Pharmacokinetic analyses of clopidogrel are hampered by the existence of multiple active metabolite isomers (H1 to H4) and their instability in blood. We sought to retest the pharmacodynamic activities of the four individual active metabolite isomers in vitro, with the ultimate aim of determining the isomers responsible for clopidogrel activity in vivo. In vitro activity was evaluated by measuring binding of [³³P]-2-methylthio-ADP on P2Y₁₂-expressing Chinese hamster ovary (CHO) cells and human platelets in platelet-rich plasma (PRP). A stereoselective method that used reverse-phase ultra high-performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS) was developed to measure individual concentrations of the stable 3'-methoxyacetophenone (MP) derivatives of H1-H4. The new method was used to analyze plasma samples from clopidogrel-treated subjects enrolled in a phase I clinical trial. In vitro binding assays confirmed the previously observed biological activity of H4 (IC₅₀: CHO-P2Y₁₂: 0.12 μM; PRP: 0.97 μM) and inactivity of H3, and demonstrated that H1 was also inactive. Furthermore, H2 demonstrated approximately half of the biological activity in vitro compared with H4. Optimisation of UHPLC conditions and MS collision parameters allowed the resolution and detection of the four derivatised active metabolite isomers (MP-H1 to MP-H4). The stereoselective assay was extensively validated, and was accurate and precise over the concentration range 0.5-250 ng/ml. Only MP-H3 and MP-H4 were quantifiable in incurred clinical samples. Based on in vitro pharmacodynamic data and found concentrations, the active metabolite isomer H4 is the only diastereoisomer of clinical relevance for documenting the pharmacokinetic profile of the active metabolite of clopidogrel.

8529 Background: We report updated data from a Phase Ib study of Isa, a CD38 monoclonal antibody, plus VCd or VRd in transplant ineligible patients (pts) with NDMM (NCT02513186). Methods: Isa-VCd: Isa (10 or 20 mg/kg; weekly [QW] cycle 1 [C1], then Q2W), V (1.3 mg/m2; twice weekly C1, then QW), C (300 mg/m2; QW C1, then Days [D] 1, 8, 15 to C12), d (20 mg; twice weekly C1, then D1, 2, 8, 9, 15, 16, 22, 23 to C12). Isa-VRd: Isa (10 mg/kg), V and d as described above; R (25 mg/day; D1–14 and D22–35). Efficacy and safety were evaluated. Conventional (M-protein levels) and minimal residual disease (MRD) IMWG response assessments were compared. MRD negativity was assessed at 10−5 by next-generation sequencing and flow. Mass Spectrometry (MS) negativity (no detectable serum M-protein) was assessed for 13 pts by Immuno-Capture and Liquid Chromatography coupled to High Resolution MS. Results: As of Nov 18, 2019, 17 pts were treated with Isa-VCd (10 mg/kg, n = 13; 20 mg/kg, n = 4), 27 with Isa-VRd; 53% and 63% remained on treatment, respectively. Infusion reactions were seen in 53% of Isa-VCd and 63% of Isa-VRd pts; Grade ≥3 infections in 23% and 37%; serious adverse events in 47% and 52%. See table for efficacy. 3 MRD positive pts were MS positive with persistent detectable M-protein ( > 10 µg/mL). 8/10 MRD negative pts were MS positive (4 at 5-10 µg/mL; 4 at > 10 µg/mL) and 2/10 were MS negative ( < 5 µg/mL). Stable residual M-protein was observed by MS up to 23 months post-MRD negativity. All pts tested by MS are still progression-free. Conclusions: Isa-VCd/VRd shows encouraging efficacy and tolerability in NDMM. MS seems to be more sensitive than MRD; low levels of M-protein were detectable even in MRD negative pts. Clinical trial information: NCT02513186 . [Table: see text]

Recent advances in multiple myeloma (MM), have seen the advent of several therapeutic monoclonal antibodies (mAb) targeting antigens on malignant plasma cells such as CD38 and SLAMF7. Evaluation of depth of response through quantification of M-Protein, by Serum Protein Electrophoresis (SPEP) and Immuno-Fixation Electrophoresis (IFE) presents a particular challenge for clinical laboratories when therapeutic mAbs are used. Specifically, therapeutic mAbs can confound SPEP and IFE measurements when they overlap with patient serum M-protein. Such interference could lead to an inaccurate determination of depth of response according to International Myeloma Working Group (IMWG) criteria Isatuximab (Isa), an IgG-kappa, anti-CD38 mAb, binds a specific epitope on CD38, which is widely expressed on MM cells. ICARIA-MM was a phase 3, prospective, randomized, open-label, active-controlled, multicenter study comparing isatuximab in combination with pomalidomide plus dexamethasone (Isa-Pd) to pomalidomide plus dexamethasone (Pd) in patients with relapsed/refractory multiple myeloma (RRMM). ICARIA-MM enrolled patients who had received ≥2 prior lines of therapy, including lenalidomide (len) and a proteasome inhibitor (PI). ICARIA-MM demonstrated that Isa-Pd significantly improved PFS, overall response and depth of response with a trend in overall survival benefit and a manageable toxicity profile in these patients. To overcome the potential interference of isatuximab with M-Protein in patients enrolled in ICARIA-MM, we developed and validated a hybrid assay using Immuno-Capture and Liquid Chromatography coupled to High Resolution Mass Spectrometry (IC-LC-HRMS). This method enriches for serum immunoglobulins by immuno-capture. Heavy chains (HC) and light chains (LC) are then dissociated and sorted using Liquid Chromatography. M-Protein and isatuximab LC are analyzed using HRMS which allows to differentiate their monoisotopic intact mass. A small number of patients (24/154; 15.6% in the Isa-Pd arm and 5/153; 3.3% in the Pd arm) fulfilled all criteria for CR (100% M-protein reduction on SPEP, bone marrow <5% plasma cells), but remained IFE positive as per IRC (near-CR category). Serial serum samples from 22 of 24 patients in the Isa-Pd arm were available for MS analysis. Separation of the M-protein and isatuximab signals was performed by IC-LC-HRMS. Using a semi-quantitative approach and applying a sensitivity threshold of 0.25g/dL for IFE-positivity (sensitivity threshold of IFE test per Covance central lab), we identified 11 of 22 patients as having M-protein levels below the threshold for IFE positivity, indicating that the true CR-rate was underestimated in the ICARIA-MM study due to interference. Of the 11 patients with MS M-protein values below the IFE sensitivity threshold, 10 were IgG (8 IgGκ, 2 IgGλ) and one was lambda LC disease with undetectable baseline HC. Baseline myeloma isotype distribution was different in patients with MS M-protein values above IFE sensitivity threshold (4 IgA, 6 IgG, 1 kappa light chain). This confirms that M-protein interference mediated by isatuximab, an IgG kappa mAb, is predominantly seen in patients with IgG isotype (mainly IgGκ), and in LC patients in whom a detectable IgG heavy chain can now be identified. At primary analysis, 4 of 11 patients with MS M-protein values above IFE sensitivity level, versus 2 of 11 patients with MS M-protein values below IFE sensitivity level had progressed. Median PFS was 13.9mo in patients with MS M-protein values above IFE sensitivity level, versus 15.21mo in patients with MS M-protein values below IFE sensitivity level. In conclusion, MS can be successfully used to separate endogenous M-protein from therapeutic isatuximab serum levels. In this subpopulation with excellent response to Isa-Pd, while most patients are still progression-free, a trend can be observed towards longer PFS in patients who would be considered IF negative after MS versus patients remaining IF positive. Disclosures Finn: Sanofi Oncology: Employment. Macé:Sanofi Oncology: Employment. Campana:Sanofi: Employment. Le-Guennec:Sanofi: Employment. Muccio:Sanofi Oncology: Employment. Tavernier:Sanofi Oncology: Employment. Rouchon:Sanofi Oncology: Employment. Roccon:Sanofi Oncology: Employment. Dai:Sanofi Oncology: Employment. Boutet:Sanofi Oncology: Employment. Mouret:Sanofi Oncology: Employment. Pradeilles:Sanofi Oncology: Employment. Hugla:Sanofi Oncology: Employment. Engelvin:Sanofi Oncology: Employment. DiNoto:Sanofi Oncology: Employment. van de Velde:Sanofi: Employment. Fedeli:Sanofi Oncology: Employment. OffLabel Disclosure: Isatuximab is an investigational agent and has not yet been approved by the US Food and Drug administration or any other regulatory agency.

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In multiple myeloma (MM) disease, malignant plasma cells produce excessive quantities of a monoclonal immunoglobulin (Ig), known as M-protein. M-protein levels are measured in the serum of patients with MM using electrophoresis techniques to determine the response to treatment. However, therapeutic monoclonal antibodies, such as isatuximab, may confound signals using electrophoresis assays. We developed a robust assay based on immunocapture and liquid chromatography coupled to high-resolution mass spectrometry (IC-HPLC-HRMS) in order to eliminate this interference. Following immunocapture of Ig and free light chains (LC) in serum, heavy chains (HC) and LC were dissociated using dithiothreitol, sorted by liquid chromatography and analyzed using HRMS (Q-Orbitrap). This method allowed the M-proteins to be characterized and the signals from isatuximab and M-proteins to be discriminated. As Mprotein is specific to each patient, no standards were available for absolute quantification. We therefore used alemtuzumab (an IgG kappa mAb) as a surrogate analyte for the semiquantification of M-protein in serum. This assay was successfully validated in terms of selectivity/specificity, accuracy/precision, robustness, dilution linearity, and matrix variability from 10.0 to 200 μg/mL in human serum. This method was used for clinical assessment of samples and eliminated potential interference due to isatuximab when monitoring patients with MM.