Molecules Of Equivalent Soluble Fluorochrome Research Articles

Co-Expression of GPRC5D, FcRH5 and BCMA Suggests That Targeting More Than One Cell Surface Marker May be a Viable Strategy in Relapsed/Refractory Multiple Myeloma (RRMM): Biomarker Results from the Phase I Study of Forimtamig, a GPRC5DxCD3 Bispecific Antibody

Background: Forimtamig is a GPRC5DxCD3 T-cell engaging bispecific antibody that induces T-cell mediated multiple myeloma (MM) cell killing and has shown promising clinical activity in a Phase I study (NCT04557150) in patients with RRMM (Carlo-Stella et al. ASH 2022). GPRC5D is known to be overexpressed in MM plasma cells (MMPCs), but little is known about its prevalence in relation to risk status and other identified MM targets like FcRH5 and BCMA. We developed a unique exploratory multiparameter flow cytometry panel to characterize GPRC5D, FcRH5 and BCMA expression on MMPCs (Lelios et al. Cyto 2023). We present data on the prevalence and co-expression of these targets, and their association with baseline patient characteristics and response to forimtamig. Methods: Baseline bone marrow aspirate samples were collected from patients who received intravenous or subcutaneous forimtamig during dose-escalation, and were analyzed with flow cytometry. Target expression was depicted as frequency of positive MMPCs and as receptor density (molecules of equivalent soluble fluorochrome). Patients were grouped by number of prior lines of therapy, ISS score, cytogenetic risk (high risk defined as presence of one or more of the following abnormalities: del(17p), t(4;14), t(14;16), and response to forimtamig (responder [≥PR] or non-responder [<PR]). All patients provided informed consent. Results: At data cut-off (June 15, 2023), 48/163 patients were biomarker evaluable (samples passing pre-defined quality standards and containing ≥100 acquired MMPCs). GPRC5D was detected at baseline in 46/48 (96%) patients, with most MMPCs expressing the target (median 95.6%; min-max: 0-100%). GPRC5D expression was more prevalent on MMPCs than FcRH5 (median 78.6%; min-max: 3.7-100%) and BCMA (median 40.3%; min-max: 0-100%; Figure). Interestingly, GPRC5D receptor density (median 20,612; min-max: 6495-208,072) was 47-100-fold higher and more homogenous than FcRH5 (median 436; min-max: 8-3921) and BCMA (median 206; min-max: 14-6657). Clinical response to forimtamig was not correlated with GPRC5D+ MMPC frequency or GPRC5D receptor density measured by flow cytometry ( Table). Percentages of GPRC5D+, FcRH5+ and BCMA+ MMPCs and receptor densities did not significantly differ between patient groups defined by ISS score, cytogenetic risk and number of prior lines of therapy. Patients previously exposed to BCMA-targeted agents (n=7) tended to have lower BCMA expression levels. GPRC5D and FcRH5 were co-expressed on 75.4% of MMPCs (min-max: 0-100%) followed by MMPCs co-expressing GPRC5D and BCMA (median 31.9%; min-max: 0-100%) or BCMA and FcRH5 (median 25.3%; min- max: 0-94.3%). In the majority of patients, double and triple negative MMPCs were rarely observed (0.5-6% of MMPCs expressed only one or none of the three targets across all analyzed patients). Conclusions: GPRC5D, FcRH5 and BCMA are highly prevalent across MM patient subgroups, including high-risk patients who may warrant targeted therapy approaches. Based on our data and previously published work for FcRH5- and BCMA-targeted T-cell bispecifics (Sumiyoshi et al. EHA 2021; Cortes-Selva et al. ASH 2022), target expression on MMPCs, as measured by flow cytometry, has not been associated with clinical response to the corresponding CD3 bispecifics, likely due to the high efficacy of T-cell mediated tumor cell killing. Co-expression of MM targets suggests that targeting more than one MM surface marker may result in double/triple hits on individual tumor cells. Our data support further testing of dual/triple MMPC targeting as a promising concept to promote fast and deep tumor clearance, and to achieve durable responses, while potentially avoiding clonal selection, in patients with RRMM. Further investigation is needed to understand the relevance of target expression differences measured by flow cytometry compared to other methods like immunohistochemistry, as well as to understand T-cell bispecific resistance mechanisms beyond target expression. Updated data will be presented.

Optimizing interpretation of survival studies of fresh and aged transfused biotin-labeled RBCs.

Ex vivo labeling with 51 chromium represents the standard method to determine red blood cell (RBC) survival after transfusion. Limitations and safety concerns spurred the development of alternative methods, including biotinylated red blood cells (BioRBC). Autologous units of whole blood were divided equally into two bags and stored under standard blood bank conditions at 2 to 6°C (N=4 healthy adult volunteers). One bag was biotinylated (15 μg/ml) on storage days 5 to 7 (fresh) and the other was biotinylated (3μg/ml) on days 35 to 42 (aged). The proportion of circulating BioRBC was measured serially, and cell-surface biotin was quantified with reference to molecules of equivalent soluble fluorochrome. Clearance kinetics were modeled by RBC age distribution at infusion (Gaussian vs. uniform) and decay over time (constant vs. exponential). Data were consistent with biphasic exponential clearance of cells of uniform age. Our best estimate of BioRBC clearance (half-life [T1/2 ]) was 49.7 ± 1.2 days initially, followed by more rapid clearance 82 days after transfusion (T1/2 =15.6 ± 0.6 days). As BioRBC aged in vivo, molecules of equivalent soluble fluorochrome declined with a T1/2 of 122 ± 9 days, suggesting gradual biotin cleavage. There were no significant differences between the clearance of fresh and aged BioRBC. Similar clearance kinetics of fresh and aged BioRBC may be due to the extensive washing required during biotinylation. Survival kinetics consistent with cells with uniform rather than Gaussian or other non-uniform age distributions suggest that washing, and potentially RBC culling, may extend the storage life of RBC products.

Standardization of neutrophil CD64 and monocyte HLA-DR measurement and its application in immune monitoring in kidney transplantation.

Infections cause high mortality in kidney transplant recipients (KTRs). The expressions of neutrophil CD64 (nCD64) and monocyte HLA-DR (mHLA-DR) provide direct evidence of immune status and can be used to evaluate the severity of infection. However, the intensities of nCD64 and mHLA-DR detected by flow cytometry (FCM) are commonly measured by mean fluorescence intensities (MFIs), which are relative values, thus limiting their application. We aimed to standardize nCD64 and mHLA-DR expression using molecules of equivalent soluble fluorochrome (MESF) and to explore their role in immune monitoring for KTRs with infection. The study included 50 KTRs diagnosed with infection, 65 immunologically stable KTRs and 26 healthy controls. The blood samples were collected and measured simultaneously by four FCM protocols at different flow cytometers. The MFIs of nCD64 and mHLA-DR were converted into MESF by Phycoerythrin (PE) Fluorescence Quantitation Kit. The intraclass correlation coefficients (ICCs) and the Bland-Altman plots were used to evaluate the reliability between the four FCM protocols. MESFs of nCD64 and mHLA-DR, nCD64 index and sepsis index (SI) with the TBNK panel were used to evaluate the immune status. Comparisons among multiple groups were performed with ANOVA one-way analysis. Receiver operating characteristics (ROC) curve analysis was performed to diagnose infection or sepsis. Univariate and multivariate logistic analysis examined associations of the immune status with infection. MESFs of nCD64 and mHLA-DR measured by four protocols had excellent reliability (ICCs 0.993 and 0.957, respectively). The nCD64, CD64 index and SI in infection group were significantly higher than those of stable KTRs group. Patients with sepsis had lower mHLA-DR but higher SI than non-sepsis patients. ROC analysis indicated that nCD64 had the highest area under the curve (AUC) for infection, and that mHLA-DR had the highest AUC for sepsis. Logistic analysis indicated that nCD64 > 3089 and B cells counts were independent risk factors for infection. The standardization of nCD64 and mHLA-DR made it available for widespread application. MESFs of nCD64 and mHLA-DR had good diagnostic performance on infection and sepsis, respectively, which could be promising indicators for immune status of KTRs and contributed to individualized treatment.

Novel 17-Plex Flow Cytometry Assay for Comprehensive Immunophenotyping and Characterization of Plasma Cells (PC) in Relapsed/Refractory (RR) Multiple Myeloma (MM) Patients

Background: Multiple Myeloma (MM) is a hematological malignancy also known as plasma cell myeloma or myeloma, a type of bone marrow cancer characterized by the proliferation of clonal malignant plasma cells (PCs). Characterization of multiple myeloma plasma cells (MMPCs) remains challenging due to their plasticity, especially in response to emerging targeted therapies such as anti-BCMA and anti-CD38. The need for novel options for comprehensive phenotyping remains high due to the evolving target expression profiles and dynamics across MM disease evolution and treatment lines. Recent progress in flow cytometry offers the opportunity to analyze more than 30 surface markers in one panel. This technology advance will help to address the current challenges, answer clinically relevant questions and generate data to enable translational research and identification of new drug targets or combination partners. Methods: We present a novel 17-plex flow cytometry assay for immunophenotyping of PCs in RRMM patients enrolled in a global Phase I multi-center clinical study (NCT04557150). The panel relies on 5 markers for the identification of malignant plasma cells: CD38, CD138, CD45, CD319 and CD229. The inclusion of less commonly used markers (CD229, CD319) allows to overcome some of the challenges in MMPCs identification, such as CD138 instability and CD38 downregulation due to prior therapies. In addition, the assay includes markers commonly used to differentiate normal from malignant PCs (CD19, CD56, CD27, CD81, CD117, CD28). The panel also includes 2 immunotherapy target ligands (PD-L1 and 4-1BBL) and offers the possibility to measure another 3 clinically relevant targets on MMPCs, including BCMA. With this configuration, the assay allows relevant target antigen density quantification via the use of molecules of equivalent soluble fluorochrome (MESF) beads in 3 channels. Results: We present examples of how to apply this strategy for a reliable identification of MMPCs in RRMM patient samples after up to 14 previous lines of therapies. The assay design permits a deep phenotypical characterization of MMPCs and pharmacodynamic changes in markers targeted by immunotherapy drugs, like anti-CD38 or anti-BCMA. As an example, we characterize BCMA and CD38 expression on the surface of MMPCs at the time of enrolment into a Phase I study. Finally, with the goal of discovering new biomarkers that could be overlooked in the traditional manual gating hierarchy applied in flow cytometry, we have visualized the results using dimensionality reduction analysis (Uniform Manifold Approximation and Projection, UMAP). Using this approach, we have observed high inter-patient heterogeneity of MMPC phenotypes. Furthermore, we could compare intra-patient differences in MMPC phenotype in two compartments, whole blood and bone marrow aspirate. Updated data will be presented. Conclusion: The multi parameter flow cytometry assay described here represents a novel tool to reliably identify MMPCs in RRMM patients independently of commonly used unstable surface markers. Furthermore, it represents an important tool in the era of surface antigen targeted therapies (e.g., anti-BCMA) helping to understand drug resistance due to target loss. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Insulin infusion decreases medium-sized extracellular vesicles in adults with metabolic syndrome.

Elevated extracellular vesicles (EVs) are associated with glucose dysmetabolism. However, the effects of insulin on EVs and subsequent relationships with insulin sensitivity, substrate oxidation, and inflammation are unknown. We tested the hypothesis that insulin would lower EVs and relate to insulin action. Fifty-one sedentary adults (54.8 ± 1.0 yr; V̇o2peak : 22.1 ± 0.6 mL/kg/min) with metabolic syndrome (MetS) and obesity (36.4 ± 0.65 kg/m2) underwent a 2-h euglycemic-hyperinsulinemic clamp (5 mmol/L; 40 mU/m2/min). Count and size (medium: 200-624 nm; larger: 625-1,000 nm) for total particle count, endothelial- (CD105+), leukocyte- (CD45+), platelet- (CD41+), and tetraspanin- (TX+: CD9/CD81/CD63), as well as platelet endothelial cell adhesion molecule- (CD31+) derived EVs were determined before and following the clamp using Full Spectrum Profiling (FSPM). Size and MESF (molecules of equivalent soluble fluorochrome) data were generated using FCMPASS Software. Fat and carbohydrate oxidation, in addition to high-sensitivity c-reactive protein (hsCRP), were measured to understand insulin effects and associations between EVs, metabolic flexibility, and inflammation. Despite low metabolic insulin sensitivity (M-Value = 2.56 ± 0.17 mg/kg/min), insulin increased carbohydrate (P = 0.015) and decreased fat oxidation (P = 0.048) and hsCRP (P = 0.016) compared with fasting. Insulin also decreased total particle count (P < 0.001), attributable to decreased medium-sized CD105+ (P = 0.052) and CD45+ EVs (P < 0.001). Elevated fasting insulin was associated with reduced insulin-stimulated changes in all EVs phenotypes (P < 0.001). Interestingly, fasting EVs were associated with increased fasting carbohydrate oxidation (all P < 0.05). These findings suggest that insulin decreases medium-sized EVs in conjunction with metabolic flexibility under euglycemic conditions in adults with MetS. More research is needed to determine how therapies alter EV phenotype/size and consequent cardiometabolic risk.NEW & NOTEWORTHY This study is one of the first to investigate the effects of insulin on medium and larger extracellular vesicles (EVs) in relation to metabolic insulin sensitivity and fuel use in adults with metabolic syndrome. Our data suggest that insulin infusion decreases the concentration of total particle counts, mainly due to reductions in medium-sized EVs. Furthermore, EVs, predominantly medium-sized, are inversely associated with metabolic flexibility.

Evaluation and validation of a novel 10-color flow cytometer.

BackgroundFlow cytometry is a powerful technique that provides information regarding cell properties. In this study, we evaluated the analytical performance of a new flow cytometer, the 10‐color BD FACSLyricTM, which could help doctors obtain reliable test results prior to clinical research.MethodsWe used SpheroTM Rainbow Calibration Particles and the SpheroTM Nano Fluorescent Particle Size Standard Kit to validate the fluorescence sensitivity and linearity. The Beckman Coulter IMMUNO‐TROL Cell was used as the quality control to evaluate the accuracy and reproducibility of surface markers detected by the flow cytometer. Furthermore, BD Calibrate APC Beads and CS&T Research Beads were applied to calculate the carry‐over contamination rate and assess the instrument stability.ResultsA linear regression equation between the molecules of equivalent soluble fluorochrome and fluorescence detection limit showed a good linear fit (R 2 > 0.99). The minimum bead size detected by side scatter was 0.22 μm. The coefficient of variation percentage of each fluorescence channel was below 2%, and the carry‐over contamination rate of the cytometer was under 0.2%. After running the BD FACSLyricTM cytometer continuously for 8 h, the median fluorescence index of particles remained close to that at the time of cytometer startup.ConclusionsThe 10‐color BD FACSLyricTM cytometer showed good performance in the evaluation performed in this study and may be trusted to provide accurate results for clinical research.

Inotuzumab Ozogamicin for Relapsed/Refractory Acute Lymphoblastic Leukemia in the INO-VATE Trial: CD22 Pharmacodynamics, Efficacy, and Safety by Baseline CD22.

We assessed the relationship between cluster of differentiation-22 (CD22) expression and outcomes of inotuzumab ozogamicin versus standard of care (SC) in INO-VATE (NCT01564784). Adults with relapsed/refractory B-cell precursor CD22-positive (by local or central laboratory) acute lymphoblastic leukemia were randomized to inotuzumab ozogamicin (n = 164) or SC (n = 162). Outcomes were analyzed by baseline CD22 positivity (percentage of leukemic blasts CD22 positive, ≥90% vs. <90%) and CD22 receptor density [molecules of equivalent soluble fluorochrome (MESF), quartile analysis]. Most patients had high (≥90%) CD22 positivity per central laboratory. The response rate was significantly higher with inotuzumab ozogamicin versus SC. Minimal/measurable residual disease negativity, duration of remission (DoR), progression-free survival, and overall survival (OS) were significantly better with inotuzumab ozogamicin versus SC in patients with CD22 positivity ≥90%. Fewer patients had CD22 positivity <90%; for whom, response rates were higher with inotuzumab ozogamicin versus SC, but DoR and OS appeared similar. Similar trends were evident in quartile analyses of CD22 MESF and CD22 positivity per local laboratory. Among inotuzumab ozogamicin-responding patients with subsequent relapse, decrease in CD22 positivity and receptor density was evident, but not the emergence of CD22 negativity. Rates of grade ≥3 hematologic adverse events (AEs) were similar and hepatobiliary AEs rate was higher for inotuzumab ozogamicin versus SC. No apparent relationship was observed between the rates of hematologic and hepatic AEs and CD22 expression. Inotuzumab ozogamicin demonstrated a favorable benefit-risk profile versus SC in patients with higher and lower CD22 expression. Patients with high CD22 expression and normal cytogenetics benefited the most from inotuzumab ozogamicin therapy.

Evaluation of Exosome Proteins by on-Bead Flow Cytometry.

Exosomes, recently re-named "small extracellular vesicles" or "sEV," are emerging as an intercellular communication system. Quantification of the molecular cargo exosomes carry by on-bead flow cytometry is needed for defining their role in information transfer and in human disease. Exosomes (sEV) isolated from cell supernatants or plasma of cancer patients by size-exclusion chromatography were captured by biotinylated antibodies specific for antigens in the exosome cargo (e.g., tetraspanins) and placed on streptavidin-labeled beads. Detection was performed with pretitered fluorochrome-labeled antibodies of desired specificity. The data were acquired in a conventional cytometer, and molecules of equivalent soluble fluorochrome (MESF) beads were used to quantify the number of fluorescent molecules bound per bead. Isotype antibody controls were obligatory. The mean fluorescence intensity (MFI) value of each sample was converted into MESF units, and the separation index (SI), which quantifies separation of stained and isotype control beads, was determined. Various proteins identified by labeled antibodies were quantified on the surface of tumor cell-derived exosomes. To identify intravesicular cargo, such as cytokines or chemokines, exosomes were lysed with 0.3% Triton-100, and the proteins in lysates were loaded on aldehyde/sulfate latex beads for flow cytometry. Examples of quantitative surface and/or intravesicular on-bead flow cytometry for exosomes produced by various cells or present in body fluids of cancer patients are provided. On-bead flow cytometry standardized for use with conventional cytometers is a useful method for protein detection and quantitation in exosomes isolated from supernatants of cell lines or plasma of patients with cancer. © 2020 International Society for Advancement of Cytometry.

Efficacy and Safety Outcomes in the Phase 3 INO-Vate Trial By Baseline CD22 Positivity Assessed By Local Laboratories

Efficacy and Safety Outcomes in the Phase 3 INO-Vate Trial By Baseline CD22 Positivity Assessed By Local Laboratories

Efficacy and Safety of Azacitidine (AZA) in Combination with the Anti-PD-L1 Durvalumab (durva) for the Front-Line Treatment of Older Patients (pts) with Acute Myeloid Leukemia (AML) Who Are Unfit for Intensive Chemotherapy (IC) and Pts with Higher-Risk Myelodysplastic Syndromes (HR-MDS): Results from a Large, International, Randomized Phase 2 Study

Efficacy and Safety of Azacitidine (AZA) in Combination with the Anti-PD-L1 Durvalumab (durva) for the Front-Line Treatment of Older Patients (pts) with Acute Myeloid Leukemia (AML) Who Are Unfit for Intensive Chemotherapy (IC) and Pts with Higher-Risk Myelodysplastic Syndromes (HR-MDS): Results from a Large, International, Randomized Phase 2 Study

Improved quantitative detection of biotin-labeled red blood cells by flow cytometry.

Biotin-labeled red blood cells (BioRBC) can be tracked after transfusion, providing a convenient and safe way to measure RBC survival in vivo. RBC survival is of interest for determining optimal blood storage conditions and for assessing the impact of genetic and biologic variants in blood donors on the survival of transfused RBCs. Here we present an improved, platform-independent assay for quantifying biotin on BioRBC. This approach is also useful for detecting BioRBC in peripheral blood samples as rare events. We optimized the signal-to-noise ratio of the detecting reagent (phycoerythrin-conjugated streptavidin [SA-PE]) by determining the SA-PE concentration yielding the greatest separation index between BioRBC and unlabeled RBCs. We calibrated the fluorescence intensity measurements to molecules of equivalent soluble fluorochrome (MESF), a quantitative metric of fluorochrome binding and therefore of biotin bound per RBC. We then characterized the limit of blank and limit of quantification (LoQ) for BioRBC labeled at different densities. Biotin-labeled RBCs at sulfo-NHS-biotin concentrations of 3 to 30 μg/mL (27-271 nmol/mL RBCs) ranged from approximately 32,000 to 200,000 MESF/RBC. The LoQ ranged from one in 274,000 to one in 649,000, depending on biotin-labeling density. Increased sensitivity to detect BioRBC may facilitate tracking over longer periods and/or reduction of the BioRBC dose. Total RBC-bound biotin dose has been shown to correlate with the likelihood of developing antibodies to BioRBC. Lowering the dose of labeled cells may help avoid this eventuality.

Comparison of CD22 Expression between Baseline, End of Treatment, and Relapse Among Patients Treated with Inotuzumab Ozogamicin Who Responded and Subsequently Relapsed in Two Clinical Trials

Introduction: Downregulation or loss of B-cell ALL surface antigens (most notably CD19) in patients with acute lymphoblastic leukemia (ALL) targeted by various therapeutic modalities including CART and bi-specific T-cell engagers (BiTEs) has been implicated in acquired resistance to those therapies. Inotuzumab Ozogamicin (InO) is a calicheamicin-conjugated antibody targeting CD22 on ALL blast cells. In the Phase 3 INO-VATE trial, patients with refractory (R/R) ALL who received InO vs standard of care chemotherapy (SC) achieved greater remission, MRD negativity, and improved survival. This study reports CD22 expression levels at baseline, end of treatment (EOT) and relapse in R/R ALL patients who received InO or SC as salvage therapy and subsequently relapsed; the goal of this analysis was to understand if relapse was associated with changes in CD22 expression.Methods: The study population consisted of R/R ALL patients who were treated with InO or SC as part of two clinical trials: Phase 1/2 Study 1010 (NCT01363297; Phase 1, Expansion Phase, + Phase 2 at a dosage of 1.8 mg/m2) and Phase 3 Study 1022 (NCT01564784). This analysis incorporated CD22 expression levels at baseline, at end of treatment, and at relapse. Central laboratory flow cytometry was used to assess CD22 expression, which was quantified as % CD22-positive leukemic blasts and as Molecules of Equivalent Soluble Fluorochrome (MESF), a quantitative measure of CD22 density on leukemic blasts. Outcomes are reported in InO (Study 1022 or 1010) and SC patients who responded to treatment (patients who achieved complete remission/complete remission with incomplete hematologic recovery [CR/CRi]) and subsequently relapsed. CR was defined by <5% marrow blasts and the absence of peripheral blood leukemic blasts; with recovery of hematopoiesis.Results: 25 InO patients (Study 1022, n=19; Study 1010, n=6) and 11 SC patients who responded to treatment and subsequently relapsed were evaluable for central-lab CD22 analysis at baseline. Among InO patients who responded to treatment, the majority had leukemic blasts that were ≥90% CD22-positive at baseline (Study 1022, 66.7%; Study 1010, 66.7%); of the evaluable patients at EOT (Study 1022, n=4; Study 1010, n=0), leukemic blast CD22-positivity was >0-<70%. At relapse, all evaluable InO patients had leukemic blasts with detectable CD22-positivity; with blasts that were predominately <90% CD22 positive (Study 1022, n=6/7; Study 1010, n=5/6 [Table 1]). In contrast, the majority of evaluable subjects who received SC and achieved CR/CRi exhibited CD22 positivity that remained ≥90% at EOT and at relapse When CD22 expression was quantified as MESF, a similar trend was evident (Table 2). In Study 1022, InO patients had a median CD22 MESF of 4085.0 at baseline, which declined to 94.0 at EOT and 418.0 at relapse. In contrast, the median CD22 MESF at EOT and at relapse (3738.0 and 3738.0, respectively) was similar to baseline (4065.0) for SC patients.Conclusions: Among R/R ALL patients who responded to InO treatment and subsequently relapsed, a decrease in CD22 positivity and receptor density was evident from baseline to EOT and relapse, but emergent CD22 negativity was not evident. These results suggest that recurrent disease is associated with decreased CD22 expression, but is not generally attributable to the outgrowth of CD22-negative clones. [Display omitted] DisclosuresStock:Jazz Pharmaceuticals: Consultancy. Cassaday:Merck: Research Funding; Jazz Pharmaceuticals: Consultancy; Incyte: Research Funding; Seattle Genetics: Other: Spouse Employment, Research Funding; Kite Pharma: Research Funding; Adaptive Biotechnologies: Consultancy; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. DeAngelo:Pfizer Inc: Consultancy, Honoraria; BMS: Consultancy; Glycomimetics: Research Funding; Shire: Honoraria; ARIAD: Consultancy, Research Funding; Blueprint Medicines: Honoraria, Research Funding; Amgen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria; Takeda: Honoraria; Incyte: Consultancy, Honoraria. Jabbour:Novartis: Research Funding; Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. O'Brien:Pharmacyclics: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; Alexion: Consultancy; Sunesis: Consultancy, Research Funding; Regeneron: Research Funding; TG Therapeutics: Consultancy, Research Funding; Celgene: Consultancy; Janssen: Consultancy; Gilead: Consultancy, Research Funding; Aptose Biosciences Inc.: Consultancy; Vaniam Group LLC: Consultancy; Pfizer: Consultancy, Research Funding; Amgen: Consultancy; Acerta: Research Funding; Abbvie: Consultancy; Astellas: Consultancy; Kite Pharma: Research Funding. Stelljes:Novartis: Honoraria; Amgen: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; MSD: Consultancy; JAZZ: Honoraria. Wang:Pfizer: Employment, Equity Ownership. Liau:Pfizer: Employment, Equity Ownership. Nguyen:Navigate BioPharma Services, Inc., a Novartis Subsidiary: Employment. Sleight:Pfizer Inc: Employment, Equity Ownership. Vandendries:Pfizer: Employment, Equity Ownership. Neuhof:Pfizer: Employment, Equity Ownership. Laird:Pfizer: Employment, Equity Ownership. Advani:Novartis: Consultancy; Amgen: Research Funding; Glycomimetics: Consultancy; Pfizer: Honoraria, Research Funding.

Exploration of Potential Relationships between CD22 and Selected Safety Outcomes in the Inotuzumab Ozogamicin Phase 3 INO-VATE Study

Introduction: CD22 is widely expressed on leukemic lymphoblasts in patients with B-cell acute lymphoblastic leukemia (ALL). Inotuzumab ozogamicin (InO), an anti-CD22 monoclonal antibody conjugated to calicheamicin, has shown significantly higher remission rates than standard of care (SC) chemotherapy in relapsed or refractory B-cell (R/R) ALL, independently of CD22 expression level. Here we report safety outcomes by CD22 expression in patients with R/R ALL receiving InO (vs SC) as salvage therapy in the INO-VATE trial (NCT01564784).Methods: Adults with CD22-positive ALL in 1st or 2nd salvage were randomized to InO (n=164; starting dose 1.8 mg/m2/cycle [0.8 mg/m2 on day 1; 0.5 mg/m2 on days 8 and 15 of a 21-28 day cycle for ≤6 cycles]) or SC (n=162; fludarabine/high-dose (HD) cytarabine (Ara-C)/granulocyte colony-stimulating factor, Ara-C plus mitoxantrone, or HD Ara-C).The InO dose was reduced to 1.5 mg/m2 per cycle in patients who achieved complete remission [CR] or CR with incomplete hematologic recover [CRi]. Last patient visit was January 4, 2017. Central flow cytometry was used to assess CD22 expression, which was quantified as % leukemic blasts CD22-positive and as Molecules of Equivalent Soluble Fluorochrome (a quantitative measure of receptor density on leukemic blasts [MESF]). Outcomes were reported in patients by baseline leukemic blast positivity (≥90% vs <90%), and in quartiles of patients (Q1-Q4) defined by CD22 MESF: Q1=lowest CD22 expression, Q4= highest CD22 expression.Results: At baseline, 142 InO patients and 117 SC patients had an evaluable sample for central-lab CD22 analysis. The majority of patients in both arms had high (≥90%) CD22 positivity (InO, 75.4%; SC, 72.6%), with a smaller proportion of patients exhibiting CD22 positivity <90% (InO, 24.6%; SC, 27.4%). The median number of completed cycles in the InO arm for the CD22 expression quartiles was n=2 for Q1, n=3 for Q2, n=2 for Q3, and n=3 for Q4; respective median overall dose was similar across quartiles: 4.2 mg/m2 (range: 1.3-9.2), 4.5 mg/m2 (range: 0.8-9.2), 4.2 mg/m2 (range: 0.8-9.3), and 4.6 mg/m2 (range: 0.8-9.6). In both arms, the most common grade ≥3 treatment-emergent adverse events (TEAEs) were neutropenia (InO, 47% vs SC, 44%), thrombocytopenia (InO, 40% vs SC, 58%), febrile neutropenia (InO, 28% vs SC, 55%), leukopenia (InO, 29% vs SC, 34%), and anemia (InO, 24% vs SC, 44%), with similar rates between CD22 MESF expression quartiles (Table 1). Grade ≥3 hepatotoxicity occurred more frequently in the InO arm (17%) vs SC (8%), occurring with similar rates in the CD22 expression quartiles (Table 1). In the InO arm, more patients with ≥90% CD22 positivity received post-treatment SCT (60 [56%] vs 12 [34%] patients with CD22 <90%); 9 (8%) patients with ≥90% CD22 positivity and 2 (6%) patients with <90% also had a pre-treatment SCT. The rate of post-treatment SCT in the CD22 MESF expression quartiles is shown in Table 2. Among patients who proceeded to SCT in the InO arm, where VOD risk was higher, post-transplant VOD occurred in 16 (27%) patients with ≥90% CD22 positivity vs 2 (17%) patients with <90%. When assessing CD22 expression by MESF, the rate of post-transplant VOD was similar across CD22 MESF quartiles (Table 2).Conclusions: The risk of developing TEAEs, including hepatic adverse events, which were more common in the InO vs SC arm, was not associated with intensity of CD22 expression in either the InO or SC arm. Among InO patients with post-treatment SCT, there was no apparent relationship between CD22 expression and the risk of developing VOD, when analyzing CD22 by % positivity or by MESF. [Display omitted] DisclosuresDeAngelo:BMS: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria; Pfizer Inc: Consultancy, Honoraria; Shire: Honoraria; Takeda: Honoraria; Blueprint Medicines: Honoraria, Research Funding; ARIAD: Consultancy, Research Funding; Amgen: Consultancy; Glycomimetics: Research Funding; Incyte: Consultancy, Honoraria. Advani:Pfizer: Honoraria, Research Funding; Amgen: Research Funding; Novartis: Consultancy; Glycomimetics: Consultancy. O'Brien:Sunesis: Consultancy, Research Funding; Acerta: Research Funding; Pfizer: Consultancy, Research Funding; Aptose Biosciences Inc.: Consultancy; GlaxoSmithKline: Consultancy; Amgen: Consultancy; Gilead: Consultancy, Research Funding; Astellas: Consultancy; Alexion: Consultancy; Abbvie: Consultancy; Kite Pharma: Research Funding; Celgene: Consultancy; Janssen: Consultancy; Vaniam Group LLC: Consultancy; Pharmacyclics: Consultancy, Research Funding; Regeneron: Research Funding; TG Therapeutics: Consultancy, Research Funding. Marks:Novartis: Consultancy; Pfizer: Consultancy; Amgen: Consultancy. Kantarjian:Orsenix: Honoraria; Novartis: Research Funding; Immunogen: Honoraria; BMS: Honoraria, Research Funding; Astex: Research Funding; ARIAD: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Actinium: Honoraria; AbbVie: Honoraria; Pfizer: Honoraria, Research Funding. Wang:Pfizer: Employment, Equity Ownership. Vandendries:Pfizer: Employment, Equity Ownership. Laird:Pfizer: Employment, Equity Ownership. Nick:Pfizer: Employment, Equity Ownership. Stelljes:Amgen: Honoraria; MSD: Consultancy; JAZZ: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Honoraria.

Quantitative Flow Cytometry to Measure Viral Production Using Infectious Pancreatic Necrosis Virus as a Model: A Preliminary Study

In recent decades, flow cytometry (FCM) has become an important tool in virology, due to its applications in viral replication and viral-cell interactions, as well as its capacity to quantify proteins (qFCM). In the present study, we have designed and evaluated a qFCM procedure for the in vitro analysis and quantification of fish viral proteins, using the infectious pancreatic necrosis virus (IPNV) as a model. We have also tested its use for viral titration and adapted the MARIS (method for analysing RNA following intracellular sorting) method for simultaneous quantification of viral RNA expression in infected cells. The procedure has proved to be repeatable and reproducible to an acceptable level, although to ensure reproducibility, the repetition of standard curves is inevitable. Regarding its use for viral quantification, a direct relationship (by a second-degree polynomial regression) between viral titres and Molecules of Equivalent Soluble Fluorochrome (MESF) was observed. Finally, the results support the use of this technology, not only for virus quantification, but also to study viral replication from a quantitative approach.

Abstract 4636: The antibody-drug conjugate D3-GPC2-PBD potently eradicates neuroblastoma patient-derived xenografts

Abstract Background: We recently developed an antibody-drug conjugate (ADC; D3-GPC2-PBD) for neuroblastoma linking a specific glypican 2 (GPC2) targeting antibody (D3) to the potent DNA damaging pyrrolobenzodiazepine (PBD) dimers (Cancer Cell, 2017). Methods: To determine the spectrum of in vivo efficacy of the D3-GPC2-PBD ADC, we utilized a panel of highly annotated neuroblastoma patient-derived xenografts (PDXs) passaged in C.B-17 scid mice. Cohorts of mice (n = 9 - 10) were stratified to treatment with either vehicle or D3-GPC2-PBD at doses of 0.5 - 3 mg/kg given once or 1 mg/kg given twice weekly x 2 weeks when mean tumor volumes reached 200-300 mm3. Co-treatment with excess D3-GPC2-IgG1, the efficacy of free PBD dimers, re-treatment of the rare ADC treated tumors that regrew, treatment of larger PDXs, and the assessment of DNA damage and apoptosis by immunohistochemistry (IHC) were also explored. PDX GPC2 cell surface expression was quantified by flow cytometry. The binding of D3-GPC2-IgG1 to mouse Gpc2 was tested by flow cytometry and mouse normal tissue Gpc2 expression was profiled utilizing IHC. Results: We first showed D3-GPC2-IgG1 binds avidly to mouse Gpc2 by flow cytometry and that murine and human GPC2 expression is similarly restricted in normal tissues by IHC. To date, the D3-GPC2-PBD ADC was tested in three neuroblastoma PDXs with a range of GPC2 expression (Molecules of Equivalent Soluble Fluorochrome [MESF]) and genomic characteristics: CHLA79 (GPC2 MESF = 825, MYCN non-amplified, ALK wild-type, TP53 wild-type), COG-N-421x (GPC2 MESF = 3301, MYCN amplified, ALK wild-type, TP53 wild-type), and SKNAS (GPC2 MESF = 672, MYCN non-amplified, ALK wild-type, TP53 mutated). Treatment with D3-GPC2-PBD resulted in complete and sustained tumor regression up to 100 days in 61% (17/28) of mice treated with 1 mg/kg ADC, 96% (27/28) of mice treated with 3 mg/kg ADC, and 95% (18/19) of mice treated with 1 mg/kg ADC given twice weekly x 2 weeks. Treatment of the CHLA79 PDX with an equivalent dose of free PBD dimers had no effect on tumor growth and co-treatment with excess D3-GPC2-IgG1 (50x) abrogated a significant amount of the ADC induced tumor regression. The rare 1 mg/kg ADC treated CHLA79 PDXs that began to regrow after 100 days (n = 2) showed complete responses to re-dosing of the ADC. Additionally, the D3-GPC2-PBD ADC dosed at 1 mg/kg ADC given twice weekly x 2 weeks induced complete regression of larger COG-N-421x PDXs (tumor volumes of 600-1000 mm3). ADC treated tumors showed significant upregulation of DNA damage (γH2AX) and apoptosis (cleaved caspase 3 and cleaved PARP) by IHC. ADC treatment was well tolerated with no discernible drug-related toxicities. Conclusions: The D3-GPC2-PBD ADC is safe and potently efficacious in a genomically diverse panel of neuroblastoma PDXs with a range of GPC2 cell surface expression. These data support the development of a PBD dimer containing GPC2 directed ADC. Citation Format: Kristopher R. Bosse, Zhongyu Zhu, Maria Lane, Daniel Martinez, John DeLong, Doncho V. Zhelev, Yang Feng, Yanping Wang, Jennifer Hwang, Dimiter S. Dimitrov, John Maris. The antibody-drug conjugate D3-GPC2-PBD potently eradicates neuroblastoma patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4636.

Additional value of quantitative expression of ZAP-70 and CD38 for prognostic factors in chronic lymphocytic leukemia.

e19006 Background: Chronic lymphocytic leukemia (CLL) has heterogeneous clinical course. Identification of prognostic markers is important for proper management of disease. Cytogenetics, IgVH mutation status, TP53 functional status, CD38, ZAP-70 and CD49d expression are accepted prognostic factors in CLL. ζ-chain-associated protein kinase 70 (ZAP-70) is associated with disease progression. However, measurement of ZAP-70 and CD38 expression relies on qualitative flow cytometry. Authors tested hypothesis whether quantitative analysis of markers can improve information about prognosis of CLL. Methods: 217 CLL patients were included; diagnosis was made from peripheral blood by morphology and immunophenotyping by flow cytometry with common antibodies. Expressions of intracellular ZAP-70 and surface CD38 were measured using quantitative fluorescence cytometry. Standardized fluorescent microparticles were used to quantify molecules of equivalent soluble fluorochrome (MESF units). Analysis of rearrangement of the IgVH gene was performed according to the European recommendation. Deletions at 11q22-q23 ( ATM), 17p13 ( TP53), 13q34 ( RB1) loci and trisomy of chromosome 12 were detected by I-FISH. Results: Patients’ ages ranged from 33 to 86 years and included all Rai stages. Expression of ZAP-70 ranged from 546 to 5,955 MESF, CD38 from 1,886 to 31,619 MESF. 48% patients embodied mutated and 52% unmutated IgVH status. In cytogenetics, del(13q) was detected in 59%, trisomy 12 in 13%, del(11q) in 21%, and del(p53) in 8% of patients. IgVH (in %) was plotted against ZAP-70 (in MESF) and patients were divided into high (HR) and low risk (LR) groups by cluster analysis based on Kaplan-Meier overall survival curves. Patients in HR group had significantly poorer prognoses (p = 0.0001) than those in LR group. When patients from HR group were clustered according to CD38 expression, very high risk (VHR) group with high CD38 and significantly worse prognosis (p = 0.035) was identified. Conclusions: We have demonstrated that quantitative ZAP-70 and CD38 expressions cannot replace IgVH analysis, but, contrary to that, addition of quantitative data can further specify prognoses of patients with CLL.

Development of a flow cytometric method for quantification of BCL-2 family members in chronic lymphocytic leukemia and correlation with sensitivity to BCL-2 family inhibitors.

We have developed a quantitative fluorescence cytometry (QFCM) method that can be used to measure BCL-2 family member proteins in cell lines and clinical samples. We described the validation of antibodies, methods development and application of the assay. We characterized and validated antibodies to BCL-2, BCL-XL , and MCL-1 in cell lines to confirm specificity for flow cytometry. Each protein was measured in a panel of leukemia/lymphoma cell lines and B-cells from chronic lymphocytic leukemia (CLL) patients treated with the BCL-2/BCL-XL inhibitor navitoclax. The cellular activity of various BCL-2 family member inhibitors alone and in combination was determined to demonstrate utility of our assay to correlate protein levels with efficacy. We identified antibodies that were highly specific for each protein. The expression profile in cell lines as determined by molecules of equivalent soluble fluorochrome was comparable to western blot. Using our assay, BCL-2, BCL-XL , and MCL-1 protein levels were shown to correlate with response to BCL-2 family inhibitors in vitro and could be measured in clinical samples. This method can quantify BCL-2 family members in a specific, highly reproducible and sensitive fashion, and requires fewer cells compared to western blot. It is particularly useful for identifying BCL-2, BCL-XL , and MCL-1 protein levels in a specific cell population within a heterogeneous population like those collected from CLL patients. These data show that our QFCM method can be used to facilitate the quantification and evaluation of biomarkers predictive of response in patients treated with BCL-2 family member inhibitors. © 2016 International Clinical Cytometry Society.

Validation of a flow cytometry-based assay to assess C5aR receptor occupancy on neutrophils and monocytes for use in drug development.

The C5a/C5a receptor (C5aR) pathway, a key component in the proinflammatory immune response, is an attractive therapeutic target since its dysregulation is implicated in a variety of autoimmune and inflammatory disorders. The objective of the present study was to validate a receptor occupancy (RO) assay for a human anti-C5aR monoclonal antibody drug candidate, NNC0215-0384 (NN0384). This flow cytometry-based assay measures the percentage (%), median fluorescence intensity (MFI), and molecules of equivalent soluble fluorochrome (MESF) of NN0384 binding to its target cells, neutrophils and monocytes, in whole blood from normal healthy donors and rheumatoid arthritis (RA) patients with clinically active disease. The validation parameters assessed included postcollection and postprocessing sample stability, intra- and interassay precision, an analyst-to-analyst comparison, a comparison of normal healthy donor and RA patient sample postcollection stability, and a laboratory-to-laboratory comparison and assay transfer. The cumulative results indicate that the assay was reproducible, met the clearly defined acceptance criteria for the validation parameters tested, and was transferable to another laboratory. In conclusion, this RO assay is suitable for use to accrue pharmacodynamic biomarker data in a multicenter, global clinical trial.

형광 영상 기반 CD4 신속 검사법 개발을 위한 항원-항체 반응 조건 정량화

Abstract. CD4+ T-cell count determines the effectiveness for antiretroviral therapy (ART) in patients with human immunodeficiency virus (HIV). Although ART slows the progression of HIV to AIDS, rapid counting of CD4+ T lymphocytes with a drop of patient’s blood sample is urgently needed to ensure timely ART treatment in rural areas. Recently point-of-care CD4 testing devices have been developed by using non-flow based imaging cytometer incorporated with a sample cartridge where CD4+ T cells are reacted with fluorescently tagged specific antibodies. Here we conducted an experimental study using a conventional fluorescence microscope-based imaging system to quantitate the interaction of CD4 antibodies with CD4+ T cells at different reaction conditions. We demonstrated that a fast and affordable point-of-care CD4 test is feasible with a far less amount of antibodies and a shorter incubation time compared with a conventional sample preparation protocol for flow cytometry. We also proposed a general method to evaluate and compare the detection limit across different CD4 counting platforms by using fluorescently labelled microbeads for intensity calibration. Key Words:CD4+ T lymphocyte(CD4 양성 T 림프구), Fluorescence(형광), Antigen-Antibody Reaction(항원-항체 반응), Point-of-Care Test(현장검사), Calibration Bead(보정입자), MESF(Molecules of Equivalent Soluble Fluorochrome)

P005 : LOW LEVEL ANTI-HLA ANTIBODIES: DETERMINING RELEVANT UNACCEPTABLE ANTIGENS BY SINGLE ANTIGEN MICROARRAY AND SPECIFIC/SURROGATE FLOW CROSSMATCHES

A challenge to any HLA laboratory is to determine the cutoff or level of anti-HLA antibody that ultimately determines the unacceptable antigens (UA) of a potential transplant patient. These UA’s are placed In UNET for virtual crossmatching. Once accomplished, this level is accepted for all HLA antigens as demonstrated by the single antigen bead arrays. The microarray assay is not quantitative and may give false positive reactions at low levels. The challenge is to determine if these antibodies would cause a positive flow T and B cell Flow crossmatch (FLXM). The object of this study is to verify that a low or intermediate Mean Fluorescence Intensity (MFI) anti HLA antibody will or will not cause a positive FLXM with extra deceased donor or normal individual’s lymphocytes. We performed 159 FLXM on 31 patient sera. Only one antibody specificity that could cause a positive FLXM was demonstrated on each of these cells by molecular typing. Our MFI cutoff was 2000 MFI (+DTT). The pronase FLXM Molecules of Equivalent Soluble Fluorochrome (MESF) cutoff was 1500 (+500) for T cell and 2500 (+500) for B cell. Twenty-three patients had negative crossmatches when cross matched by at least five different cells and their serum. The false positive UA as demonstrated by a negative FLXM were removed from UNET. The MFI values for the UA’s varied from 1000 to 5000. There was no correlation of MFI levels to outcome of FLXM. One patient received a renal transplant and two were removed from the wait list. In conclusion: the use of FLXM to demonstrate relevant levels of anti-HLA antibody for each individual patient and allowed us to remove false positive UA’s. This type of analysis gives a potential kidney recipient a greater chance to receive a renal transplant.