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

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.

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

Blood ◽

10.1182/blood.v126.23.4092.4092 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4092-4092 ◽

Cited By ~ 2

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.

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

Journal of Experimental Medicine ◽

10.1084/jem.182.6.1997 ◽

1995 ◽

Vol 182 (6) ◽

pp. 1997-2006 ◽

Cited By ~ 40

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.

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CD33 Splicing Polymorphism Is a Strong Predictor of Therapeutic Efficacy of Gemtuzumab Ozogamicin in De Novo AML: Report from COG-AAML0531 Study

Blood ◽

10.1182/blood.v128.22.2743.2743 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2743-2743

Author(s):

Jatinder K Lamba ◽

Lata Chauhan ◽

Michael R. Loken ◽

Jessica Pollard ◽

Yi-Cheng Wang ◽

...

Keyword(s):

Cell Surface ◽

Binding Site ◽

Surface Expression ◽

Gemtuzumab Ozogamicin ◽

Genotype Frequency ◽

Cell Surface Expression ◽

Exon 2 ◽

Transcript Levels ◽

Risk Patients ◽

Splice Isoform

Abstract Gemtuzumab ozogamicin (GO), a CD33-targeted immunoconjugate, is a re-emerging as therapeutic for AML. We have previously discovered polymorphisms in CD33 coding region that might be associated with outcome in patients treated with GO. One particular coding polymorphism, CD33-SNP rs12459419-C>T (Ala14Val), is located within the splice enhancer region of exon-2, leading to expression of an alternate splice isoform lacking exon-2. This alternate splice isoform (D2-CD33), would encode a protein product lacking the IgV domain, which is the binding site for GO and most if not all CD33 antibodies used for diagnostic immunophenotyping. We therefore hypothesized that the SNP rs12459419 genotype would be associated with differential expression of the D2-CD33 transcript levels and differential cell surface CD33 expression leading to genotype determined differential response to GO. We evaluated the genotype frequency and functional significance of rs12459419, its association with CD33 cell-surface expression on leukemic blasts, and clinical response in 816 children and young adults with AML randomized to GO vs. No-GO in the COG trial AAML0531. The CD33 SNP rs12459419 genotype frequency was-CC=51%, CT=39% and TT=10% in patients, similar to the observed frequency in the general population. Correlation of SNP allele frequency with CD33 transcript levels and surface CD33 expression (as determined by p67.6 antibody) demonstrated that the T-allele was significantly associated with higher levels of D2-CD33 transcript (P=4.7e-11, Figure 1) and with lower diagnostic leukemic cell CD33 surface intensity (P=1.93e-29). Clinical outcome based on the SNP genotype demonstrated that patients with CC-genotype had significantly lower RR of 26%±7% in the GO arm whereas those in the No-GO arm had a RR of 49%±9% (HR=0.468, P<0.001). The corresponding DFS for CC genotype patients in the GO and No-GO arms was 65%±7% and 46%±9%, respectively (HR=0.597, P=0.004; Figure 1). In contrast, in those with heterozygous CT or homozygous TT genotype, GO exposure provided no clinical benefit in RR (CT: 38%±9% vs. 37±10%, P=0.975; TT: 46%±20% vs. 46%±20%, P=0.798) nor DFS (CT: 56%±9% vs. 60%±10% GO vs. No-GO, P=0.821; TT: 51%±20% vs. 54%±18%, GO vs. No-GO, P=0.972, Figure 1). We further evaluated the impact of the CD33 genotype on the efficacy of GO in different risk groups as well as in high vs. low CD33 expression cohorts. Patients in the low-risk (LR) group with the CC genotype treated with GO had a RR of 10%±8% vs. 37%±13% (P< 0.001) from remission. Standard-risk patients with CC genotype had a RR of 41±12% vs. 59±12% (P=0.056) and high-risk patients had a RR of 36%±27% vs. 70%±32% (P=0.073) for the GO and No-GO arms. In contrast there was no benefit of addition of GO in patients with the CT or TT genotypes within each risk group. Since CD33 expression has been recently associated with GO efficacy, we evaluated the association of rs12459419 genotypes in patients with low (N=153) and high CD33 (N=436) cell surface expression quartile-1 and quartiles-2-4, respectively. Significant improvement in RR was observed in patients with the CC genotype in the GO arm over those in the No-GO arm (P=0.001) within quartiles 2-4, but not with the CT/TT genotype (P=0.112). Within lower CD33 expression (quartile-1) cohort, a similar trend towards improvement in RR compared with those in the No-GO arm (P=0.055) was observed although the CC genotype was less frequent. Multivariate cox regression analysis that included genotype, risk status, and CD33 expression demonstrated that CD33 CC genotype was an independent predictor of response to GO (HR= 0.45, P<0.001 for RR and HR=0.57, P=0.003 for DFS). The rs12459419 genotype mediates expression of the GO binding site and informs on which patients should receive GO. The knowledge of CD33 genotype and prediction of response to GO provides opportunities to use patient genotypes for selecting CD33-targeted therapies. Given only half of the patients are expected to have a response to GO, we propose that all prior GO containing studies be re-evaluated for response based on patient CD33 genotype. The efficacy of GO in patients with an appropriate antibody binding domain also raises the possibility of developing next-generation CD33 immunoconjugates with epitopes targeted to regions not affected by alternative splicing and SNPs. Disclosures Loken: Hematologics: Employment, Equity Ownership.

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Targeting B-Cell Maturation Antigen (BCMA) with CC-93269, a 2+1 T Cell Engager, Elicits Significant Apoptosis in Diffuse Large B-Cell Lymphoma Preclinical Models

Blood ◽

10.1182/blood-2019-126115 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1580-1580 ◽

Cited By ~ 1

Author(s):

Patrick R. Hagner ◽

Michelle Waldman ◽

Falon D. Gray ◽

Renee Yura ◽

Sarah Hersey ◽

...

Keyword(s):

T Cell ◽

Cell Surface ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Plasma Cells ◽

Surface Expression ◽

Employment Equity ◽

Car T ◽

Equity Ownership

B-cell maturation antigen (BCMA), a member of the TNF receptor superfamily, serves as the cell surface receptor for B-cell activating factor (BAFF). Upon binding of BAFF to BCMA, an intracellular signaling cascade is initiated resulting in upregulation of JNK pathway signaling events and NFkB mediated transcription. The canonical expression pattern of BCMA begins on germinal center B-cells and becomes maximally expressed on mature cells such as plasma cells. Given the high degree of expression of BCMA on multiple myeloma (MM) cells, the malignant counterpart to plasma cells, it has become a target of interest for CAR T and antibody mediated modalities such as antibody drug conjugates and bispecific molecules. Recent clinical data from clinical trials employing BCMA targeted CAR T cells or T cell engager (TCE) antibodies have demonstrated significant responses in heavily pretreated myeloma patients with overall response rates ranging from 70% to 80%. As BCMA is known to be expressed in earlier B-cell lineages, we sought to explore the expression of BCMA in non-Hodgkin lymphoma (NHL) and its sensitivity to CC-93269, a 2+1 TCE currently being clinically investigated in MM. NHL is a heterogeneous collection of lymphomas that can be classified into two major subgroups; aggressive lymphomas of which diffuse large B-cell (DLBCL) is the most prevalent subtype and indolent lymphomas of which follicular lymphoma is the largest constituent. We first sought to directly quantitate cell surface expression of BCMA utilizing a flow cytometry system based on a logarithmic dilution of phycoerythrin beads of a known quantity. In a panel of 43 NHL cell lines, we determined that BCMA expression ranged from 43 to 17,048 molecules per cell (median, 420). An isogenic pair of K562 that is null for BCMA expression and K562 constitutively overexpressing BCMA (K562-BCMA) (15,866 molecules/cell) served as negative and positive controls, respectively. Additionally, the MM cell line H929 was profiled to serve as an additional control with a BCMA expression level of 7,065 molecules/cell. Next, utilizing quantitative PCR we found that relative BCMA mRNA expression in the lymphoma cell lines ranged from 0.001 to 0.17-fold when normalized to the H929 MM cell line. Furthermore, we were able to determine that in the lymphoma cells there is a poor correlation between protein expression (mean fluorescent intensity) and mRNA expression (r2, 0.33). We next examined if there was any correlation between BCMA surface expression and T-cell mediated cytotoxicity after administration of CC-93269 in a co-culture assay. We selected 11 DLBCL cell lines with a surface expression ranging from 45 molecules to 17,000 molecules per cells and incubated them in a co-culture system with a defined 1:5 target:effector ratio with CC-93269 (0-200 ng/ml) for five days. Significant apoptosis as measured by annexin V and ToPro-3 staining of CFSE positive target cells was observed in 10 of the 11 cell lines profiled with an IC50 of 0.1 to 38 ng/ml for CC-93269. As controls, the K562 isogenic pair were also profiled with the K562-BCMA cell line exhibiting an IC50 of 0.5 ng/ml and no activity observed against the parental K562 cell line. Additionally, a bispecific antibody where the two binding domains for BCMA was altered to target HEL (hen egg lysozyme) demonstrated no activity against any of the cell lines profiled at a defined dose of 200 ng/ml. No association between CC-93269 activity and BCMA expression or cell of origin was found. To determine the expression of BCMA in primary DLBCL biopsy samples, we developed a novel monoclonal BCMA immunohistochemistry antibody (clone: G12). The antibody and IHC staining protocol were validated to have good on-target specificity in both cell lines and tissues, including MM and DLBCL biopsies, with a range of stain intensity (1-3+) observed in both the golgi and on the plasma membrane. A proof of concept study on a cohort of 110 commercial DLBCL samples is currently underway. Cumulatively, our data demonstrate that BCMA is expressed on the cell surface of a broad panel of NHL cell lines and in primary DLBCL lymph node biopsies. Additionally, the expression levels of BCMA in these preclinical cell line models was sufficient to elicit significant CC-93269 mediated cytotoxicity. These data highlight the potential for the treatment of DLBCL patients with a 2+1 T-cell engager targeting BCMA. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Waldman:Celgene: Employment, Equity Ownership, Patents & Royalties. Gray:Celgene: Employment, Equity Ownership. Yura:Celgene: Employment, Equity Ownership. Hersey:Celgene: Employment, Equity Ownership. Chan:Celgene: Employment, Equity Ownership. Zhang:Celgene: Employment, Equity Ownership. Boss:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.

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Epor Is Not Expressed or Functional in Primary Tumor Cells Isolated Directly From Human Primary and Metastatic Epithelial Tumors Including Breast, Non-Small Cell Lung, Colorectal, and Ovarian Tissues

Blood ◽

10.1182/blood.v118.21.4781.4781 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4781-4781

Author(s):

Ian McCaffery ◽

John M Rossi ◽

Katherine L Paweletz ◽

Yanyan Tudor ◽

Steve Elliott ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

Tumor Cells ◽

Human Tumor ◽

Small Cell ◽

Small Cell Lung ◽

Employment Equity ◽

Equity Ownership ◽

And Function ◽

Epor Expression

Abstract Abstract 4781 Several clinical trials have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents (ESA) in patients with cancer. One hypothesis proposes that ESAs stimulate tumor proliferation and or survival directly through binding and activating cell-surface receptors (EpoR). Expression and function of cell surface EpoR in human tumor tissue is controversial and has not been extensively investigated using validated reagents. Methods EpoR expression and function was analysed in disaggregated primary tumor cells obtained from surgically resected human tumor tissues from 186 patients: colorectal (n=46), breast (n=34, including 8 Her2+ tumors defined by HercepTest™), non small cell lung (n=41), ovarian (n=35), Head and Neck (n=5) and other tumor types (n=8). Metastatic tissues were also analysed: colon (n=2), ovary (n=3), lung (n=2), pancreatic (n=1) as well as tumors from pre-treated patients: colorectal (oxaliplatin, bevacizumab; n=1), breast (cyclophosphamide, methotrexate, fluorouracil; n=2), breast (radiation, docetaxel cyclophosphamide, Paclitaxel; n=1), non small cell lung (radiation; n=2), ovarian (carboplatin and Paclitaxel; n=2). To examine if cell surface EpoR is expressed in the tumor cell compartment, flow cytometry analysis was carried out using a validated, EpoR-specific antibody (mAb 307). To evaluate if tumor cells respond functionally to recombinant human Epo (rHuEpo), levels of phosphorylated downstream signaling proteins (Akt, Erk1/2 and STAT5) were analysed by intracellular flow cytometry using phospho-specific antibodies following ex vivo stimulation with a titration of rHuEpo (0U [vehicle] to 300U/mL for 5 and 30 min). Expression of known tumor growth factor receptors (IGF-1R, c-Met and EGFR) was analyzed as positive controls. Additionally, the response of Akt, Erk and STAT pathways to stimulation with a tumor growth factor cocktail (“GF”: EGF, HGF and IGF-1), was used as a control for pathway integrity. The sensitivity of all flow based assays was validated through the analysis of EpoR expression and function in erythroid precursor cells (EPCs) across a time course of differentiation from primary human bone marrow samples. Results Though tumor samples exhibited cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface EpoR was detected in tumor cells from any of the 186 tumors examined by flow cytometry. Mean expression relative to negative control: EGFR = 2.80 (95% CI, 1.63–3.97), c-Met = 5.28 (95% CI, 4.39–6.17), IGF-1R = 8.03 (95% CI, 6.81–9.24) and EpoR (0.69 (95% CI, 0.64–0.73). No EpoR was detected by Western blot analysis of tumor lysates. As shown in Table 1, treatment with the GF cocktail stimulated phosphorylation of Akt and Erk in all cohorts. However, no activation of these pathways (or STAT5) by rHuEpo was observed in any of tumors even at high supra-pharmacological concentrations of rHuEpo (up to 300U/mL) and extended stimulation times (up to 30 mins). Analysis of EpoR expression and function during differentiation in-vitro of EPCs revealed detectable EpoR cell surface expression (2.13-fold above baseline) and function (e.g. pSTAT5: 1.39-fold relative to vehicle at 300 U/mL rHuEpo) as early as day 1 when EPCs (CD36+/CD34-) comprised only 6.9% of the population. By day 8, (>90% EPCs) EpoR expression was detectable at 6.36-fold relative to baseline and pSTAT5 activation was observed 5.6-fold relative to vehicle at 300U/mL rHuEpo. Conclusion Using sensitive methods no evidence of EpoR expression or function in primary human tumor cells isolated directly from primary and metastatic tumors was observed in multiple epithelial tumor types. Expression and functional activation of EGFR, c-Met and IGF1R was readily detectable. Therefore, this study suggests that it is unlikely that rHuEpo acts as a growth factor for primary human tumor cells. Disclosures: McCaffery: Amgen: Employment, Equity Ownership. Rossi:Amgen: Employment, Equity Ownership. Paweletz:Amgen: Employment, Equity Ownership. Tudor:Amgen: Employment, Equity Ownership. Elliott:Amgen Inc: Employment. Fitzpatrick:Amgen Inc.: Employment, Equity Ownership. Patterson:Amgen: Employment, Equity Ownership.

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Cell-surface expression of PrPC and the presence of scrapie prions in the blood of goats

Journal of General Virology ◽

10.1099/vir.0.039032-0 ◽

2012 ◽

Vol 93 (5) ◽

pp. 1127-1131 ◽

Cited By ~ 8

Author(s):

Rohana P. Dassanayake ◽

David A. Schneider ◽

Lynn M. Herrmann-Hoesing ◽

Thomas C. Truscott ◽

William C. Davis ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

Whole Blood ◽

Surface Expression ◽

Infected Sheep ◽

Cell Surface Expression ◽

Prion Infectivity ◽

Infected Goat ◽

Scrapie Infection ◽

Relative Differences

Although host-encoded prion protein (PrPC) expression in ovine PBMCs and prion infectivity in scrapie-infected sheep blood have been demonstrated, such studies have not been reported in goats. Therefore, this study characterized cell-surface expression of PrPC on PBMC subsets derived from normal goats and sheep, by flow cytometry, and determined prion infectivity in blood from a scrapie-infected goat using a transfusion bioassay in goat kids. Cell-surface PrPC expression was detected on all subsets of goat PBMCs. The highest PrPC cell-surface expression was found in CD2+ T lymphocytes in goats. Transmission of infection was detected in all three recipients who received whole blood from a goat with classical scrapie. It was concluded that caprine PBMCs express PrPC similarly to sheep but with relative differences among PBMCs subsets, and that blood-borne infectious prions can be detected in scrapie-infected goats. Thus, similar to sheep, goat blood may be a suitable diagnostic target for the detection of scrapie infection.

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The rs35112940 CD33 Polymorphism Reduces CD33 Internalization and Efficacy of CD33-Directed Gemtuzumab Ozogamicin

Blood ◽

10.1182/blood-2021-150927 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2247-2247

Author(s):

Mohammed O Gbadamosi ◽

Vivek M. Shastri ◽

Soheil Meshinchi ◽

Jatinder K. Lamba

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

Conflicts Of Interest ◽

Surface Expression ◽

Gemtuzumab Ozogamicin ◽

Cell Surface Expression ◽

Specific Cell ◽

Antibody Drug Conjugate ◽

Critical Feature ◽

The Impact

Abstract Background CD33 is a myeloid-specific cell surface protein widely expressed on acute myeloid leukemia (AML) cells making it an excellent immunotherapeutic target. Current CD33-directed immunotherapeutic treatment strategies include gemtuzumab ozogamicin (GO), an antibody-drug conjugate (ADC) which was approved for the treatment AML in 2017 and has demonstrated promising results thus far. The mechanism of action of GO begins with recognition of CD33 by the antibody portion of GO, followed by internalization of the CD33-GO complex, and finally delivery of free calicheamicin molecules to the cell to induce cellular apoptosis. As such, modifications that impact these steps on any level presumably impact the response and overall efficacy of GO. Indeed, previous studies from our group have identified germline variations in CD33 that are associated with differences in CD33 structure, CD33 cell surface expression levels, and clinical outcomes in response to GO. Among these germline variations is rs35112940 (G>A; Arg304Gly), a missense polymorphism which is located in exon five of CD33 adjacent to the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) domain, a critical feature for CD33 internalization. While our previous work identified statistical associations between the A allele of rs35112940 and lower CD33 expression and reduced benefit from treatment using GO, these results are yet to be validated functionally. Additionally, it still remains unknown if the impact of the rs35112940 variation is due to reduced CD33 expression alone or if the rs35112940 variation also impacts CD33 internalization thereby modulating CD33 efficacy. Methods To functionally validate the effect of the rs35112940 variant, we used CRISPR/cas9 to knockout CD33 in HL60 cells and subsequently engineered the HL60-CD33 KO cells to express either wildtype CD33 (HL60-CD33 FL) or CD33 encoding the rs35112940 variant (HL60-CD33 FL-rs35112940). The engineered cells were then treated with GO for 48 hours to capture the impact of the rs35112940 variation on the efficacy of GO. To assess the impact of the rs35112940 variation on CD33 internalization, we performed a flow cytometry-based internalization assay using secondary antibodies to capture the remaining amounts of CD33 present on the cell surface after 4 hours allowing us to determine the internalization of CD33 over time. Results All engineered cells expressed CD33 with less than 1-log fold difference in median fluorescence intensity (MFI) (HL60-CD33 FL MFI vs HL60-CD33 FL-rs35112940 MFI: 22536 vs 24882, Figure 1) and thus we were able to characterize the impact of the rs35112940 variant independent of its impact on CD33 cell surface expression. After 48-hour treatment with 250 ng/mL of GO, we observed that HL60-CD33 FL-rs35112940 cells were more resistant to GO than HL60-CD33 FL cells (66.4% vs 46.5% cell viability, P = 0.02, Figure 2A). Similar results were observed at multiple concentrations of GO. Given the proximity of the rs35112940 loci to the ITIM domain of CD33, we hypothesized that the rs35112940 variation may impact CD33 internalization as well. In a flow cytometry-based internalization assay over a 4-hour window, we observed that that HL60-CD33 FL-rs35112940 cells had an approximate 10% reduction in CD33 internalization in comparison to HL60-CD33 FL cells (Figure 2B). Taken together these results provide insight into the effect of the rs35112940 variant on GO efficacy and CD33 biology, corroborating our previous findings, and support the use of CD33 polymorphisms to guide patient selection for treatment with GO. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Ex Vivo High-Throughput Flow Cytometry Screening Identifies Subsets of Responders to Differentiation Agents in Individual AML Patient Samples

Blood ◽

10.1182/blood.v128.22.5206.5206 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5206-5206 ◽

Cited By ~ 2

Author(s):

Sacha L. Prashad ◽

Leylah Drusbosky ◽

Hassan Sibai ◽

Mark D. Minden ◽

Stephen J. Western ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

Ex Vivo ◽

Surface Marker ◽

Cell Surface Marker ◽

Employment Equity ◽

Surface Marker Expression ◽

Cd163 Expression ◽

Differentiating Agents ◽

Equity Ownership

Abstract Background: Prognoses for acute promyelocytic leukemia (APL) patients improved drastically upon the introduction of differentiation therapy with all-trans-retinoic acid (ATRA) in combination with conventional chemotherapy. Unfortunately, this therapeutic approach has not translated to other genetic subtypes of acute myeloid leukemia (AML) where patients demonstrate marked heterogeneity to differentiating agents. To provide improved detection of drug-induced differentiation in AML patients, we have developed a high-throughput, flow cytometry-based personalized medicine platform. Methods: Total white blood cells were isolated from each patient sample by red cell lysis, plated in serum-free media in 384-well format and incubated with drugs for 3 days. Viable cells remaining after each drug treatment were identified and quantified using cell surface marker expression, cell membrane integrity, and morphology (FSC/SSC) to determine the compound's efficacy and specificity against the blast population. Changes in cell surface marker expression and shifts in morphology indicative of blast differentiation were also evaluated with each compound. As a control for ex vivo differentiation, two APL patient samples were treated ex vivo with ATRA and we observed the blasts gaining CD66b expression indicating granulocytic differentiation. Results: A refractory AML patient was identified whose leukemic blasts exhibited a strong differentiating response to dexamethasone treatment ex vivo. This resulted in loss of CD34 expression (a marker of immature blast cells), gain of CD163 expression (a marker of monocytic/macrophage maturation) and a significant change in cellular size and granularity. After being enrolled in a clinical trial (REB: 13-6962-C) the patient was treated based on the assay for 1 week (40 mg/day) with dexamethasone. Post-treatment samples from the peripheral blood and bone marrow of the patient exhibited the same morphological and cell surface marker changes predicted by the ex vivo assay. The CD163+ cells in the patient also gained additional markers of myeloid differentiation (CD11b, CD14, CD16). After additional cytarabine and fludarabine treatment, the patient remains in remission 4 months post-treatment. Conclusions: Following this initial study, we have continued to identify subgroups of both AML and Myelodysplastic Syndrome patients where blasts differentiate in response to dexamethasone, calcitriol, ATRA or other known differentiating agents using unique cell surface markers of monocytic and myeloid maturation. Flow cytometry expression changes correlated with changes in morphology as observed by May-Grunwald Giemsa staining. In the patient described above this included an increase in cytoplasm and vacuoles consistent with monocytic/macrophage differentiation, which positively correlates with CD163 expression. We aim to apply our assay towards the identification of subgroups of AML patients who respond to differentiation therapies and develop clinical trials to combine differentiating agents with chemotherapy. This approach has the potential to extend the clinical success of APL differentiation therapy to AML patients. Disclosures Prashad: Notable Labs: Employment, Equity Ownership. Western:Notable Labs: Consultancy. Biondi:Notable Labs: Employment. Shah:Notable Labs: Employment. Liu:Notable Labs: Employment, Equity Ownership. Nguyen:Notable Labs: Employment, Equity Ownership. Warnock:Notable Labs: Employment, Equity Ownership. Quinzio:Notable Labs: Employment, Equity Ownership. De Silva:Notable Labs: Employment, Equity Ownership. Schimmer:Novartis: Honoraria. Heiser:Notable Labs: Employment, Equity Ownership.

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