scholarly journals Development of a Hydrashift 2/4 Isatuximab Assay to Mitigate Interference with Monoclonal Protein Detection on Immunofixation Electrophoresis in Vitro Diagnostic Tests in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-15
Author(s):  
Greg Finn ◽  
Sandrine Macé ◽  
Ruiyin Chu ◽  
Helgi van de Velde ◽  
Samia Menad ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Outcomes ◽  
Normal Serum ◽  
M Protein ◽  
Efficient Removal ◽  
Serum Samples ◽  
In Vitro Diagnostic ◽  
Acid Violet ◽  
Monoclonal Components

Introduction Evaluation of multiple myeloma (MM) response through quantification of M-protein, by serum protein electrophoresis (SPEP) and immuno-fixation electrophoresis (IFE) is challenging for clinical laboratories because therapeutic monoclonal antibodies (mAbs) can confound IFE when they converge with serum M-protein. This can be misleading when interpreting patients' response to therapy. Isatuximab (Isa), an IgG-kappa anti-CD38 mAb is approved based on the pivotal ICARIA-MM study in combination with pomalidomide (P) and dexamethasone (d), in the United States, the European Union, Canada, Australia, Switzerland and Japan for the treatment of adult patients with relapsed/refractory MM who have received at least two prior therapies including lenalidomide and a proteasome inhibitor. Recently, the Phase 3 IKEMA study evaluating Isa plus carfilzomib (K) and d met its primary endpoint at the first planned interim analysis, demonstrating significantly prolonged progression free survival (PFS) compared with Kd alone in patients with relapsed MM. Methods To overcome the interference mediated by Isa on the IFE test, we have developed an Isa-specific assay "HYDRASHIFT 2/4 Isa" using a highly specific rabbit, anti-Isa mAb. The HYDRASHIFT 2/4 Isa test is used in conjunction with the regular Hydragel IF procedure and the semi-automated Hydrasys 2 electrophoresis apparatus. The HYDRASHIFT 2/4 Isa test displaces Isa (IgG kappa) interference during the electrophoresis process by forming Isa/anti-Isa complex which is moved out of the gamma zone toward the alpha globulin fractions. Results: Sensitivity of the HYDRASHIFT 2/4 Isa assay was demonstrated on serum samples, including normal serum samples. Isa control and serum samples with different monoclonal components spiked with Isa (final concentrations in the range of 0.1 and 3.0 g/L) were analyzed with the HYDRASHIFT 2/4 Isa procedure used in conjunction with Hydragel 4 IF Acid Violet. The detection limit of Isa and/or the Isa / anti-Isa antibody complex visualized was 0.2 g/L. Importantly, efficient removal of Isa from the gamma globulin zone, with no trace signal evident, was demonstrated for all Isa concentrations tested, even for 3 g/L. Reproducibility of the HYDRASHIFT 2/4 Isa assay was also demonstrated between gels, between product lots, between instruments and on different test days. Ten different serum samples, including 1 normal serum sample and 9 serum samples with monoclonal components spiked with Isa at 1 g/L, were run using the HYDRASHIFT 2/4 Isa procedure in conjunction with Hydragel 4 IF Acid Violet. All samples gave 100% concordant results between gels on the different instruments and with different HYDRASHIFT 2/4 Isa lots. To evaluate specificity, 50 serum samples from MM patients were spiked with 1 g/L Isa and the anti-Isa mAb shifted Isa specifically with no impact on the patients' M-spike, demonstrating 100% specificity. Finally, the HYDRASHIFT 2/4 Isa test was evaluated on 15 samples from treated patients enrolled in Isa clinical trials at different therapy cycles. The evaluated samples demonstrated efficient removal of Isa from G and Kappa tracks on the gamma globulins zone and visualization of the Isa/anti-Isa complex on alpha zone and/or Isa on ELP track. For the pre-treatment samples, there was as expected no Isa detection, and no impact on M-protein was observed, further confirming the specificity of the assay toward Isa. Conclusions Therapeutic mAb inclusion in MM treatment regimens offer patients significant improvements in clinical outcomes. With rapid evolution of therapeutic options in MM, there is a clear need for a standardized and reliable method to ensure authentic IFE-based clinical assessment. Development of the Isa-specific HYDRASHIFT 2/4 Isa assay offers the advantage of high clinical utility due to the simplicity of the method as an add-on to the conventional IFE In vitro diagnostic test. Consequently, this assay will facilitate the correct assessment of clinical outcomes for patients receiving Isa as part of their MM treatment. Submission for global regulatory clearance for Isa-specific HYDRASHIFT 2/4 assay is planned in 2020. Disclosures Finn: Sanofi: Current Employment. Macé:Sanofi: Current Employment. Chu:Sanofi: Current Employment. van de Velde:Sanofi: Current Employment, Current equity holder in publicly-traded company. Melki:Sebia: Current Employment.

scholarly journals Seroprevalence of Anti-SARS-CoV-2 IgG and IgM among Adults over 65 Years Old in the South of Italy

Diagnostics ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 483
Author(s):  
Immacolata Polvere ◽  
Alfredina Parrella ◽  
Giovanna Casamassa ◽  
Silvia D’Andrea ◽  
Annamaria Tizzano ◽  
...  
Keyword(s):  
Antibody Response ◽  
Immunoglobulin M ◽  
Molecular Testing ◽  
Elisa Assay ◽  
The South ◽  
Serum Samples ◽  
Serological Screening ◽  
Rna Detection ◽  
In Vitro Diagnostic

SARS-CoV-2 is a zoonotic betacoronavirus associated with worldwide transmission of COVID-19 disease. By the beginning of March, WHO reported about 113,820,000 confirmed cases including more than 2,527,000 deaths all over the world. However, the true extent of virus circulation or its real infection/fatality ratio is not well-estimated due to the huge portion of asymptomatic infections. In this observational study, we have estimated the prevalence of specific immunoglobulin M and G directed towards SARS-CoV-2 antigen in a cohort of 1383 adult volunteers aged over 65 years old, living in the district of Benevento, in the South of Italy. Serological screening was carried out on capillary blood in September 2020, seven months after pandemic outbreak in Italy, to evaluate virus circulation and antibody response among elderly adults, in which severe symptoms due to viral infection are more common. The overall seroprevalence of anti-SARS-CoV-2 antibodies was 4.70% (CI 3.70%–5.95%) with no statistically significant differences between sexes. Among these, 69.69% (CI 55.61%–77.80%) tested positive to IgM, 23.08% (CI 14.51%–34.64%) to IgG and 9.23% (CI 4.30%–18.71%) was positive for both. All patients that were positive to IgM underwent molecular testing through RT-qPCR on oral-rhino pharyngeal swabs and only one specimen was positive for SARS-CoV-2 RNA detection. Instead, the presence of IgG from screened volunteers was confirmed by re-testing serum samples using both an ELISA assay validated for in vitro diagnostic use (IVD) and a recently published synthetic peptide-based ELISA assay. In conclusion, our report suggests that (1) early restrictions were successful in limiting COVID-19 diffusion in the district of Benevento; (2) rapid serological analysis is an ideal testing for both determining real seroprevalence and massive screening, whereas detection of viral RNA remains a gold standard for identification of infected patients; (3) even among people without COVID-19 related symptoms, the antibody response against SARS-CoV-2 antigens has individual features.

scholarly journals Gaps in the Traceability Chain of Human Growth Hormone Measurements

2013 ◽  
Vol 59 (7) ◽  
pp. 1074-1082 ◽  
Author(s):  
Sébastien Boulo ◽  
Katja Hanisch ◽  
Martin Bidlingmaier ◽  
Cristian-Gabriel Arsene ◽  
Mauro Panteghini ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Human Growth Hormone ◽  
Human Growth ◽  
Higher Order ◽  
Serum Samples ◽  
Traceability Chain ◽  
Eu Directive ◽  
In Vitro Diagnostic ◽  
Relative Differences

BACKGROUND Human growth hormone (hGH) is measured for the diagnosis of secretion disorders. These measurements fall under the EU Directive 98/79/EC on in vitro diagnostic medical devices requiring traceability of commercial calibrator values to higher-order reference materials or procedures (Off J Eur Communities 1998 Dec 7;L 331:1–37). External quality assessment schemes show large discrepancies between results from different methods, even though most methods provide results traceable to the recommended International Standard (IS 98/574). The aim of this study was to investigate possible causes for these discrepancies. METHODS We investigated the commutability and recovery of hGH in reconstituted IS 98/574. We tested different reconstitution protocols and used 4 different serum matrices for spiking. These IS preparations were measured together with serum samples. We quantified hGH by 5 different methods in 4 different laboratories. RESULTS Results from the different methods correlated well for the serum samples. Mean discrepancies between results from different methods were ≤20%. None of the IS preparations was commutable for all the method comparisons. The recovery of hGH in preparations of IS 98/574 depended on the reconstitution protocol (>10-fold differences) and BACKGROUND matrix (relative differences ≤17% for different serum matrices). CONCLUSIONS The use of different protocols for reconstitution and spiking of hGH reference preparations affects quantification by immunoassays, potentially leading to a bias between commercial methods, despite the use of calibrators with values claimed to be traceable to the same higher-order reference material.

scholarly journals IgA Pyroglobulin, Hyperviscosity Syndrome and Coagulation Abnormality in a Patient With Multiple Myeloma

Blood ◽  
1972 ◽  
Vol 39 (2) ◽  
pp. 224-237 ◽  
Author(s):  
Susumu Sugai
Keyword(s):  
Multiple Myeloma ◽  
Blood Clotting ◽  
Guanidine Hydrochloride ◽  
Signs And Symptoms ◽  
High Serum ◽  
M Protein ◽  
Sulfhydryl Reagents ◽  
Blurred Vision ◽  
Intermolecular Reaction

Abstract A 48-yr-old woman with a bleeding diathesis and complaining of blurred vision, headache, paresthesias, and vomoting was diagnosed as having IgA, kappa type multiple myeloma. Markedly increaesed serum viscosity was noted. After plasmapheresis the signs and symptoms of high serum viscosity disappeared. Pyroglobulin was found in the M protein. The M protein had a prominent 15.5S peak and smaller 17.0 and 20S peaks, which decreased to a 6.7S peak after in vitro reduction with 0.1 M 2-mearcptoethanol, 2-mercaptoethylamine hydrochloride, or glutathione. The serum viscosity was decreased by treatment with these reducing agents. Pyroglobulin formation was presumably a noncovalent intermolecular reaction, as it was inhibited by 5.0 M guanidine hydrochloride, acidification and alkalinization, but not by reduction with sulfhydryl reagents. The M protein also had an antithrombin activity causing interference with the conversion of fibrinogen to fibrin and the phenomenon of gelification noted on blood clotting.

scholarly journals Distinguishing Drug from Disease by Use of the Hydrashift 2/4 Daratumumab Assay

2019 ◽  
Vol 3 (5) ◽  
pp. 857-863 ◽  
Author(s):  
Katie L Thoren ◽  
Matthew J Pianko ◽  
Youssef Maakaroun ◽  
C Ola Landgren ◽  
Lakshmi V Ramanathan
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
False Positive ◽  
Protein Electrophoresis ◽  
Response To Therapy ◽  
M Protein ◽  
Oligoclonal Bands ◽  
Serum Samples ◽  
The Status ◽  
Pretreatment Serum

Abstract Background Daratumumab, a monoclonal antibody used to treat relapsed or refractory multiple myeloma, can interfere with protein electrophoresis and immunofixation assays. False-positive immunofixation results due to daratumumab can cause uncertainty regarding the status of a patient's disease and lead to potential misclassification of their response to therapy. The Hydrashift 2/4 Daratumumab assay (Sebia) was recently cleared by the Food and Drug Administration for resolving daratumumab interference on immunofixation. Here, we evaluate the performance of the Hydrashift assay in multiple myeloma patients receiving treatment with daratumumab-based regimens. Methods Waste serum samples from multiple myeloma patients (n = 40) receiving daratumumab were analyzed by standard immunofixation and the Hydrashift assay. Results from these tests were compared and were evaluated along with pretreatment serum protein electrophoresis and immunofixation results, if available. Results The Hydrashift assay shifted the migration of daratumumab in patient samples. In 27 cases, the patient's M protein was distinguishable from daratumumab by standard immunofixation. In these cases, the Hydrashift assay confirmed that the IgGκ band was daratumumab and helped identify the presence of treatment-related oligoclonal bands. There were 11 instances in which the patient's IgGκ M protein comigrated with daratumumab. In all 11 cases, the Hydrashift assay confirmed the presence of residual M protein. Finally, in 2 patients whose pretreatment immunofixation results were not available, the Hydrashift assay confirmed that the IgGκ band visible on immunofixation was due to daratumumab alone. Conclusions The Hydrashift 2/4 Daratumumab assay is a useful tool to clarify the source of an IgGκ band on immunofixation and allow a patient's M protein to be viewed without interference.

A Phase I, Multi-Dose, Dose Escalation Study of SGN-40 (anti-huCD40 mAb) in Patients with Refractory or Recurrent Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2413-2413
Author(s):  
Mohamad A. Hussein ◽  
Ruben Niesvizky ◽  
Nikhil Munshi ◽  
James C. Berenson ◽  
Kenneth C. Anderson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Phase I ◽  
Dose Escalation ◽  
Transmembrane Protein ◽  
M Protein ◽  
Type I ◽  
Antitumor Effects ◽  
Grade 3 ◽  
Dose Levels

Abstract CD40 is a type I transmembrane protein that upon binding to CD40 ligand regulates important biologic effects in the immune system. CD40 is also highly expressed on hematologic tumors, which has raised interest in the potential for its use as a tumor target for antibody-based cancer therapy. SGN-40 is a humanized monoclonal antibody that selectively binds to human CD40 and induces apoptosis and growth inhibition of a wide variety of B-cell derived cancer cell lines in vitro. Our preclinical work has confirmed the in vitro cytotoxicity of SGN-40 against human multiple myeloma (MM) cells via several mechanisms. These include induction of cytotoxic ligands of TNF superfamily; suppression of IL-6-induced proliferative and anti-apoptotic effects, as well as antibody-dependent cell-mediated cytotoxicity (Tai, et al, Cancer Research64, 2846–2852, April 15, 2004). Since ≥ 90% of MM cells express CD40, targeting CD40 using SGN-40 presents a potential novel treatment strategy. Based on these preclinical data, a phase I study is being conducted to define the toxicity profile, characterize the pharmacokinetics (PK), and evaluate antitumor effects of SGN-40 in patients with refractory or recurrent MM. Four weekly doses ranging from 0.5 to 16 mg/kg are planned to be administered to groups of at least three patients per cohort. Patients will be followed for up to 6 weeks post their last dose. Currently, a total of seven patients have been treated with SGN-40 at dose levels of 0.5 and 1.0 mg/kg. No grade 3 or 4 non-hematologic dose limiting toxicities have been observed. One patient experienced a transient Grade 3 decrease in hemoglobin. Decrease in CD19 positive B-cells were noted for patients treated at both dose levels. Changes in serum and urine M protein levels were measured to estimate potential anti-tumor effects of SGN-40. Of the seven patients evaluated, one patient at 0.5 mg/kg dose had stable disease, based on serum M protein, over the 10 week study period. Clinical evaluation with dose escalation of this agent continues and updated safety, PK and antitumor data will be presented.

Safety and Efficacy of Daratumumab with Lenalidomide and Dexamethasone in Relapsed or Relapsed, Refractory Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 84-84 ◽  
Author(s):  
Torben Plesner ◽  
Hendrik-Tobias Arkenau ◽  
Henk M. Lokhorst ◽  
Peter Gimsing ◽  
Jakub Krejcik ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Dose Escalation ◽  
Research Funding ◽  
M Protein ◽  
Open Label ◽  
Advisory Committees ◽  
Dose Escalation Study ◽  
Human Dose

Abstract Background: Daratumumab (DARA) (HuMax™-CD38), a human IgG1κ monoclonal antibody effectively mediates destruction of CD38-expressing malignant plasma cells. In the first-in-human dose-escalation study, 42% of heavily pretreated patients with relapsed, or relapsed, refractory (RR) multiple myeloma (MM) treated with DARA alone (≥4mg/kg) achieved partial response (PR) and 25% had minimal response (MR) (modified IMWG guidelines) (1). In preclinical studies, DARA + lenalidomide (LEN) enhanced killing of MM cells in vitro (2). We evaluated safety, pharmacokinetics (PK) and efficacy of DARA + LEN + low-dose dexamethasone (DEX) in patients with relapsed or RR MM. Methods: This ongoing phase I/II open-label multicenter study consisted of 2 parts: Part1 was dose-escalation study in which patients (≥ 18 years old) with life expectancy ≥3 months and ECOG status 0, 1 or 2 received DARA+LEN+DEX (DARA [2-16 mg/kg] per week [8 weeks], twice a month [16 weeks], then, once monthly until disease progression, unmanageable toxicity or 24 months in total; LEN [25 mg PO day 1 through 21 of 28-days cycles]; DEX [40 mg] once weekly). Part 2 was cohort expansion study which explored the testing of maximum tolerated DARA dose (MTD) (16 mg/kg) determined in part 1 along with LEN (25 mg mg PO day 1 through 21 of 28-days cycles) and DEX (40 mg) once weekly. Results: Data from 22 patients (13 patients [fully enrolled] from part 1 and 9 patients from part 2, [ongoing enrollment]) were presented at ASCO earlier this year (3). These results demonstrated that the most frequent (>30% patients) adverse events (AEs) were neutropenia and diarrhea; no dose limiting toxicities (DLTs) were reported. Infusion reactions (grade 1 and 2) were reported in 4 patients. 8 serious AEs were reported, all assessed as unrelated to DARA. MTD was not reached. DARA+LEN+DEX PK-profile was similar to DARA alone suggesting LEN and DEX do not affect the DARA PK-profile. Available preliminary efficacy data from 20 patients demonstrated marked decrease in M-protein in all patients; 15/20 patients achieved PR or better, 3/20 with CR, 6/20 with VGPR. Median time to response was 4.3 weeks (range: 2.1-11.3). Overall response rate (ORR) was 75% (15/20) combining all patients in part 1 and 2 and 92.3% (12/13) for part 1 patients, who had at least 2 months of follow-up or discontinued earlier. Conclusions: DARA+LEN+DEX has favorable safety profile with manageable toxicities in relapsed and RR MM. Encouraging early activity is seen with marked reduction in M-protein and majority of the patients (~75%) achieved PR or better. Results of approximately 30 patients from part 2 with at least 2 months of treatment exposure and 10 patients (out of 30 patients) with shortened duration of infusion will be presented. References Lokhorst et. al., EHA 2013 abstract #8512 van der Veer et. al., Haematologica 2011;96(2):284-90 Plesner et. al. J Clin Oncol 32:5s, 2014 (suppl; abstr 8533). Disclosures Plesner: Genmab: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; Celegene: Membership on an entity's Board of Directors or advisory committees. Lokhorst:Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Minnema:Janssen: Consultancy, Honoraria. Laubach:Onyx: Research Funding; Novartis: Research Funding; Millenium: Research Funding; Celgene: Research Funding. Ahmadi:Janssen: Employment. Yeh:Janssen: Employment. Guckert:Janssen: Employment. Feng:Janssen: Employment. Brun:Genmab: Employment. Lisby:Genmab: Employment. Basse:Genmab: Employment. Palumbo:Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding.

A Novel Bcma-Specific, Centyrin-Based CAR-T Product for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2127-2127 ◽  
Author(s):  
David L. Hermanson ◽  
Burton Earle Barnett ◽  
Srinivas Rengarajan ◽  
Rebecca Codde ◽  
Xinxin Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
M Protein ◽  
Manufacturing Processes ◽  
Protein Levels ◽  
Car T

Abstract Chimeric-antigen receptor (CAR)-T cell immunotherapies have been remarkably effective in treating acute lymphoblastic leukemia. However, current strategies generally suffer from difficult, inefficient and costly manufacturing processes, significant patient side effects and poor durability of response in some patients. Here, we report for the first time a CAR-T cell therapeutic comprising a non-immunoglobulin alternative scaffold Centyrin molecule (a "CARTyrin") manufactured with a novel non-viral piggyBacTM (PB) transposon-based system. Our lead candidate, P-BCMA-101, encodes a CARTyrin that targets the B cell maturation antigen (BCMA) for the treatment of multiple myeloma (MM) and has several unique aspects that improve upon earlier CAR-T products. First, P-BCMA-101 is manufactured using only in vitro transcribed mRNA and plasmid DNA without the need for lentivirus or g-retrovirus, resulting in time and cost savings. Importantly, PB is also safer than viral systems due to a less mutagenic insertional profile and is non-oncogenic. Furthermore, PB can efficiently deliver transgenes as large as several hundred kilobases, and, once inserted, transgenes demonstrate more stable, prolonged and higher expression when compared to those delivered by virus. Second, a mutein of the dihydrofolate reductase (DHFR) gene is included in the P-BCMA-101 transgene that can be used in combination with the non-genotoxic drug methotrexate (MTX) to provide a simple and effective method of CARTyrin+ cell enrichment and reduces variability in patient product material. Third, P-BCMA-101 incorporates a safety switch for optional depletion in vivo in case of adverse events. Lastly, the CARTyrin is comprised of a BCMA-specific Centyrin, which are based on a human tenascin fibronectin type III (FN3) consensus sequence. Centyrins have similar binding affinities to the antibody-derived single chain variable fragments (scFv), but are smaller, more thermostable and predicted to be less immunogenic. Importantly, no signs of tonic signaling leading to T cell exhaustion have been observed with CARTyrins unlike scFv-based CAR molecules, which can interact with each other on the surface causing non-specific CAR signaling. The manufacture process of P-BCMA-101 from primary human T cells is straightforward, employs no cytokines, and easily produces enough CARTyrin+ cells to treat patients. Within 18 days of electroporation of purified T cells, we demonstrate > 95% of the cell product is positive for CARTyrin expression and ready to be administered. Notably, our manufacturing process results in > 60% of CARTyrin+ T cells exhibiting a stem-cell memory phenotype (i.e. CD45RA+ CD62L+). P-BCMA-101 cells exhibit specific and robust in vitro activity against BCMA+ tumor targets, ranging from high to very low levels of BCMA, as measured by target-cell killing and CARTyrin-T cell proliferation. Importantly, proliferating P-BCMA-101 cells were highly sensitive in vitro to activation of the safety switch. Finally, we have evaluated the anti-tumor efficacy of P-BCMA-101 in a model of human MM. NSG™ mice were injected IV with 1.5x106 luciferase+ MM.1S cells, an aggressive human MM-derived cell line. After the tumor cells were allowed to grow for 21 days, animals received a single IV administration of 5x106 P-BCMA-101 cells. All untreated control animals demonstrated a marked increase in serum M-protein levels, rapid growth of tumor cells demonstrated by bioluminescent imaging (BLI), and death within four weeks. In stark contrast, 100% of animals that received P-BCMA-101 rapidly eliminated tumors within 7 days as measured by BLI and serum M-protein levels and improved survival out to at least 60 days post-treatment. P-BCMA-101 is the first-in-class of Centyrin-based CAR therapeutics. The CARTyrin, combined with our advanced manufacturing processes, represents a significant improvement over first generation, immunoglobulin-based and virally-transduced CAR-T products. P-BCMA-101 exhibited an advantageous stem-cell memory phenotype and demonstrated specific and potent anti-tumor efficacy against BCMA+ myeloma cells both in vitro and in vivo. Based on these results, we plan to initiate a phase I clinical trial of P-BCMA-101 for the treatment of patients with relapsed and/or refractory MM. Disclosures Hermanson: Poseida Therapeutics: Employment. Barnett:Poseida Therapeutics: Employment. Rengarajan:Poseida Therapeutics: Employment. Codde:Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Martin:Poseida Therapeutics: Employment. Smith:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

scholarly journals Detection of M-Protein in Acetonitrile Precipitates of Serum Using MALDI-TOF Mass Spectrometry: A Novel Methodology

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-37
Author(s):  
Nikita Mehra ◽  
Gopal Gopisetty ◽  
Jayavelu S ◽  
Arivazhagan Rajamanickam ◽  
Shirley Sundersingh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mass Spectra ◽  
The Other ◽  
M Protein ◽  
Light Chains ◽  
Serum Samples ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms ◽  
Apparently Healthy

Background: Multiple myeloma (MM) and plasmacytoma(s) belong to a group of clonal plasma cell dyscrasias. In 97-98% of all cases, they are characterised by the detection of a monoclonal protein (M-protein) in the blood, and sometimes in the urine. MALDI-TOF-mass spectrometry (MS) has demonstrated excellent analytical sensitivity for the screening and detection of M-protein. We present the results of a novel methodology for M-protein analysis by MALDI-TOF MS. Patients and Methods: Blood samples from patients and controls were collected after obtaining Institutional Ethics Committee approval. The work was carried out in accordance with the Declaration of Helsinki after obtaining written informed consent. Patients with confirmed multiple myeloma or plasmacytoma and M-protein detected by serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (IFE), and serum free light chains (FLC) were included for MALDI-TOF MS analysis. IFE and FLC analysis were sent to independent laboratories for external validation of the MALDI-TOF MS results. Reagent-based extraction The serum fraction was separated from whole blood by centrifugation at 5000 rpm for 15 minutes and stored at -80oC until further analysis. Twenty-five μL of the serum sample was mixed with 50% acetonitrile (ACN) to form a precipitate. After precipitation and incubation, the mixture was centrifuged. The protein precipitate was washed with 20% ACN. After centrifugation, the supernatant was discarded, and the precipitate was reconstituted in a buffer comprising 10% formic acid (FA) and 50 mmol/L tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The MALDI-TOF MS results were validated using immunoenrichment by anti-kappa (κ) and anti-lambda (λ) biotin-labelled antibodies immobilised on streptavidin magnetic beads. MALDI-TOF MS measurements were obtained for intact proteins using alpha-cyano-4-hydroxycinnamic acid as a matrix. The images obtained were overlaid on apparently healthy serum samples to confirm the presence of M-protein. The samples were then analysed using UltraflexTM LT, Bruker MALDI/TOF-TOF mass spectrometer. The mass spectra for each sample was exported to FlexAnalysis 3.3 (Bruker Daltonics) and background subtracted. A sample was considered positive for M-protein if there was a sharp or broad peak within the κ or λ mass/charge (m/z) range- κ m/z- [M+2H]2+: 11550-12300 Da; [M+H]+: 23100-24600 Da), and λ m/z- [M+2H]2+: 11100-11500 Da; [M+H]+: 22200-23100 Da. All the images were acquired at a m/z range of 10000-29000 Da. Mass measurement was analysed with a summation of 500-5000 shots depending on the intensity of the M-peak. Results: Twenty-seven patient samples: 24- multiple myeloma, and 3- plasmacytoma with an M-protein identified by other biochemical tests, were chosen for ACN precipitation and analysed by MALDI-TOF MS. The median age was 62 years (range:44-72); males-12 (44%). A mass spectrometrist, S.J was blinded to the IFE and FLC results- blinded analyst. N.M was the unblinded analyst. Neat sample (without dilution) was spotted on the MALDI plate for all the control and patient samples. The Gaussian distribution of κ and λ light chains were obtained by analysing 20 serum samples of apparently healthy blood donors. All the 27 samples (100%) with M-protein confirmed by the other biochemical techniques, demonstrated a peak suggestive of M-protein with mass/charge (m/z) falling within the κ or λ range on MALDI-TOF MS: 24 patients with κ peak, and 3 with λ peak. (Fig. 1a and 1b) Immunoenrichment was performed on two samples- 1 with κ peak, and the other with λ peak, analysed by MALDI-TOF-MS by ACN precipitation. The mass spectra by immunoenrichment and ACN precipitation were found to be identical with the light chain m/z falling within their respective range. (Fig. 2 and 3) Three samples were labelled as confounders due to low peak intensity. However, their peaks matched their corresponding IFE and FLC reports. Concordance between MALDI-TOF MS and IFE was observed in 21/23 patients (91%); concordance between MALDI-TOF MS and FLC was observed in 23/24 patients (96%). Conclusions: We report the results of a low-cost, reagent-based extraction process using ACN precipitation to enrich for κ and λ light chains, which can be used for screening and for qualitative analysis of M-protein. Further studies are required to identify the immunoglobulin isotype, and to quantify the M-protein by this methodology. Disclosures No relevant conflicts of interest to declare.

scholarly journals Separation of human myeloma cells from bone marrow aspirates in multiple myeloma and their proliferation and M-protein secretion in vitro

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 562-566 ◽  
Author(s):  
K Iwato ◽  
M Kawano ◽  
H Asaoku ◽  
O Tanabe ◽  
H Tanaka ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Protein Secretion ◽  
Secretion Rate ◽  
M Protein ◽  
Cell Fraction ◽  
Bone Marrow Mononuclear Cell ◽  
Myeloma Cells ◽  
Human Myeloma Cells

Abstract Human myeloma cells were purified from bone marrow aspirates from patients with multiple myeloma (MM) by Percoll discontinuous density- gradient centrifugation, E rosette formation and treatment with antimyelomonocytic antibody (Leu M1), plus complement. Thus, the purified cell fraction consisted of greater than 90% myeloma cells, even when as little as 15% myeloma cells were contained in bone marrow mononuclear cell fraction, determined by morphological and immunologic examinations. With highly purified myeloma cells from 29 patients with IgG type MM, biologic characteristics such as spontaneous proliferation (3H-TdR uptake) and M-protein secretion rate in vitro were evaluated. Both activities varied among patients within stage I and III, and a 3H- TdR uptake of 255–24, 132 cpm/4 x 10(4) cells, and an M-protein secretion rate of 9 to 72 pg/cell/day, respectively, were recorded. However, in each patient, there was no correlation between 3H-TdR uptake and M-protein secretion rate. These results thus suggest that 3H- TdR uptake and M-protein secretion rate of highly purified myeloma cells are independent biologic parameters, not associated with the clinical stages, and the purification of myeloma cells we describe can contribute to further studies on the biologic characteristics and to understanding of the pathophysiology involved in MM.

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