Mono/Oligoclonal T and NK Cells Are Common in Philadelphia Chromosome Positive (Ph+) Leukemia Patients at Diagnosis and Expand During Successful Tyrosine Kinase Inhibitor Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 856-856
Author(s):  
Anna Kreutzman ◽  
Vesa Juvonen ◽  
Veli Kairisto ◽  
Marja Ekblom ◽  
Leif Stenke ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Gene Rearrangements ◽  
Imatinib Treatment ◽  
Healthy Controls ◽  
Clonal Rearrangement

Abstract Abstract 856 Introduction. Central to current treatment of Ph+ leukemia patients are tyrosine kinase inhibitors (TKIs), which predominantly target the BCR-ABL1 kinase in malignant cells. However, broader-spectrum 2nd generation TKIs, such as bosutinib, dasatinib and nilotinib, also inhibit off-target kinases with important physiological functions. Several in vitro studies have implied that TKIs may have immunosuppressive effects by suppressing activation and proliferation of effector lymphocytes. In contrast, we recently observed immunostimulation during dasatinib therapy in the form of marked expansion of clonal cytotoxic lymphocytes (T- and NK cells) resulting in chronic LGL-type lymphocytosis in peripheral blood (PB). The prevalence, detailed molecular background and clinical implications of clonal lymphocytes during TKI therapy are currently unknown. The aim of this study was to comprehensively analyze clonality and evolution of lymphocyte clones during TKI therapy. Patients and methods. The study population included patients with Ph+ leukemia, both CML (n=28) and ALL patients (n=4) on dasatinib (n=23) and imatinib (n=9) therapies. In addition, samples from 12 healthy controls and diagnostic samples from the nine imatinib treated patients were analyzed. Lymphocyte clonality was determined by analysis of PB mononuclear cells (MNC) for clonal T cell receptor (TCR) γ and δ gene rearrangements by 18 primer pairs covering most known clonal TCR γ and δ rearrangements. Upon positive reaction in heteroduplex analysis, the purified PCR products were sequenced. If clonal rearrangement was observed, allele-spesific PCR primers were designed to allow for quantitative follow-up of lymphocyte clones in each patient. Results. Sequencing-confirmed clonal TCR γ rearrangement was observed only in 1 of 12 healthy controls and no TCR δ gene rearrangements were found in this group. Surprisingly, 7 of 9 (78%) CML patients showed clonal TCR rearrangements at diagnosis. In 3 patients the clonal rearrangement was detected in the TCR δ genes, in 7 patients in the TCR γ genes and 3 patients had rearrangemens both in TCR δ and γ genes. After one year of imatinib treatment the same clones could be detected in 5 of the 7 patients (71%). Although clonal cells were observed, none of the imatinib patients had signs of a concomitant lymphoproliferative disorder and the distribution of lymphocyte subclasses was normal. Next, 23 patients treated with dasatinib were studied, 10 without (LGLneg) and 13 with PB LGL lymphocytosis (LGLpos) including T- or NK-cell expansions. In all LGLpos dasatinib patients (including patients with a CD3neg NK-cell expansion) clonal TCR γ or δ rearrangements were found. In LGLneg dasatinib patients the prevalence of TCR rearrangements was 80%. LGLpos patients had more often clonal rearrangements in TCR δ genes (62%) than LGLneg patients (10%). No differences in clonal rearranged TCR γ genes (77% vs. 80%) were detected. Most patients displayed more than one clonal TCR rearrangement. Quantitative follow-up of LGLpos patients revealed that the expansion of a single predominant lymphocyte clone accounted for LGL lymphocytosis. Intriguingly, quantitative follow-up of lymphocyte clones by PCR showed that the observed clones existed at low levels already before start of dasatinib therapy during imatinib treatment, but no lymphocyte expansions were then seen. Sorting of lymphocytes showed that clonal cells resided in the CD8+ and CD4+ T-cell populations and, strikingly, also among CD16/56+CD3neg NK cells. All dasatinib patients with NK cell expansions (n=3) showed TCR δ rearrangements in their NK cells. Conclusions. Clonal lymphocytes could rarely be found in healthy controls. In contrast, they were frequently present in CML patients at diagnosis and persisted during TKI therapy. In a distinct subgroup of dasatinib treated patients, clonal cells massively expanded during successful therapy. Clonal TCR rearrangements were detected in CD4+, CD8+ and, unexpectedly, also in NK cells. The epitopes and function of clonal, CML-associated lymphocytes are under investigation. Previous studies showed that clonal expansions during dasatinib were associated with excellent, long-lasting therapy responses in advanced leukemia. We therefore hypothesize, that the clonal lymphocytes present at CML diagnosis may be anergic anti-leukemic cells and part of the immune escape mechanisms inherent to leukemogenesis and that dasatinib therapy can reverse this anergy. Disclosures: Ekblom: BMS: Honoraria. Seggewiss:BMS: Honoraria. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:BMS: Honoraria.

scholarly journals Immunological Monitoring of CML Patients during First-Line Bosutinib and Imatinib Treatment

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3069-3069
Author(s):  
Anna Kreutzman ◽  
Perttu Koskenvesa ◽  
Kasanen Tiina ◽  
Ulla Olsson-Strömberg ◽  
Jesper Stentoft ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Nk Cell ◽  
T Cell Subsets ◽  
Imatinib Treatment ◽  
First Line

Abstract Background: Tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia (CML) are not entirely selective for the BCR-ABL1 kinase but also inhibit a variety of other kinases, sometimes triggering unpredicted biological effects. As an example, the TKIs dasatinib and bosutinib both inhibit Src-kinases, which are important mediators of T-cell function. Earlier in vitro data has shown that dasatinib can suppress activation and proliferation of T and NK cells, but it can also elicit signs of immunostimulation in patients, including rapid mobilization of lymphocytes and LGL lymphocytosis. No extensive analyses of the immunological in vivoeffects of bosutinib have been performed thus far. Therefore, we aimed at characterizing T and NK cell phenotypes and functional features in CML patients in a clinical setting in the context of first-line bosutinib and imatinib treatment. Methods:Peripheral blood samples were obtained from newly diagnosed CML CP patients enrolled in the BFORE clinical trial (NCT02130557), receiving bosutinib (n=13) or imatinib (n=20) as frontline TKI treatment. Samples were drawn at diagnosis and following 3 and 12 months of therapy. Detailed immunophenotyping of NK and T cells was performed with multicolor flow cytometry. In addition, mononuclear cells were used to study the function of NK and T cells (CD107ab degranulation upon stimulation with K562 cells and detection of IFN-γ/TNF-α secretion after stimulation with anti-CD3/anti-CD28 antibodies, respectively). Moreover, blood differential counts were taken before and 2 hours after drug intake at 3 and 12 months to examine the direct effects on lymphocyte counts (mobilization). Results: No significant changes were observed in absolute white blood cell or lymphocyte counts directly (2 hours) after bosutinib or imatinib intake, in contrast to what has been observed in dasatinib treated patients. Analysis of T cell subsets during bosutinib treatment revealed that the proportion of CD4+ cells increased after the start of treatment (median dg. 60.0% vs. 3 months 62.0% p=0.06; vs. 12 months 72.8% p=0.03), but no significant changes were observed in the phenotype. Correspondingly, the proportion of CD8+ T-cells decreased moderately (dg. 31.6% vs. 3 months 25.5% p=0.01) after the therapy start. Interestingly, the proportion of PD1+ (dg. 19.6% vs. 3 months 11.9%, p=0.06; vs. 12 months 14.3%, p=0.11) and DNAM+ CD8+ T-cells decreased (dg. 73.1% vs. 3 months 66.2% p=0.004; vs. 12 months 64.6% p=0.02). No changes in the cytokine production of any of the studied subgroups of T-cells was observed. Moreover, the proportion, phenotype and function of NK-cells were not affected by bosutinib treatment. In contrast, during imatinib treatment the proportion of CD56+CD16+ NK-cells significantly increased (dg 4.3% vs. 3 months 9.9% p=0.0005; vs 12 months 14.4% p=0.002; 8.1% in bosutinib treated patients). Moreover, in imatinib patients NK-cells downregulated CD27 (dg 9.0% vs. 3 months 5.2% p=0.004; vs. 12 months 4.9%; p=0.002). Further, NK-cells from imatinib-treated patients expressed more CD107ab upon stimulation with K562 at 3 and 12 months, when compared to samples from diagnosis (dg 13.0% vs. 3 months 16.1%, p=0.01; vs. 12 months 23.2%, p=0.008). The proportion of CD4+ T-cells increased 3 months after the start of imatinib treatment (dg 60.1% vs. 3 months 63.5% p=0.01), whereas the percentage of CD8+ T-cells decreased (dg. 38.6% vs. 3 months 31.5% p=0.02). Decreased expression of DNAM (dg 73.5% vs. 3 months 67.9% p=0.0008; vs. 12 months 62.4% p=0.002) was observed in the CD4+ T-cells. Similarly as in bosutinib treated patients, the proportion of PD1+ CD8+ cells decreased during imatinib treatment (dg 18.2% vs. 3 months 14.7%, p=0.02; vs. 12 months 14.8%, p=0.03). Both CD4+ and CD8+ T-cell subsets from imatinib-treated patients secreted less cytokines after the start of treatment when compared to the pre-treatment samples. Conclusions: Despite of the Src-kinase inhibitory profile of bosutinib, no major changes were observed in T- or NK-cell phenotype or function during first-line bosutinib treatment. In contrast, in imatinib treated patients the proportion of NK-cells increased and their degranulation responses were significantly higher than in untreated CML patients. Comparison of these data with the clinical variables and treatment outcome is warranted. Disclosures Stentoft: Novartis: Research Funding; Bristol-Myers-Squibb: Research Funding; Pfizer: Research Funding; Ariad: Research Funding. Gjertsen:BerGenBio AS: Consultancy, Research Funding. Janssen:Pfizer: Honoraria; Novartis: Research Funding; Ariad: Honoraria; BMS: Honoraria. Brümmendorf:Pfizer: Research Funding; Novartis: Research Funding. Richter:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Large Granular Lymphocyte (LGL) Expansions Comprising Oligoclonal T Cell or NK Cell Populations in Dasatinib Treated Patients Are Associated with HLA-A*0201, CMV Reactivation and Enhanced Anti-Leukemic Control.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1123-1123 ◽  
Author(s):  
Satu Mustjoki ◽  
Anna Kreutzman ◽  
Carolin Dix ◽  
David A Price ◽  
Kristin Ladell ◽  
...  
Keyword(s):  
T Cell ◽  
Cytotoxic Activity ◽  
Expression Profile ◽  
Research Funding ◽  
Nk Cell ◽  
Large Granular Lymphocyte ◽  
Healthy Controls ◽  
Cmv Reactivation

Abstract Abstract 1123 Poster Board I-145 Introduction Immunosuppressive effects of the second generation tyrosine kinase inhibitor dasatinib (Sprycel®) on T cells and NK cells have been described in vitro. In contrast, in some CML or Ph+ ALL patients receiving dasatinib, the development of a chronic, oscillating lymphocytosis has been observed in vivo. We previously showed that dasatinib-induced lymphocytosis typically comprises of oligoclonal NK or CD8+ cytotoxic T cell large granular lymphocyte (LGL) expansions and was associated with HLA-A*0201, CMV reactivation and enhanced, long-lasting therapy responses in patients with advanced leukemia. Patients and Methods To further elucidate the mechanisms underlying LGL expansions during dasatinib, we analyzed T cell and NK cell effector functions directly ex vivo. Specifically, we evaluated activation, proliferation, cytotoxic activity, cytokine secretion, degranulation, KIR expression profile and apoptosis/necrosis susceptibility. Results We observed decreased NK cell cytotoxic activity against the cell line K562 only in patients with NK cell expansions, potentially due to exhaustion based on excessive in vivo proliferation. This reduced lytic ability was restored after pre-treatment with IL-2 for 18h. No correlation between functionality and KIR expression profile was found. Furthermore, patients with LGL expansions exhibited elevated IP10/RANTES levels in the plasma compared to non-LGL patients and healthy controls. Strikingly, within the CD8+ T cell population specific for the CMV epitope NLVPMVATV (pp65, residues 495-503) restricted by HLA-A*0201, we observed distinct CD8high and CD8low fractions in patients with LGL expansions (n=5) but not in patients without LGL expansions (n=4) or in healthy controls (n=3). The CD8high subpopulation was more resistant to inhibition by dasatinib compared to other cell fractions; in addition, dasatinib effectively abrogated down-regulation of both TCR and CD8 in the CD8high subpopulation in vitro. Conclusions Thus, we hypothesize that CMV reactivation is intimately linked to the observed LGL expansions in dasatinib-treated patients. The expanded cytotoxic cells may target both CMV-infected and leukemic cells by virtue of epitope sharing and thus explain the enhanced anti-leukemic control we observed. Further investigations based on the application of polychromatic flow cytometry and analysis of T cell clonality are underway to establish the nature of this association in more detail. Dasatinib and derivatives may prove to be useful in modulating anti-leukemic/anti-host immune responses in vivo. Disclosures Mustjoki: BMS: Honoraria. Ekblom:BMS: Honoraria. Porkka:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Seggewiss:BMS: Honoraria.

T Cell Exhaustion and Downregulation of Cytotoxic NK Cells – an Immune Escape Mechanism in Adult Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3781-3781
Author(s):  
Eolia Brissot ◽  
Sawa Ito ◽  
Kit Lu ◽  
Carly Cantilena ◽  
B. Douglas Smith ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Research Funding ◽  
Immune Escape ◽  
Nk Cell ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Healthy Donors

Abstract Adult acute lymphoblastic leukemia (ALL) remains a therapeutic challenge with less than 40% long term survival. There is growing evidence that malignant diseases exert an “immune editing” effect which blocks antitumor immunity and permits tumor growth through immune evasion. Such tumor escape represents an obstacle for anticancer immunotherapy. In ALL such immune escape mechanisms are not well characterized. We therefore profiled cellular immunity in ALL, by characterizing the subsets of T cells, regulatory T cells (Treg), natural killers (NK) cells and γd T cells, using various functional markers including T cell exhaustion and NK cell activating or inhibitory molecules. Forty ALL patients were included in the study. The median age was 39 y (range, 18-75). Thirty-six presented with B-lineage ALL and 4 with T-lineage ALL. Mononuclear cells were isolated from blood (n=19) or bone marrow (n=21) at the onset of leukemia or at relapse. The median infiltration of blasts was 85% (range 24-96%). Healthy donor peripheral blood (n=12) and bone marrow (n=9), from age and gender matched population, were simultaneously analyzed as controls. Extra-and intra cellular staining were performed using using antibodies directed against CD3, CD4, CD8, CD45, CD45, CD45RA, CD45RO, CCR7, CD95, CD27, CD19, CD14, CD127, CD25, Foxp3, Helios, αβTCR, HLA-DR, CD117, CD20, CD10, CD22, CD34, LAG3, PD1, PDL1, CD56, NKG2A, NKG2C, NKG2D, KIR2DL1, KIR2DL3, CD57, CD33, CD11b, CD15, CD38 and CD24. Data were acquired on a BD LSRFORTESSA flow cytometer. The expression of programmed cell death 1 (PD-1, CD279) receptor on CD8+T cells was significantly increased in blood and bone marrow of ALL patients compared to healthy donors (p<0.0001 and p=0.004, respectively) (Fig. 1). Focusing on the different subsets, CD8+ effector memory T cells significantly over-expressed PD-1 in blood and bone marrow of ALL patients compared to healthy donors (p=0.008 and p=0.04, respectively). Moreover, there was a significant positive correlation between PD-1 expression on CD8+ effector memory T cells and blast infiltration (R2=0.23, 95%CI 0.026-0.76, p=0.04). Expression of the co-inhibitory receptor lymphocyte-activation gene 3 (LAG-3, CD223) was similar in ALL patients compared to healthy donors. A significantly higher frequency of T regulators (CD25+, CD127 low, Foxp3+) was found in bone marrow microenvironment in ALL patients (4.3% versus 1.6%, p=0.02). Concerning γd T cells, frequency was similar in blood and bone marrow of ALL patients compared with healthy donors. There was a significantly lower frequency of CD56dimNKG2A+KIR-CD57- (p=0.02) in the bone marrow of ALL patients indicating a maturation arrest. Interestingly, expression of the activating receptor NKG2D which plays an important role in triggering the NK cell–mediated tumor cell lysis was significantly reduced in NK cells of ALL patients while no difference in NK cell expression of NKG2C was found(Fig. 2). Adult patients with ALL show evidence of immune-editing of T cells and NK cells. This global immunosuppressive mechanism may contribute to the eventual escape of ALL from immune control. PD-1, overexpression, described in acute myeloid leukemia and chronic myeloid leukemia has been implicated in T-cell exhaustion and subsequent tumor immune evasion. Our data suggests similar immune escape mechanisms pertain in ALL. Effective antileukemia immunotherapy will require targeting one or more of these immunosuppressive pathways to achieve optimum results. Disclosures Fathi: Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.

Early Disease Relapse after Tyrosine Kinase Inhibitor Treatment Discontinuation in CML Is Related Both to Low Number and Impaired Function of NK-Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 812-812 ◽  
Author(s):  
Mette Matilda Ilander ◽  
Ulla Olsson-Strömberg ◽  
Hanna Lähteenmäki ◽  
Kasanen Tiina ◽  
Perttu Koskenvesa ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Treatment Discontinuation ◽  
Disease Relapse ◽  
Cell Counts ◽  
Tnf Α ◽  
Ifn Γ

Abstract Background: Recent reports suggest that approximately 40% of CML patients who have achieved sustained complete molecular remission are able to stop TKI treatment without disease relapse. However, there are no predictive markers for successful therapy discontinuation. Therefore, we set up an immunological sub-study in the ongoing pan-European EURO-SKI stopping study. Our aim was to identify predictive biomarkers for relapse/non-relapse and to understand more on the mechanisms of immune surveillance in CML. Methods: The EURO-SKI study started in 2012, and patients included were at least three years on TKI and at least one year in MR4 or deeper before the study entry. Basic lymphocyte immunophenotyping (the number of NK-, T- and B-cells) was performed at the time of therapy discontinuation and 1, 6, and 12 months after the TKI stop and in case of relapse (defined as loss of MMR, BCR-ABL1>0.1% IS). In addition, from a proportion of patients more detailed immunophenotypic and functional analyses (cytotoxicity of NK-cells and secretion of Th1 type of cytokines IFN-γ/TNF-α) were done at the same times. Results: Thus far 119 Nordic patients (imatinib n=105, dasatinib n=12, nilotinib n=2) who have discontinued TKI treatment within the EURO-SKI study have been included in the lymphocyte subclass analysis (results are presented from patients who have reached 6 months follow-up). Immunophenotyping analysis demonstrates that imatinib treated patients who were able to maintain remission for 6 months (n=36) had increased NK-cell counts (0.26 vs. 0.15x109cells/L, p=0.01, NK-cell proportion 18.9% vs. 11%, p=0.005) at the time of drug discontinuation compared to patients who relapsed early (before 5 months n=22). Furthermore, the phenotype of NK-cells was more cytotoxic (more CD57+ and CD16+cells and less CD62L+cells), and also their IFN-γ/TNF-α secretion was enhanced (19.2% vs. 13%, p=0.02). Surprisingly, patients who relapsed more slowly (after 5 months, n=16) had similar baseline NK-cell counts (0.37x109cells/L), NK-cell proportion (21.2%), and phenotype and function as patients, who were able to stay in remission. No differences in the NK-cell counts were observed between patients who had detectable or undetectable BCR-ABL1 transcripts at the baseline (0.22 x109cells/L vs. 0.31 x109cells/L, p=0.61). Interestingly, NK-cell count was higher in patients with low Sokal risk score than in patients with intermediate risk (0.33 x109cells/L vs. 0.20 x109cells/L, p=0.04). Furthermore, there was a trend that male patients had a higher proportion of NK-cells than females (21.6% vs. 15.7%, p=0.06). Pretreatment with IFN-α or the duration of imatinib treatment did not have an effect on NK-cell count or proportion. In comparison to the imatinib group, dasatinib treated patients had higher NK-cell counts at the baseline (median 0.52x109cells/L vs. 0.26x109cells/L, p=0.02), and also the proportion of CD27 (median 50% vs. 16%, p=0.01) and CD57 expressing (median 79% vs. 74%, p=0.05) NK-cells was higher. The follow-up time of dasatinib treated patients is not yet long enough to correlate the NK-cell counts with the success of the treatment discontinuation. The absolute number of T-cells or their function did not differ significantly between relapsing and non-relapsing patients at the time of treatment discontinuation. However, both CD4+ and CD8+ T-cells tended to be more mature in patients who stayed in remission compared to patients who relapsed early (CD4+CD57+CD62L- median 5.7% vs. 2.4%, p=0.06, CD8+CD62L+CD45RA+ 13% vs. 26.7%, p=0.05). The analysis of follow-up samples showed that in patients who stayed in remission the Th1 type cytokine (IFN-γ/TNF-α) secretion of CD8+T-cells increased at 6 months compared to baseline (23.6 vs. 18.5%, p=0.07). Same phenomenon was observed in the late relapsing group at relapse compared to baseline (37.9 vs. 13.5%, p=0.03). No similar increase was observed in the early relapsing group. Conclusions: Low NK-cell numbers and poor cytokine secretion may predict early disease relapse after TKI discontinuation. However, patients who relapse later have high numbers of normally functioning NK-cells. Further research (detailed phenotypic analysis of NK- and T-cells including activating and inhibitory receptors and immune checkpoint molecules) and correlation of biomarker data with clinical parameters are ongoing to understand the ultimate determining factors of relapse. Disclosures Själander: Novartis: Honoraria. Hjorth-Hansen:Novartis: Honoraria; Bristol-myers Squibb: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

A Novel HIV Envelope Bi-Specific Killer Engager Enhances Natural Killer Cell Mediated ADCC Responses Against HIV-Infected Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2517-2517 ◽  
Author(s):  
Zachary B. Davis ◽  
Todd Lenvik ◽  
Louis Hansen ◽  
Martin Felices ◽  
Sarah Cooley ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Mhc I ◽  
Variable Heavy ◽  
Infected Cells ◽  
T Cell Lines ◽  
Hiv Envelope

Abstract Natural Killer (NK) cells, a critical component of the immune response to viral infection, recognize and destroy cells with diminished expression of major histocompatibility class-I (MHC-I) molecules and expression of ligands for activating NK receptors such as NKG2D. Down-modulation of MHC-I is a hallmark of viral infection, as it allows infected cells to evade a CD8 T-cell response. Stalling of the cell cycle to enhance viral replication induces NK activation ligands such as the NKG2D ligands unique long binding proteins (ULBP)-1 and -2 which could trigger NK destruction of infected cells. Unfortunately, incomplete down-modulation of MHC-I by HIV leaves HLA-C on the cell surface, which inhibits the majority of NK cells from killing infected targets. CD16, the low affinity Fc receptor, is the most potent NK cell activating receptor. It mediates antibody dependent cell-mediated cytotoxicity (ADCC), and can override inhibition by MHC-I. We designed a series of bi-specific killer-engager (BiKE) constructs to direct NK cell ADCC against an HIV-infected target. We linked the Fab portions of broadly neutralizing (bn)Abs to a novel llama-derived nanobody EF91 that binds CD16 at high affinity and signals strong activation. We chose to use EF91 as its structure is unique compared to the use of a single chain variable fragment (scFv). Rather than being composed of a variable heavy (VH) and variable light (VL) chain, the nanobody is composed of a single variable heavy (VHH) domain. A distinct advantage to using a CD16 nanobody over a scFv is in the purity of the generated product. During protein folding it is not uncommon for the wrong VH to associate with the wrong VL; the result of which is a nonfunctional product. Since the nanobody is single VHH, and does not require association with another domain, there is less risk of a misfolded product. Nanobodies are also known to have similar, if not increased, affinity for their target molecules. In the case of EF91, this may result in more robust activation of NK cells than with a traditional scFv. We tested a BiKE constructed with the bnAb, VRC01, which recognizes the CD4 binding domain of HIV-Env. The specificity of our novel anti-CD16 nanobody was demonstrated by binding of our BiKE construct to CD16+ NK cells (Figure 1A). Function of our BiKE construct was tested by incubating it with chronically infected T-cell lines (HIV-IIIB and ACH-2) or with their respective uninfected counterparts (H9 and CEM). We only observed binding to infected cells (Figure 1B), demonstrating HIV-Env binding specificity to the HIV strains ACH-2 (LAI strain) and HIV-IIIB. The ability of the anti-Env BiKE construct to mediate ADCC and IFNγ production was tested against two uninfected CD4 T-cell lines or their infected counterparts. While NK cells degranulated when incubated with the infected cell lines (50% against HIV-IIIB and 20% against LAI), this response was markedly enhanced when co-incubated with the HIV-Env specific BiKE (80% against HIV-IIIB and 60% against LAI) (Figure 1C). Furthermore, the HIV-Env BiKE enhanced IFNγ production against HIV-infected T-cell lines compared to responses in the absence of BiKE (28% against HIV-IIIB compared to 36% with BiKE; 15% against ACH-2 compared to 37% with BiKE) (Figure 1D). Our data demonstrate that a BiKE construct containing the Fab of an HIV bnAb and an anti-CD16 component can eliminate HIV-infected targets that express the HIV-envelope on their surface. The reservoir of latently infected CD4 T cells lack expression of any recognizable virus protein on the cell surface, we plan to combine our BiKE strategy with cellular activation using IL-15. Alternatively, we can construct a tri-specific engager (TriKE) with an IL-15 segment that may activate CD4 T cells while enhancing NK cell killing. Disclosures Cooley: Fate Therapeutics: Research Funding. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

scholarly journals Single-Cell Multi-Omic Analysis Uncovers Comprised Immune Function and Primary Resistance Mechanism in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 378-378
Author(s):  
Jianbiao Zhou ◽  
Jonathan Adam Scolnick ◽  
Stacy Xu ◽  
Melissa Ooi ◽  
Priscella Shirley Chia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Single Cell ◽  
Research Funding ◽  
Immune Escape ◽  
Nk Cell ◽  
Primary Resistance ◽  
Bm Niche

Abstract Background: Approximately 20% of AML patients do not respond to induction chemotherapy (primary resistance) and 40-60% of patients develop secondary resistance, eventually leading to relapse followed by refractory disease (RR-AML). Diversified molecular mechanisms have been proposed for drug resistance and RR phenotype. However, we still cannot predict when relapse will occur, nor which patients will become resistant to therapy. Single-cell multi-omic (ScMo) profiling may provide new insights into our understanding of hematopoietic stem cell (HSC) differentiation trajectories, tumor heterogeneity and clonal evolution. Here we applied ScMo to profile bone marrow (BM) from AML patients and healthy controls. Methods: AML samples were collected at diagnosis with institutional IRB approval. Cells were stained with a panel of 62 DNA barcoded antibodies and 10x Genomics Single Cell 3' Library Kit v3 was used to generate ScMo data. After normalization, clusters were identified using Uniform Manifold Approximation and Projection (UMAP) and annotated using MapCell (Koh and Hoon, 2019). We analyzed 23,933 cells from 4 adult AML BM samples, and 39,522 cells from 2 healthy adults and 3 sorted CD34+ normal BM samples. Gene set enrichment analysis (GSEA) and Enrichr program were used to examine underlying pathways among differentially expressed genes between healthy and AML samples. Results: We identified 16 cell types between the AML and normal samples (Fig 1a) amongst 45 clusters in the UMAP projection (Fig 1b). Comparative analysis of the T cell clusters in AML samples with healthy BM cells identified an "AML T-cell signature" with over-expression of genes such as granzymes, NK/T cell markers, chemokine and cytokine, proteinase and proteinase inhibitor (Fig 2a). Among them, IL32 is known to be involved in activation-induced cell death in T cells and has immunosuppressive role, while CD8+ GZMB+ and CD8+ GZMK+ cells are considered as dysfunctional or pre-dysfunctional T cells. Indeed, Enrichr analysis showed the top rank of phenotype term - "decreased cytotoxic T cell cytolysis". We next examined whether NK cells, are similarly dysfunctional in the AML ecosystem. The "AML NK cell signature" includes Fc Fragment family, IFN-stimulated genes (ISGs), the effector protein-encoding genes and other genes when compared to normal NK cells (Fig 2b). GSEA analysis revealed "PD-1 signalling" among the top 5 ranked pathways in AML-NK cells, though no increase in PD-1 protein nor PDCD1 gene were identified in these cells. Inhibitory receptor CD160 was expressed higher in AML samples along with exhaustion (dysfunction) associated genes TIGIT, PRF1 and GZMB (Fig 2c). Enrichr analysis uncovered enrichment of "abnormal NK cell physiology and "impaired natural killer cell mediated cytotoxicity". Similarly, the "AML monocyte signature" was significantly enriched with genes in "Tumor Infiltrating Macrophages in Cancer Progression and Immune Escape" and "Myeloid Derived Suppressor Cells in Cancer Immune Escape". We also analyzed HSPC component in one pair of cytogenetically matched, untreated complete remission (CR) /RR AML pair (Fig 2d). Notably, half of the 10 genes overexpressed in RR-AML, CXCR4, LGALS1, S100A8, S100A9, SRGN (Serglycin), regulate cell-matrix interaction and play pivotal roles in leukemic cells homing bone marrow niche. The first 4 of these genes have been demonstrated as prognostic indicators of poor survival and associated with chemo-resistance and anti-apoptotic function. Furthermore, single-cell trajectory analysis of this CR/RR pair illustrated a change in differentiation pattern of HSPCs in CR-AML to monocytes in RR-AML. We are currently analyzing more AML samples to validate these findings. Conclusions: Our ScMo analysis demonstrates that the immune cells are systematically reprogrammed and functionally comprised in the AML ecosystem. Upregulation of BM niche factors could be the underlying mechanism for RR-AML. Thus, reversing the inhibited immune system is an important strategy for AML therapy and targeting leukemic cell-BM niche interaction should be considered for cases with high expression of these molecules on AML HSPCs. Note: J.Z. and J.A.S. share co-first authorship. Figure 1 Figure 1. Disclosures Scolnick: Proteona Pte Ltd: Current holder of individual stocks in a privately-held company. Xu: Proteona Pte Ltd: Current Employment. Ooi: Jansen: Honoraria; Teva Pharmaceuticals: Honoraria; GSK: Honoraria; Abbvie: Honoraria; Amgen: Honoraria. Lovci: Proteona Pte Ltd: Current Employment. Chng: Aslan: Research Funding; Takeda: Honoraria; Johnson & Johnson: Honoraria, Research Funding; BMS/Celgene: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Antengene: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria.

scholarly journals Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 341-341
Author(s):  
Lucila Kerbauy ◽  
Mecit Kaplan ◽  
Pinaki P Banerjee ◽  
Francesca Lorraine Wei Inng Lim ◽  
Ana Karen Nunes Cortes ◽  
...  
Keyword(s):  
T Cell ◽  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Nk Cell ◽  
Ex Vivo

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

scholarly journals CD94 1A transcripts characterize lymphoblastic lymphoma/leukemia of immature natural killer cell origin with distinct clinical features

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3567-3574 ◽  
Author(s):  
Chung-Wu Lin ◽  
Ting-Yun Liu ◽  
Shee-Uan Chen ◽  
Kun-Teng Wang ◽  
L. Jeffrey Medeiros ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Killer Cell ◽  
Cell Lineage ◽  
Lymphoid Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Gene Rearrangements

AbstractMost lymphoblastic lymphomas (LBLs) are regarded as neoplasms of immature T cells because they express cytoplasmic CD3 and frequently carry T-cell receptor (TCR) gene rearrangements. Immature natural killer (NK) and T cells, however, have a common bipotent T/NK-cell precursor in the thymus, and NK cells also express cytoplasmic CD3. Thus, some LBLs could arise from immature NK cells. Mature NK cells express 2 CD94 transcripts: 1A, induced by interleukin 15 (IL-15), and 1B constitutively. Because immature NK cells require IL-15 for development, CD94 1A transcripts could be a marker of NK-LBL. To test this hypothesis, we used laser capture microdissection to isolate IL-15 receptor α+ lymphoid cells from the thymus and showed that these cells contained CD94 1A transcripts. We then assessed for CD94 transcripts in 21 cases of LBL that were cytoplasmic CD3+, nuclear terminal deoxynucleotidyl transferase positive (TdT+), and CD56-, consistent with either the T-cell or NK-cell lineage. We found that 7 LBLs expressed CD94 1A transcripts without TCR gene rearrangements, suggesting NK-cell lineage. Patients with NK-LBL were younger than patients with T-LBL (15 years versus 33 years; P = .11) and had a better 2-year survival (100% versus 27%; P &lt; .01). These results improve the current classification of LBL and contribute to our understanding of NK-cell differentiation.

scholarly journals CD5-CD56+ T-cell receptor silent peripheral T-cell lymphomas are natural killer cell lymphomas [see comments]

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1466-1473 ◽  
Author(s):  
JF Emile ◽  
ML Boulland ◽  
C Haioun ◽  
P Kanavaros ◽  
T Petrella ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
T Cell Receptor ◽  
Nk Cell ◽  
Cell Receptor ◽  
Cell Markers ◽  
Clonal Rearrangement ◽  
T Cell Lymphomas ◽  
Peripheral T Cell Lymphomas

Non-Hodgkin's lymphomas are divided into B- and T-cell neoplasms. The existence and the clinical relevance of lymphomas derived from the third lymphocyte lineage, ie, natural killer (NK) cells are still controversial. NK cells are lymphocytes that mediate cytotoxicity without prior sensitization. NK cells also have phenotypic and genotypic characteristics: they express the NK-related antigen CD56, T- cell markers such as CD2 and CD7, but do not express CD5 and T-cell receptor (TCR) proteins, and their TCR locus is not rearranged. Therefore, if NK cell lymphomas exist, they should express some T-cell markers, but not alpha beta or gamma delta TCR proteins. Such lymphomas are actually called TCR silent peripheral T cell lymphomas (PTCL). To detect and characterize NK cell lymphomas, we investigated the immunophenotype and immunogenotype of 35 patients with TCR silent PTCL. The first group included 16 patients with a lymphoma of CD5-CD56+ phenotype, which is identical to normal NK cells. These patients had either a nasal/nasopharyngeal lymphoma (11 cases) or a lymphoma with predominant non-nasal/non-nodal initial involvement (five cases). Eight of the nine cases for which immunogenotypic data were available lacked clonal rearrangement of the TCR gamma genes. Thus, these tumors are likely to be NK cell lymphomas. The second group of 15 cases had a CD5+ phenotype (14 were CD56-, and 1 was CD56+) and clonal rearrangement of TCR gamma genes, indicating that they were true PTCL with unproductive TCR rearrangement. The four remaining cases were CD5- CD56- lymphomas and disclosed either a clonal (two cases) or no clonal (two cases) rearrangements of the TCR gamma genes. Altogether these findings show that CD5-CD56+ so-called “TCR silent PTCL” bear the immunophenotype and immunogenotype of normal NK cells and display peculiar clinical features distinct from true PTCL.

scholarly journals Preliminary Clinical Data from a Phase 1 Trial with CPI-818, a Selective ITK Inhibitor That Preferentially Blocks the Growth of T Lymphoma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1571-1571
Author(s):  
Patrick P. Ng ◽  
Mehrdad Mobasher ◽  
Kitman S. Yeung ◽  
Andrew N. Hotson ◽  
Craig M. Hill ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Phase 1 ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Introduction ITK is a tyrosine kinase critical to T cell receptor (TCR) signaling. Overexpression of this gene has been reported in cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL). Genomic analyses have demonstrated the contribution of aberrant TCR signaling in the pathogenesis of T-cell lymphomas (TCL). RLK, a closely related kinase, is co-expressed with ITK in T and NK cells, and is partially functionally redundant with ITK signaling. In NK cells, ITK has been shown to be involved in FcγRIII signaling and antibody-dependent cellular cytotoxicity (ADCC). However, the relative contribution of ITK vs RLK in ADCC is not well understood. Thus, selective inhibition of ITK, but not other signal transduction components such as RLK, may be an effective strategy to treat TCL while preserving normal T and NK cell functions. CPI-818 is an orally bioavailable, covalent inhibitor of ITK with &gt;100-fold selectivity over RLK and BTK. It was well tolerated and exhibited anti-tumor activity in companion dogs with spontaneous TCL (2019 AACR Annual Meeting Abstract #1313). A phase 1/1b trial with CPI-818 in human TCL has been initiated (NCT03952078). Here we present preclinical evidence that CPI-818 inhibits the proliferation of human malignant T cells with relative sparing of normal lymphocytes and report early results from the clinical trial. Methods Eligible patients for the dose-escalation/expansion trial of CPI-818 have relapsed/refractory TCL (PTCL, CTCL and others). Starting dose of CPI-818 is 100 mg BID continuously. The objectives of the study are to evaluate the safety and tolerability of CPI-818 in ascending dose levels; evaluate pharmacokinetics/pharmacodynamics and potential biomarkers. In in vitro studies, T cells from the blood of Sézary syndrome patients were stimulated for 6 days with αCD3/CD28. Sézary cells were identified by antibodies to specific TCR Vβ. For assays of ADCC, αCD20-coated lymphoma B cells were cultured with NK cells from multiple healthy donors for 18 h with inhibitors. In animal studies, mice received control or CPI-818-formulated diet (300 mg/kg/day). C57BL/6 mice were vaccinated with keyhole limpet hemocyanin (KLH) or subcutaneously implanted with the TCL line EL4. MRL/lpr mice began treatment at 9 weeks old. Lymph nodes were calipered weekly. Spleens and lungs were harvested at 22 weeks. Results Mouse models were studied to assess the impact of CPI-818 on normal, autoreactive and malignant T cells in vivo. No changes in total blood cell counts or T, B, NK cell subsets in lymphoid organs were seen in normal mice receiving daily doses of CPI-818 sufficient to continuously inhibit ITK for 28 days. Immune responses to antigen re-challenge were not affected in these mice, as determined by levels of antibody or CD4 T cell response to vaccination with KLH. In mice with established EL4 lymphoma, administration of CPI-818 reduced the growth of tumors at the primary site and in the draining lymph nodes (P values &lt;0.033). CPI-818 also reduced lymphadenopathy and expansion of autoreactive T cells in the spleens of MRL/lpr mice (P values &lt;0.0001), without affecting CD4 or CD8 cells. Sézary cells from 3 of 3 patients tested in vitro were more sensitive to growth inhibition with CPI-818 than autologous normal CD4 or CD8 cells, or T cells from a healthy donor (Figure 1). CPI-818 showed minimal inhibition of NK-mediated ADCC (5%), whereas CP-2193, an ITK/RLK dual inhibitor with an IC50 for ITK comparable to CPI-818, reduced ADCC by 50%. CPI-818 has been administered to two patients at the first dose level cohort (100 mg BID) with no DLTs, and with no changes to B, T, and NK cell counts in blood during the first dosing cycle (21 days). Pharmacokinetic and occupancy studies have revealed 80% and 50% occupancy of ITK at peak and trough drug levels, respectively in peripheral blood T cells. Conclusions CPI-818 is a selective covalent ITK inhibitor that has greater antiproliferative effects on malignant and autoreactive T cells compared to normal T cells. The drug has a minimal impact on NK mediated ADCC compared with a less selective inhibitor that also blocks RLK. Preliminary data from a phase 1/1b study shows CPI-818 at 100 mg BID was tolerable with acceptable bioavailability and ITK occupancy. Further dose escalation is ongoing. Disclosures Ng: Corvus Pharmaceuticals, Inc.: Employment, Equity Ownership. Mobasher:Corvus Pharmaceuticals: Employment, Equity Ownership. Yeung:Corvus Pharmaceuticals: Employment, Equity Ownership. Hotson:Corvus Pharmaceuticals: Employment, Equity Ownership. Hill:Corvus Pharmaceuticals: Employment, Equity Ownership. Madriaga:Corvus Pharmaceuticals: Employment, Equity Ownership. Dao-Pick:Corvus Pharmaceuticals: Employment, Equity Ownership. Verner:Corvus Pharmaceuticals: Employment, Equity Ownership. Radeski:Corvus Pharmaceuticals: Research Funding. Khodadoust:Corvus Pharmaceuticals: Research Funding. Kim:Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Horizon: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Galderma: Research Funding; Elorac: Research Funding; Soligenix: Research Funding; Medivir: Honoraria, Membership on an entity's Board of Directors or advisory committees; miRagen: Research Funding; Forty Seven Inc: Research Funding; Neumedicine: Research Funding; Portola Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Corvus: Honoraria, Membership on an entity's Board of Directors or advisory committees; Trillium: Research Funding. Miller:Corvus Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Buggy:Corvus Pharmaceuticals: Employment, Equity Ownership. Janc:Corvus Pharmaceuticals: Employment, Equity Ownership.

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