) cell population was not affected, but MDSCs significantly decreased (p<0.0001). Treatment with Acala, but not Ibr restores total MDSCs. However, MDSC impairment occurs in the Gr- but not M- MDSC population and both Acala and Ibr restores this population (Figure 1a). When we examined the spleen, treatment with both Ibr (p<0.001) and Acala (p<0.001) reduced CD5+CD19+ cells, whereas neither BTKi affected the overall myeloid (CD19-CD11b+) cell population. Gr-MDSCs were restored by both treatments whilst M-MDSCs were only restored after Ibr treatment (p<0.001 in each case). In part 2 of this experiment we observed that treatment with all CAR-T cell groups provokes the clearance of all CD19+CD5+ cells. The overall CD19-CD11b+ population stays the same across all mice groups 35 days after treatment in PB with any group of CAR and untransduced T cells. Overall MDSC population is maintained following all CAR T cells compared to CLL-bearing mice (p<0.0001) and it is the Gr- but not the M- MDSC population which is recovered in PB (Figure 1b). These parts of the experiments can of course be influenced by treatment with cyclophosphamide. We conclude that novel therapies for CLL treatment have an effect not only in CLL cells but also in non-malignant cell components of the TME. In this animal model of CLL, the rapid expansion of CLL cells in PB and secondary lymphoid organs provokes loss of MDSC, particularly the Gr-MDSC subpopulation is affected. Treatment with BTKi and CAR T cells provokes clearance of CLL cells in PB and spleen allowing MDSC recovery; suggesting this may be BTK and ITK independent. We continue to explore secondary lymphoid organs to further characterize the shift of the CLL microenvironment from an immunosuppressive to an immune effective one and its impact on immune function in this model. Disclosures Sanderson: Kite/Gilead: Honoraria. Gribben:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

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Single-cell transcriptomics identifies multiple pathways underlying antitumor function of TCR- and CD8αβ-engineered human CD4+ T cells

Science Advances ◽

10.1126/sciadv.aaz7809 ◽

2020 ◽

Vol 6 (27) ◽

pp. eaaz7809 ◽

Cited By ~ 1

Author(s):

Jan A. Rath ◽

Gagan Bajwa ◽

Benoit Carreres ◽

Elisabeth Hoyer ◽

Isabelle Gruber ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Cell Function ◽

Xenograft Model ◽

Cell Receptor ◽

Target Cells ◽

Antigen Specificity ◽

Molecular Features ◽

Mouse Xenograft Model

Transgenic coexpression of a class I–restricted tumor antigen–specific T cell receptor (TCR) and CD8αβ (TCR8) redirects antigen specificity of CD4+ T cells. Reinforcement of biophysical properties and early TCR signaling explain how redirected CD4+ T cells recognize target cells, but the transcriptional basis for their acquired antitumor function remains elusive. We, therefore, interrogated redirected human CD4+ and CD8+ T cells by single-cell RNA sequencing and characterized them experimentally in bulk and single-cell assays and a mouse xenograft model. TCR8 expression enhanced CD8+ T cell function and preserved less differentiated CD4+ and CD8+ T cells after tumor challenge. TCR8+CD4+ T cells were most potent by activating multiple transcriptional programs associated with enhanced antitumor function. We found sustained activation of cytotoxicity, costimulation, oxidative phosphorylation– and proliferation-related genes, and simultaneously reduced differentiation and exhaustion. Our study identifies molecular features of TCR8 expression that can guide the development of enhanced immunotherapies.

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Inhibitory Effects of Lactic Acid on Human Antigen-Specific CD8+ T-Cells.

Blood ◽

10.1182/blood.v104.11.3844.3844 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3844-3844

Author(s):

Marina Kreutz ◽

Karin Fischer ◽

Petra Hoffmann ◽

Simon Volkl ◽

Matthias Edinger ◽

...

Keyword(s):

T Cells ◽

Lactic Acid ◽

T Cell ◽

Cd8 T Cells ◽

Acid Production ◽

Cell Function ◽

Immune Escape ◽

Lactic Acid Production ◽

Target Cells ◽

Thymidine Uptake

Abstract A characteristic feature of inflammatory lesions or tumor sites is local acidosis, which is attributed to the local increase in lactic acid production. We studied the effect of such an acidic environment on the immune functions of antigen-specific CD8+ T-cells by incubating the cells in the presence of various concentrations of lactic acid for up to 48h. CD8+ T-cells were isolated from healthy donors and expanded by weekly stimulation with autologous dendritic cells pulsed with a mutated HLA-A2-binding Melan-A (ELAGIGILTV) peptide. The obtained T cell population consisted of at least 90% CD8+ and about 60% Melan-A specific T cells, as determined by Melan-A multimer staining. Incubation of CD8+ T cells with up to 20mM lactic acid for 24h did not cause T-cell apoptosis or cell death as determined by combined annexin/propidium iodide staining. However, the proliferative capacity of CD8+ T cells, as determined by 3H-thymidine uptake, was strongly inhibited. Similar results were obtained when we determined cytokine production and cytotoxic activity of the cells after a 24h culture period in 5-20 mM lactic acid. Production of both, IL-2 and IFN-gamma was strongly diminished in comparison to untreated cells, as determined by intracellular staining after stimulation with PMA/ionomycin for 5h in the presence of monensin. Analysis of the antigen-specific cytolytic capacity revealed that CD8+ T cells pre-cultured with lactic acid were less effective in killing antigen-loaded T2 target cells as compared to untreated CD8+ T cells. In parallel, the intracellular contents of the cytotoxic effector molecules granzyme-B and perforin was diminished. Re-adjusting the pH of the medium to a physiological value of pH7.4 could partially revert the effect of lactic acid. Treatment of CD8+ T cells with sodium lactate instead of lactic acid had no inhibitory effect. We conclude, that lactic acid is an important modulator of CD8+ T-cell function and may contribute, together with other factors, to immune escape mechanisms in the tumor environment.

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Specificity of cytotoxicity T cells directed to influenza virus hemagglutinin.

Journal of Experimental Medicine ◽

10.1084/jem.149.4.856 ◽

1979 ◽

Vol 149 (4) ◽

pp. 856-869 ◽

Cited By ~ 40

Author(s):

T J Braciale

Keyword(s):

T Cells ◽

T Cell ◽

Cytotoxic T Cells ◽

Type A ◽

Cross Reactivity ◽

Cytotoxic T Cell ◽

Target Cells ◽

Effector Cells ◽

Cell Induction

Purified type A influenza viral hemagglutinin stimulates an in vitro cell-mediated cytotoxic cell response that exhibits a high degree of specificity for the immunizing hemagglutinin. The response magnitude is proportional to the hemagglutinin dose used for stimulation. The lytic activity of the effector cells is H-2 restricted. Analysis of the specificity of the response indicated that these cytotoxic T cells readily distinguish target cells expressing serologically unrelated hemagglutinin from target cells bearing hemagglutinins serologically related to the stimulating hemagglutinin. Further analysis of the fine specificity of cytotoxic T-cell recognition with serologically cross-reactive type A influenza hemagglutinins revealed a hierarchy of cross-reactivity among these hemagglutinins that was the converse of the serologic hierarchy. These results are discussed in terms of possible differences and similarities in the specificity repertoire of cytotoxic T cells and antibodies. Possible implications of these findings from the standpoint of cytotoxic T-cell induction are also discussed.

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