A Recombinant Antibody to Siglec-8 Shows Selective ADCC Activity Against Mast Cells from Systemic Mastocytosis Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4092-4092 ◽  
Author(s):  
Rustom Falahati ◽  
Jessica Bright ◽  
Alejandro Dorenbaum ◽  
Christopher Bebbington ◽  
Nenad Tomasevic ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Systemic Mastocytosis ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Adcc Activity ◽  
Employment Equity ◽  
Effector Cells ◽  
Equity Ownership ◽  
Kit D816v

Abstract Background: Systemic mastocytosis (SM) is a rare myeloproliferative neoplasm characterized by the accumulation of neoplastic mast cells (MC) in one or more extracutaneous organs. In all forms of SM, anti-mediator drugs are used to control symptoms of MC degranulation. In advanced forms of SM, organ damage is common and patients (pts) exhibit reduced life expectancy. In these individuals, cytoreductive agents such as cladribine and interferon-alpha have been used off-label, and inhibitors of KIT D816V are under investigation. A significant unmet need exists for these patients. Siglec-8 is an inhibitory receptor of the CD33-related family of sialic acid-binding, Ig-like lectins (Siglecs) that is expressed selectively on the surface of mature MC, eosinophils, and basophils. Engagement of Siglec-8 with monoclonal antibodies has been previously shown to inhibit IgE-mediated MC degranulation and to induce apoptosis of cytokine-activated eosinophils. Thus this receptor is a potential target for antibody therapy of SM with or without associated eosinophilia. Anti-Siglec-8 antibodies do not directly affect MC viability but antibodies with effector function can induce antibody cell-mediated cytotoxicity (ADCC). Here we show that a recombinant anti-Siglec-8 IgG1 monoclonal antibody can elicit ADCC activity against MC derived from SM patients ex vivo. Methods: Bone marrow (BM) aspirates from SM patients were evaluated for Siglec-8 cell-surface expression on CD117+ FcεRI+ MC or CD25+ MC by flow cytometry. For ADCC assays, BM MC enriched using CD117-targeting magnetic beads or a Siglec-8 transfected Ramos cell line were used as target cells. Peripheral blood leukocytes (PBL) or NK cells purified from peripheral blood were used as effector cells at an effector:target ratio of 10:1. Recombinant anti-Siglec-8 antibody or an isotype control antibody was added at various concentrations and the percent viable CD117+ FcεRI+ MC remaining after 48 hours of culture was determined by flow cytometry. Results: Samples from 9 pts with SM were included in the analysis (ISM, n=1; SSM, n=1; SM-CMML, n=3; SM-MDS, n=1; SM-CEL, n=1; ASM, n=1; MCL, n=1). Eight pts were KIT D816V positive. At the time of sample collection, treatments included midostaurin (n=3); cladribine (n=1); corticosteroids (n=1); and 4 pts were not receiving any biologic or cytoreductive therapy. All BM samples showed detectable CD117+ MC. Robust and selective cell-surface expression of Siglec-8 was observed in all 6 cases evaluated and 100% of CD117+ FcεRI+ MC were Siglec-8 positive by FACS, including CD25+ MC. Levels of Siglec-8 were comparable to or higher than levels on mature MC isolated from normal skin. In this limited sample size, no difference in Siglec-8 expression was observed between patients receiving different therapies or no therapy. To evaluate the ADCC activity of recombinant anti-Siglec-8 antibody on MC, enriched BM MC were incubated with anti-Siglec-8 antibody or isotype-matched control antibody at 1 μg/ mL in the presence of purified NK effector cells. In two patients evaluated, significant anti-Siglec-8-mediated ADCC activity on MC was observed using non-autologous NK cells (69% reduction, 1 pt) or autologous NK cells (76% reduction, 1 pt) indicating that anti-Siglec-8 has the potential to reduce MC burden in these patients. ADCC activity has been reported to be defective in some cancer patients. To evaluate the ability of effector cells in SM patients to mediate ADCC, an assay was developed using a Siglec-8 transfected target cell line to screen blood samples for ADCC activity induced by anti-Siglec-8 antibody. Using PBL as effector cells, ADCC was observed in all samples tested (5/5). Titration of antibody was performed on 2 samples and potent ADCC activity was observed in both, with an EC50 for target depletion of 49 and 65 ng/mL of anti-Siglec-8 antibody, respectively. Conclusion: These data provide a strong rationale for evaluating the effect of an antibody to Siglec-8 with ADCC activity in patients with SM. Disclosures Falahati: Allakos, Inc.: Employment, Other: Options for Equity Owernship. Bright:Allakos, Inc.: Employment, Other: Options for Equity Owernship. Dorenbaum:Allakos, Inc.: Employment, Equity Ownership. Bebbington:Allakos, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tomasevic:Allakos, Inc.: Employment, Equity Ownership. George:Allakos: Research Funding; Novartis: Consultancy. Gotlib:Allakos, Inc.: Consultancy.

scholarly journals CD33 SNP Genotype and Splice Variation Are Associated with CD33 Cell Surface Expression and SGN-CD33A Pharmacokinetics

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3942-3942
Author(s):  
Katherine Tarlock ◽  
Zixing Wang ◽  
Rory Rohm ◽  
Travis Biechele ◽  
Rhonda E. Ries ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Drug Exposure ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Set Domain ◽  
Employment Equity ◽  
Exon 2 ◽  
Splice Variation ◽  
Equity Ownership

Abstract The cell surface antigen CD33 is expressed on the majority of AML blasts and is appropriate for immunotherapeutic targeting with antibody drug conjugates (ADCs). Expression of CD33 is in part mediated by splicing of the CD33 transcript, and has been demonstrated to be one of the factors that may mediate response to the ADC gemtuzumab ozogamicin, which results in significant benefit in some patients but lacks responses in others. Splicing of the CD33 transcript is in part regulated by a single nucleotide polymorphism (SNP) in exon 2 (e2) that causes a C>T substitution and the resultant skipping of e2. CD33 thus exists in 2 main isoforms, as either a full length (FL) transcript or a truncated version missing e2 (Δe2), which includes the IgV binding domain that is the epitope for diagnostic and therapeutic antibodies (Ab). The CC genotype encodes the FL isoform at a higher rate compared to the CT or TT, and the TT genotype encodes the short isoform at a higher rate compared to CT or CC. SGN-CD33A is a CD33-directed ADC, utilizing a pyrolobenzodiazepine (PBD) dimer. SGN-CD33A has been evaluated in multiple clinical trials as either monotherapy or in combination. We hypothesized that the patient's CD33 genotype would impact CD33 expression as well as response characteristics following treatment with SGN-CD33A. We analyzed CD33 genotype variation in bone marrow (BM) or peripheral blood (PB) samples from patients treated with SGN-33A as either monotherapy (NCT01902329; n=133) or in combination with hypomethylating agents (HMAs; NCT02785900; n=83). CD33 SNP genotyping was determined on gDNA using RFLP PCR with 2 restriction enzymes recognizing cut sites generated by the C and T polymorphisms and genotype confirmed using fragment length analysis (CC=108, CT=86, TT=22). CD33 surface expression on the AML blasts was determined by flow cytometric analysis using the human anti-CD33 monoclonal Abs HIM3-4 and H212, which bind to the membrane-proximal C2-set and V-set domain, respectively. HIM-34 measured CD33 levels independent of SNP-driven splice variation. The h2H12 epitope is within e2, thus its binding may be susceptible to splice variation. We subsequently evaluated the association of CD33 genotype with pharmacokinetic (PK), clinical and other variables using a generalized regression model. Patients classified as CC genotype had significantly higher surface CD33 expression as determined by flow cytometry in both BM and PB. In accordance with observed differences in CD33 expression, we also found drug exposure demonstrated an inverse relationship according to CC genotype in both mono and combination therapy trials. For monotherapy, compared to patients with CC and CT genotypes, patients with TT genotypes had significantly higher drug exposure following SGN-CD33A. Patients with TT had higher AUCs following the first and last doses of SGN-33A (p < 10-4 -; Fig 1). Cmax following SGN-CD33A exposure was higher in TT genotype patients compared to the CT and CC (p< 10-1.5 for Cmax following the first dose and p<10-1.6 for Cmax over all treatments;Fig 1). In combination with HMAs, the TT genotype was also associated with significantly higher SGN-CD33A AUC and Cmax (p-values ranging from 10-3.3 - 10-9.7). We next examined expression and subcellular localization of CD33 to elucidate the mechanism by CD33 variation contributes to cell surface presentation. Transfection of cDNA encoding the FL CD33 transcript resulted in increased cell surface expression, as indicated by flow cytometry with both HIM3-4 and h2H12. In contrast, both Abs failed to detect cell surface CD33 following transfection with cDNA encoding the Δe2 variant. Examination of the intracellular compartment revealed that HIM3-4, but not 2H12, binds to the Δe2 variant in a pattern localized proximal to the nucleus. Taken together, our findings suggest that the Δe2 splice CD33 variant lacks the portion of the V-set domain required for h2H12/SGN-CD33A binding and does not efficiently traffic to the cell surface. We show that CD33 SNP genotype is associated with CD33 expression, with CC patients demonstrating higher CD33 as detected by flow cytometry; and that CD33 SNP genotype affects the PK profile of SGN-CD33A, with TT patients having higher levels of drug exposure. Our findings suggest that the CD33 genotype can impact CD33 expression, PK profile, and trafficking of bound agents and thus may impact therapeutic targeting of CD33-directed agents. Disclosures Wang: Seattle Genetics: Employment, Equity Ownership. Rohm:Seattle Genetics: Employment, Equity Ownership. Biechele:Seattle Genetics: Employment, Equity Ownership. Means:Seattle Genetics: Employment, Equity Ownership. Thurman:Seattle Genetics: Employment, Equity Ownership. Arthur:Seattle Genetics: Employment, Equity Ownership.

Correlation of CD30 Expression on Neoplastic Mast Cells in Systemic Mastocytosis Assessed By Immunohistochemistry Versus Multiparameter Flow Cytometry and Correlation to Clinical Parameters

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1616-1616
Author(s):  
Frauke Bellos ◽  
Karl Sotlar ◽  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mast Cell ◽  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Melting Curve ◽  
Normal Karyotype ◽  
Multiparameter Flow Cytometry ◽  
Employment Equity ◽  
Equity Ownership ◽  
Kit D816v

Abstract Background: Systemic mastocytosis (SM) is rarely diagnosed and presents with highly variable clinical manifestation from taking rather indolent to very aggressive courses, mast cell leukemia being the most aggressive variant. Expression of CD30 (Ki-1 antigen) has been detected on some neoplastic mast cells (MC) by immunohistochemical staining (IHC) in bone marrow (BM) biopsies and presence of CD30 has been reported to be correlated to more aggressive variants of SM. Assessmentof CD30 expression by multiparameter flow cytometry (MFC) might not only contribute to improved diagnostic accuracy but correlation of the hereby detected CD30 expression with clinical SM parameters might also give further insights into disease biology. Aims: Comparison of CD30 expression detected by either MFC or IHC on MC of patients with SM and correlation of results with patient and disease characteristics. Correlation of MFC detected CD30 expression with cytogenetics (CG) determined by chromosome banding analysis and molecular genetics (MG). Methods: For this study, CD30 expression was analyzed in BM samples from 93 patients with SM by MFC using a five color staining assay with monoclonal antibodies against CD30, CD45, CD117, CD2 and CD25. We identified MC based on CD45 positivity and bright expression of CD117. Based on aberrant coexpression of CD2 and/or CD25 neoplastic MC were identified. On those MC, mean and median fluorescence intensities (MFI, medFI) of CD30 were determined and related to CD30 MFI and medFI in lymphocytes to derive CD30 index. Moreover, data on MC infiltration and CD30 expression by IHC was assessed in 22 patients and examination of CG and MG was done in 44 and 80 patients, respectively. KIT D816V mutation was analyzed using melting curve-based DNA mutation analysis applying PNA-mediated PCR clamping according to Sotlar et al. [Am J Pathol. 162: 737-746, 2003]. Results of MFC detected CD30 expression was correlated to those of CD30 expression examined with IHC and to the results of CG and MG. Results: 42 patients were female and 51 male. Median age was 59 years (20-87 years). While we found normal karyotypes in 41 patients, 3 patients showed aberrant karyotypes. KIT D816V mutation was seen in 74/80 patients (93%). Diagnosis of concurrent hematological non-mast cell disease (AHNMD) was made in 14/93 patients (15%). Mean (±SD) MC infiltration was 20%±26% (range, 1.5%-85%) by IHC and 0.4%±1.8% (range, 0.01%-17%) by MFC. Mean (±SD) CD30 index was 19±20 (range, 3-154), mean (±SD) CD30 expression by IHC was 9%±17% (range, 0%-70%). Percentages of MC infiltration detected by IHC and MFC correlated significantly (p=0.002, r=0.819). No correlation of MFC CD30 index (MFI and medFI) with age, sex, concomitant AHNMD, grade of MC infiltration or percentage of CD30 positive MC by IHC was found. Interestingly, a significantly higher medFI CD30 index and a trend to higher MFI CD30 index were seen in patients with normal karyotype (12.2±6.2 vs 6.3±2.7, p=0.037 and 15.1±9.4 vs. 11.6±10.3, n.s., respectively) versus those with an aberrant karyotype. We also detected a trend to higher MFI and medFI CD30 index in patients with KIT D816V mutation (19.8±21.8 vs. 10.2±7.4, n.s. and 24.0±89.0 vs 10.0±5.8, n.s., respectively). Conclusions: Assessment of CD30 expression as a dynamic parameter on neoplastic MC in patients with SM can be reliably performed by MFC. A stronger expression of CD30 expression on neoplastic MC harbouring an aberrant karyotype was found. CD30 expression seems also stronger on MC from patients harbouring KIT D816V mutations compared to those who do not. CD30 expression on neoplastic MC in patients with SM should be further analyzed combining analyses by MFC and IHC to substantiate the present findings. Disclosures Bellos: MLL Munich Leukemia Laboratory: Employment. Sotlar:Ludwig-Maximilians-University: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals Pharmacokinetic and Pharmacodynamic Study of NKTR-255, a Polymer-Conjugated Human IL-15, in Cynomolgus Monkey

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2952-2952
Author(s):  
Takahiro Miyazaki ◽  
Peiwen Kuo ◽  
Mekhala Maiti ◽  
Palakshi Obalapur ◽  
Murali Addepalli ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cynomolgus Monkey ◽  
Memory T Cells ◽  
Granzyme B ◽  
Employment Equity ◽  
Equity Ownership ◽  
Dose Dependent

Abstract Introduction IL-15 is a common gamma chain cytokine that activates and provides a survival benefit to T-cells and NK cells and has long been recognized as having potential as an immunotherapeutic agent for the treatment of cancer. Therapeutic use of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic and safety properties. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the biological effects of NKTR-255 in naïve cynomolgus monkey. Methods In vitro monkey whole blood was treated with NKTR255 and the percentage of pSTAT5 positive populations in each NK, CD4 T and CD8 T cells was determined by flow cytometry. In an PK/PD study, monkeys received single IV doses of 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg NKTR-255. Blood samples were collected to determine the plasma concentrations of NKTR-255 and to assess the effects of NKTR-255 on NK and CD8 T cells at multiple time points; flow cytometry was used to measure STAT5 phosphorylation, Ki-67 expression and frequency of cell populations. Granzyme B expression was assessed in NK and CD8 T cells by flow cytometry. Results NKTR-255 induced dose-dependent phosphorylation of STAT5 in monkey whole blood (EC50 values NK cells: 6.9 ng/ml, CD8 T cells: 39 ng/ml, CD4 T cells: 53 ng/ml). The half-life and clearance of NKTR-255 were 26x longer and 38x lower, respectively, than IL-15. NKTR-255 engaged the IL-15 signaling pathway, in vivo, demonstrating both robust and sustained STAT5 phosphorylation in lymphocytes. NKTR-255 drove the proliferation of total CD8 T cells and NK cells in a dose-dependent manner, with dramatic and durable increases observed in Ki67 positive population and absolute cell numbers (NK cells: 6.1 fold; CD8 T cells: 7.8 fold from baseline on day 5 at 0.1 mg/kg). These effects were strongly biased towards CD8 T cells and NK cells, with substantially less induction of CD4 T cells. The Ki67 response analyses of the T cell subpopulation revealed a higher response of memory populations than for naive T cells. Among memory T cells, effector memory T cells showed the highest response over stem cell memory T cells and central memory T cells. Finally, NKTR-255 also increased the expression of Granzyme B in both NK and CD8 T cells, concomitant with an enhancement in target cell lysis. Conclusions Nektar has generated a novel and potent molecule in NKTR-255 that not only preserves the relevant biology of IL-15, but additionally provides enhanced PK and PD properties relative to the native IL-15 cytokine. NKTR-255 is being developed as an immune-stimulatory agent to target NK and CD8 T cell biology for the treatment of cancer. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kuo:Nektar Therapeutics: Employment, Equity Ownership. Maiti:Nektar Therapeutics: Employment, Equity Ownership. Obalapur:Nektar Therapeutics: Employment, Equity Ownership. Addepalli:Nektar Therapeutics: Employment, Equity Ownership. Rubas:Nektar Therapeutics: Employment, Equity Ownership. Sims:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

scholarly journals Immune Microenvironment Analysis of Bone Marrow By Mass Cytometry and RNA Sequencing in Multiple Myeloma Patients Treated with Daratumumab and Durvalumab

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3296-3296 ◽  
Author(s):  
Frances Seymour ◽  
Mary H Young ◽  
Mark Tometsko ◽  
Jamie Cavenagh ◽  
Ethan G. Thompson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Research Funding ◽  
Cell Populations ◽  
Mass Cytometry ◽  
Immune Microenvironment ◽  
Employment Equity ◽  
Equity Ownership

Abstract Introduction Relapsed and refractory multiple myeloma (RRMM) remains a challenging disease to treat due to its heterogeneity and complexity. There is an urgent need for novel combination strategies, including immunotherapy. The study of the tumour and immune microenvironment before and after treatment with combination therapy is a crucial part of understanding the underpinning of disease response. Methods Longitudinal samples of bone marrow aspirates and whole blood were collected from a phase II clinical trial, MEDI4736-MM-003 (NCT02807454) where daratumumab and durvalumab naïve patients were exposed simultaneously to both these drugs. A combination of mass cytometry (CyTOF), RNAseq and flow cytometry were performed on a subset of samples from these subjects. Specifically, paired bone marrow mononuclear cells (BMMC) samples from nine patients taken at screening and six weeks post-treatment were analysed by mass cytometry (CyTOF) using a 37-marker pan-immune panel that included both lineage and functional intracellular/extracellular markers. In addition, whole blood sample specimens were collected at screening and on treatment (8, 15, 30, and 45 days after treatment) and analysed by flow cytometry. Flow cytometry panels were designed to allow interrogation of the abundance and activation status of immune cell subsets. Finally, RNA from bone marrow aspirates at screening and C2D15 were analysed by RNA sequencing. Expression profiles from the aspirates were used to estimate cell proportions by computational deconvolution. Individual cell types in these microenvironments were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results In a heavily pre-treated population with RRMM, treatment with durvalumab and daratumumab leads to shifts in a number of key immunological populations when compared to pre-treatment. In the bone marrow, CD8 and CD4 populations rise (by CyTOF and RNAseq), while NK, DC and B cell populations fall (by CyTOF). In the bone marrow within CD8+ T lymphocyte populations, we observed a post-treatment rise in markers of degranulation (granzyme p=0.0195, perforin p=0.0078, Wilcoxon signed-rank test). This is also accompanied by a fall in PD1 expression (p=0.0078) and rise in the co-stimulatory receptor DNAM1 (p=0.0273). These changes are most marked on cells with an effector memory CD45RA+ CD8+ T cell phenotype. In the blood, similar to the bone marrow, CD8+ T cells proliferate over the course of treatment (flow cytometry). A fall in both naïve and active NK cell populations is seen following treatment in bone marrow. NK cells express high levels of CD38 and are therefore depleted by daratumumab. Those NK cells which remain have an active phenotype with increased expression of TNFa (p=0.0039) and IFNg (p=0.0195) following treatment. Across the time points sampled in peripheral blood, NK cells were also decreased and those that remained were proliferating. Dendritic cells with a tolerogenic phenotype can be identified prior to treatment and are seen to fall in abundance following treatment with durvalumab and daratumumab. Conclusions The combination of durvalumab and daratumumab leads to several immune microenvironment changes that biologically portend clinical effect. We see increases in the abundance of cell populations with functional anti-tumour activity, including granzyme B+ CD8 T cells and a reduction in PD1high T cells. Despite the treatment expectedly reducing NK cell numbers, many functionally competent NK cells remain, as evidenced by the presence of anti-tumour cytokines. This combination strategy also reduces immunosuppressive tolerogenic DCs, which suppress CD4 and CD8 T cell activity. Taken together, this suggests that this chemotherapy free, doublet treatment has the potential to up-regulate anti-tumour immunological responses, which may restore immunosurveillance mechanisms critically needed in these highly refractory patients. Disclosures Seymour: Celgene: Research Funding. Young:Celgene Corporation: Employment, Equity Ownership. Tometsko:Celgene Corporation: Employment, Equity Ownership. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Thompson:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Danziger:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Dervan:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Gribben:Acerta Pharma: Honoraria, Research Funding; Cancer Research UK: Research Funding; TG Therapeutics: Honoraria; Roche: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Unum: Equity Ownership.

Targeting Immune Suppressive Microenvironment By Immune Checkpoint Blockade in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 27-27 ◽  
Author(s):  
Gullu Topal Gorgun ◽  
Kristen Cowens ◽  
Steven Paula ◽  
Mehmet Kemal Samur ◽  
Hiroto Ohguchi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Growth ◽  
Immune Responses ◽  
Tumor Cell ◽  
Nk Cells ◽  
Effector Cell ◽  
Cell Surface Expression ◽  
Tumor Cell Growth ◽  
Flow Cytometry Analysis ◽  
Effector Cells

Abstract Bidirectional interaction between MM cells and accessory cells regulates tumor development on the one hand, while transforming the BM microenvironment into a tumor promoting and immune suppressive milieu on the other. Recent developments in targeted therapies have indicated that generation of the most effective therapeutic strategies requires not only targeting tumor or stroma, but also methods to overcome blockade of anti-tumor immune responses. Tumor associated immune suppressor cells such as Treg and myeloid derived suppressor cells (MDSC) can effectively block anti-tumor immune responses, thereby representing an important obstacle for immunotherapy. Co-inhibitory molecules programmed cell death-1 (PD-1) and its ligand (PD-L1) play a fundamental role in tumor immune escape by inhibiting immune effector functions. Since PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell mediated anti-tumor immune responses, we here assessed the impact of PD-1/PD-L1 blockade, alone or in combination with lenalidomide (Len), on accessory and immune cells function, as well as tumor cell growth of MM in the BM milieu. Methods: PD-L1 gene expression was determined in IFM MM data set (n=170). Cell surface expression of PD-L1 in CD138+ MM cells, stroma, and MDSCs; as well as PD-1 expression on effector cells (CD4T, CD8T, NK, NKT and monocytes/macrophages) were determined in the bone marrow (BM) and peripheral blood (PB) of patients with MGUS (n=3), newly diagnosed (ND-MM, n=5) and relapsed/refractory (RR-MM, n=11) compared to healthy donors (HD, n=10). PD-1/PD-L1 signaling was assessed in the autologous cocultures of patient MM cells or MM cell lines with stroma (BMSC) or MDSCs and effector cells, in the presence or absence of PD-1/PD-L1 blockade and Len. Len effect on PD-1 expression on effector cells and PD-L1 expression on tumor, BMSC and MDSCs was determined by flow cytometry analysis. BMSC and MDSC induced MM growth/viability was measured by MTT, 3H-Thy and CFSE flow cytometry analysis. Effector cell-mediated MM cytotoxicity was measured by CFSE/PI flow cytometry. The effect of PD-1/PD-L1 blockade with or without Len on cytokine pattern was determined by intracellular cytokine flow cytometry analysis. Results: Statistical analysis of IFM MM data demonstrated that the majority of patient MM cells have increased PD-L1 mRNA compared to HD (p=0.0064). Cell surface expression of PD-L1 was also significantly increased in ND-MM cells (median 65%) and even higher in RR-MM cells. Correspondingly, there was a significant increase in PD-1 expression on CD8T and NK cells in ND-MM and RR-MM. Moreover, PD-L1 expression was significantly higher on both mMDSC and nMDSC than APCs in ND-MM and RR-MM. Coculture with BMSC significantly increased expression of PD-L1 on MM cells. PD-1/ PD-L1 blockade overcame BMSC-induced tumor cell growth in both patient MM cells and MM cell lines. Importantly, Len significantly reduced PD-L1 expression on MM cells; and combined blockade of PD-1/PD-L1 with Len further decreased BMSC-induced MM growth. Immunomodulatory effects of PD-1/PD-L1 blockade were also evaluated in autologous cocultures of immune effector cells with MM cells. Even though there was no change in effector cell proliferation, PD-1/PD-L1 blockade significantly induced cytotoxic activity of autologous T cells, NK cells, and macrophages cultured with MM cells; and Len further enhanced effector cell-mediated cytotoxicity. PD-1/PD-L1 blockade induced intracellular expression of cytotoxic cytokines IFNg and Granzyme B (Gzm B) in CD4T cells, CD8T cells, NK cells and Macrophages. Furthermore, MDSC-mediated MM growth was significantly decreased by PD-1/ PD-L1 blockade. Finally, PD-1/PD-L1 blockade induced intracellular expression of IFNg and Gzm B in T cells, NK cells and NKT cells cultured with autologous MDSC; and Len further enhanced this effector cell activation. Conclusion: Our data demonstrated that immune checkpoint signaling plays an important role in providing the tumor promoting, immune suppressive microenvironment in MM. Blockade of PD-1/PD-L1 signaling induces anti-MM immune responses that can be enhanced by Len. Targeting checkpoint signaling using PD-1 and PD-L1 blocking antibodies, particularly in combination with Len, therefore represents a promising novel immune-based therapeutic strategy to both inhibit tumor cell growth and restore host immune function in MM. Disclosures Kikuchi: The ITO Foundation for the Promotion of Medical Science: Research Funding. Hideshima:Acetylon Pharmaceuticals: Consultancy. Raje:novartis, Amgen, Celgene, Millenium, Onyx: Consultancy; Eli Lilly, Acetylon: Research Funding. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

First Selective KIT D816V Inhibitor for Patients with Systemic Mastocytosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3217-3217 ◽  
Author(s):  
Erica K. Evans ◽  
Brian L. Hodous ◽  
Alexandra Gardino ◽  
Julia Zhu ◽  
Adam Shutes ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Systemic Mastocytosis ◽  
Tumor Growth Inhibition ◽  
Employment Equity ◽  
Equity Ownership ◽  
Kit D816v

Abstract Systemic mastocytosis is a disease characterized by the abnormal proliferation and accumulation of mast cells. In aggressive cases, these mast cells accumulate in organs such as bone marrow, liver and spleen and result in compromised organ function with average patient survival only 3 to 5 years after diagnosis. The mast cells of nearly all systemic mastocytosis patients harbor a heterozygous D816V mutation in the activation loop of KIT conferring constitutive, ligand-independent activation of this receptor tyrosine kinase, suggesting this mutation is a driver of disease. While KIT D816V can be targeted by small molecules such as dasatinib and midostaurin, these agents have activity against many human kinases resulting in dose limiting toxicities in the clinic that prevent complete suppression of KIT D816V activity in vivo. In vitro, their potent activity against multiple kinases leads to uncertainties regarding their mechanism of action. Thus far, selective inhibition of the KIT D816V mutation has not been achieved. However starting with a novel chemical library optimized for kinase selectivity, we have identified BLU-285, a small molecule inhibitor targeting KIT exon 17 mutants including the activated KIT D816V kinase. BLU-285 potently disrupts KIT D816V oncogenic signaling as measured by inhibition of both KIT D816V autophosphorylation and phosphorylation of the downstream substrates Akt and Stat3 in the human mast cell leukemia cell line HMC1.2. In vitro, BLU-285 inhibits proliferation and induces apoptosis in the mouse mastocytoma cell line P815. In vivo, BLU-285 is a well-tolerated, orally bioavailable agent that achieves dose dependent tumor growth inhibition in a P815 mouse xenograft model with tumor regression observed at 30 mg/kg once daily dosing. Tumor growth inhibition correlates with inhibition of KIT autophosphorylation; greater than 80% target suppression throughout the 24-hour dosing period is required for effective tumor growth inhibition. Prolonged target suppression is achievable with BLU-285 but not dasatinib, even when dosed at the MTD in mouse. Furthermore, to more closely mimic the nature of systemic mastocytosis, we have developed a disseminated model of disease whereby the in vivo growth of P815-luciferase expressing cells inoculated intravenously can be measured by whole body bioluminescence. Treatment of mice with systemic disease leads to dose dependent inhibition of disease, with a 3-fold increase in survival time when dosed 30 mg/kg QD. In addition, as anticipated by its selectivity profile, BLU-285 is very well tolerated in vivo with no impact on body weight at efficacious doses. Our data demonstrate that selective inhibition of KIT D816V with BLU-285 achieves complete and prolonged inactivation of the disease-driving kinase and suggests that BLU-285 may provide a compelling new therapy for patients with systemic mastocytosis. Disclosures Evans: Blueprint Medicines: Employment, Equity Ownership. Hodous:Blueprint Medicines: Employment, Equity Ownership. Gardino:Blueprint Medicines: Employment, Equity Ownership. Zhu:Blueprint Medicines: Employment, Equity Ownership. Shutes:Blueprint Medicines: Employment, Equity Ownership. Davis:Blueprint Medicines: Employment, Equity Ownership. Kim:Blueprint Medicines: Employment, Equity Ownership. Wilson:Blueprint Medicines: Employment, Equity Ownership. Wilson:Blueprint Medicines: Employment, Equity Ownership. Zhang:Blueprint Medicines: Employment, Equity Ownership. Kohl:Blueprint Medicines: Employment, Equity Ownership. Guzi:Blueprint Medicines: Employment, Equity Ownership. Lengauer:Blueprint Medicines: Employment, Equity Ownership.

Impact of β2 integrin deficiency on mouse natural killer cell development and function

Blood ◽  
2011 ◽  
Vol 117 (10) ◽  
pp. 2874-2882 ◽  
Author(s):  
Karine Crozat ◽  
Céline Eidenschenk ◽  
Baptiste N. Jaeger ◽  
Philippe Krebs ◽  
Sophie Guia ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Maturation ◽  
Cell Surface Expression ◽  
Mcmv Infection ◽  
Β2 Integrin

Abstract Natural killer (NK) cells are innate immune cells that express members of the leukocyte β2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that β2 integrin–deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit+ cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, β2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

scholarly journals Physical dissociation of the TCR-CD3 complex accompanies receptor ligation.

1995 ◽  
Vol 182 (6) ◽  
pp. 1997-2006 ◽  
Author(s):  
H Kishimoto ◽  
R T Kubo ◽  
H Yorifuji ◽  
T Nakayama ◽  
Y Asano ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Signal Transduction Pathway ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Levels ◽  
Before And After ◽  
Physical Dissociation ◽  
Receptor Ligation

Recent studies indicate that there may be functional uncoupling of the TCR-CD3 complex and suggest that the TCR-CD3 complex is composed of two parallel signal-transducing units, one made of gamma delta epsilon chains and the other of zeta chains. To elucidate the molecular mechanisms that may explain the functional uncoupling of TCR and CD3, we have analyzed their expression by using flow cytometry as well as immunochemical means both before and after stimulation with anti-TCR-beta, anti-CD3 epsilon, anti-CD2, staphylococcal enterotoxin B, and ionomycin. We present evidence that TCR physically dissociates from CD3 after stimulation of the TCR-CD3 complex. Stimulation with anti-CD3 resulted in down-modulation of TCR within 45 min whereas CD3 epsilon was still expressed on the cell surface as detected by flow cytometry. However, the cell surface expression of TCR and CD3 was not affected when cells were stimulated with anti-TCR-beta under the same conditions. In the case of anti-CD3 treatment of T cells, the TCR down-modulation appeared to be due to the internalization of TCR, as determined by immunoelectron microscopy. Immunochemical analysis of cells after stimulation with either anti-TCR or anti-CD3 mAbs revealed that the overall protein levels of TCR and CD3 were similar. More interestingly, the dissociation of the TCR-CD3 complex was observed with both treatments and occurred in a manner that the TCR and the associated TCR-zeta chain dissociated as a unit from CD3. These results provide the first report of physical dissociation of TCR and CD3 after stimulation through the TCR-CD3 complex. The results also suggest that the signal transduction pathway triggered by TCR may differ from that induced by CD3.

Kaposi’s sarcoma-associated herpesvirus ubiquitin ligases downregulate cell surface expression of l-selectin

2021 ◽  
Vol 102 (11) ◽  
Author(s):  
Mizuho Kajikawa ◽  
Nanae Imaizumi ◽  
Shiho Machii ◽  
Tomoka Nakamura ◽  
Nana Harigane ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Immune Evasion ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Ubiquitin Ligases ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic etiological factor for Kaposi’s sarcoma and primary effusion lymphoma in immunocompromised patients. KSHV utilizes two immune evasion E3 ubiquitin ligases, namely K3 and K5, to downregulate the expression of antigen-presenting molecules and ligands of natural killer (NK) cells in the host cells through an ubiquitin-dependent endocytic mechanism. This allows the infected cells to evade surveillance and elimination by cytotoxic lymphocytes and NK cells. The number of host cell molecular substrates reported for these ubiquitin ligases is limited. The identification of novel substrates for these ligases will aid in elucidating the mechanism underlying immune evasion of KSHV. This study demonstrated that K5 downregulated the cell surface expression of l-selectin, a C-type lectin-like adhesion receptor expressed in the lymphocytes. Tryptophan residue located at the centre of the E2-binding site in the K5 RINGv domain was essential to downregulate l-selectin expression. Additionally, the lysine residues located at the cytoplasmic tail of l-selectin were required for the K5-mediated downregulation of l-selectin. K5 promoted the degradation of l-selectin through polyubiquitination. These results suggest that K5 downregulates l-selectin expression on the cell surface by promoting polyubiquitination and ubiquitin-dependent endocytosis, which indicated that l-selectin is a novel substrate for K5. Additionally, K3 downregulated l-selectin expression. The findings of this study will aid in the elucidation of a novel immune evasion mechanism in KSHV.

scholarly journals Ex Vivo Activity of Immunotherapeutic Approaches Targeting CD38 Against Daratumumab-Resistant Multiple Myeloma Patient Samples

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1848-1848
Author(s):  
Maria Karvouni ◽  
Heyue Zhou ◽  
Arnika Kathleen Wagner ◽  
Qiangzhong Ma ◽  
Alamdar H. Baloch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Nk Cells ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Phase 1 ◽  
Employment Equity ◽  
Multiple Myeloma Patient ◽  
Myeloma Cells ◽  
Car T ◽  
Equity Ownership

Background: Multiple myeloma (MM) is a plasma cell malignancy that remains incurable. The identification of CD38, a transmembrane glycoprotein overexpressed on MM cells, led to the development of target-specific therapeutics such as the FDA approved monoclonal antibody (mAb) Daratumumab (DARA). Although a valuable treatment option for refractory/relapsed (R/R) MM patients, DARA has a limited response rate of below 50%, which highlights the clinical need for novel therapeutics. Aims: Aiming to further exploit the therapeutic potential of CD38 in the MM setting, immunotherapies based on the novel anti-CD38 mAb CD38A2 were tested. Methods: For the first approach, the CD38A2 mAb -that binds to a unique, distinct from DARA's, CD38 epitope- was conjugated with either the alkylating agent Duomycin (ADC-136) or the microtubulin binder Duostatin (ADC-129). The ADCs were compared to DARA, in cultures of primary MM cells from patients refractory to DARA treatment. In a second approach, a chimeric antigen receptor (CAR) consisting of the CD38A2 scFv and the intracellular domains of CD28 and CD3ζ was used to transduce primary T and NK cells from R/R MM patients. The functionality of the CAR-T and CAR-NK cells was assessed in cytotoxicity assays against autologous myeloma cells. Results: ADC-136 demonstrated the most potent cytotoxicity against the MM cells with an IC50 of 6pM at day 6 following a single dose treatment. ADC-129 showed cell killing with an IC50 of 30pM, while DARA did not exhibit appreciable cytotoxicity. Regarding the cell therapy approach, patients' T and NK cells were effectively transduced, showing a CD38A2-CAR expression ranging between 11-68%. In functional assays, CAR-T and CAR-NK cells were assayed against autologous myeloma cells, where they exhibited an increase in target cell cytotoxicity, compared to the untransduced cells. Summary/Conclusion: Altogether, our preliminary findings demonstrate that CD38 targeting using CD38A2-based immunotherapies could be a viable therapeutic approach in R/R MM patients previously exposed to DARA. Currently, an anti-CD38 CAR-T therapy based on CD38A2 is being evaluated in Phase 1 studies in R/R MM patients by Sorrento Therapeutics, Inc. Disclosures Zhou: Sorrento Therapeutics Inc: Employment, Equity Ownership. Ma:Sorrento Therapeutics Inc: Employment, Equity Ownership. Zhu:Sorrento Therapeutics Inc: Employment, Equity Ownership. Zhang:Sorrento Therapeutics Inc: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

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