Bioanalytical Pharmacokinetics (PK) methods are designed for robust performance under rigorous regulatory compliance requirements to ensure the generated data is reliable and maintains integrity. In a phase 1 dose-finding clinical study, aberrant PK profiles of two co-administered biologics drugs were observed. Unexpectedly, we discovered high fill levels in collection tubes from the majority of samples. This led to the hypothesis that the highly filled tubes might cause difficulty in achieving complete sample thaw and thorough mixing at the time of sample analysis, potentially contributing to the abnormalities observed in the PK dataset. Evaluation of the impact of sample fill levels and processing procedure can be challenging since PK concentrations of study samples were unknown. Therefore, a systematic approach was employed to conduct a thorough examination using mock samples. The data illustrate a correlation between sample thawing and mixing process and the variability in the PK data. The concentrations from properly filled mock samples that underwent complete thawing and mixing showed 100

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.

Tiragolumab is a first-in-class, fully human IgG1/kappa anti-TIGIT monoclonal antibody that blocks the binding of TIGIT to CD155 (the poliovirus receptor). We summarize the pharmacokinetics (PK) data from the phase 1a/1b GO30103 study of Q3W (every 3 weeks) sequential dosing of tiragolumab (2, 8, 30, 100, 400, 600, or 1200 mg) followed by atezolizumab (1200 mg), Q4W (every 4 weeks) sequential dosing (tiragolumab 840 mg followed by atezolizumab 1680 mg), and Q4W co-infusion (tiragolumab 840 mg plus atezolizumab 1680 mg). Serum samples were collected at multiple time points following tiragolumab and atezolizumab intravenous infusion in patients with solid tumors for PK and immunogenicity assessment. The serum PK profile of tiragolumab appeared to be biphasic, with a rapid distribution phase followed by a slower elimination phase when administered alone or in combination with atezolizumab. In phase 1a, across doses of tiragolumab ranging from 2 to 1200 mg (cycle 1), the geometric mean (GM), coefficient of variation (CV%), serum tiragolumab Cmax ranged from 0.682 to 270 µg/mL (18.6% to 36.5%) and Cmin ranged from 0.0125 to 75.3 µg/mL (0.0% to 24.2%). The GM systemic exposure (area under the plasma drug concentration-time curve, AUC0-21) ranged from 310 to 2670 µg day/mL (20.5% to 27.0%); interindividual variability in AUC0-21 ranged from 20.5% to 43.9%. Tiragolumab exposure increased in an approximately dose-proportional manner when administered alone or with atezolizumab at doses ≥100 mg. Postbaseline, 4/207 patients (1.9%) were positive for treatment-emergent antidrug antibodies (ADA) against tiragolumab, each at a single time point. Tiragolumab combined with atezolizumab demonstrated desirable PK properties, with no drug-drug interactions or immunogenicity liability. There were no meaningful differences in tiragolumab or atezolizumab exposure between the Q4W co-infusion and sequential dosing cohorts. ClinicalTrials.gov: NCT02794571 (date of registration June 6, 2016).

Immunogenicity evaluation is a critical part of drug development. Regulatory guidelines from multiple health agencies provide recommendations for the development and validation of anti-drug antibody (ADA) assays to assess immunogenicity in clinical trials. These recommendations primarily describe an ADA method run in one bioanalytical laboratory supporting a biotherapeutic molecule; however, there are increasing instances that may necessitate the support of the ADA method being run in more than one laboratory. A program can rapidly expand into multiple clinical studies within one or multiple countries, where the most appropriate way to support the program is by having multiple laboratories perform ADA sample analysis. In addition, there may be certain country-specific challenges that may make it infeasible to transport samples outside of the country for analysis. China for example has a lengthy sample exportation process that has potential to negatively impact study timelines. If multiple laboratories analyze samples using the same ADA method, comparable method performance should be established. Here, we describe a three-way assessment of ADA assay comparability between two US-based bioanalytical laboratories and one based in China.

BackgroundNeurofilament light chain (NfL), a neuronal cytoskeletal protein that is released upon neuroaxonal injury, is associated with multiple sclerosis (MS) relapsing activity and has demonstrated some prognostic ability for future relapse-related disease progression, yet its value in assessing non-relapsing disease progression remains unclear.MethodsWe examined baseline and longitudinal blood NfL levels in 1421 persons with relapsing MS (RMS) and 596 persons with primary progressive MS (PPMS) from the pivotal ocrelizumab MS trials. NfL treatment-response and risk for disease worsening (including disability progression into the open-label extension period and slowly expanding lesions [SELs] on brain MRI) at baseline and following treatment with ocrelizumab were evaluated using time-to-event analysis and linear regression models.FindingsIn persons from the RMS control arms without acute disease activity and in the entire PPMS control arm, higher baseline NfL was prognostic for greater whole brain and thalamic atrophy, greater volume expansion of SELs, and clinical progression. Ocrelizumab reduced NfL levels vs. controls in persons with RMS and those with PPMS, and abrogated the prognostic value of baseline NfL on disability progression. Following effective suppression of relapse activity by ocrelizumab, NfL levels at weeks 24 and 48 were significantly associated with long-term risk for disability progression, including up to 9 years of observation in RMS and PPMS.InterpretationHighly elevated NfL from acute MS disease activity may mask a more subtle NfL abnormality that reflects underlying non-relapsing progressive biology. Ocrelizumab significantly reduced NfL levels, consistent with its effects on acute disease activity and disability progression. Persistently elevated NfL levels, observed in a subgroup of persons under ocrelizumab treatment, demonstrate potential clinical utility as a predictive biomarker of increased risk for clinical progression. Suppression of relapsing biology with high-efficacy immunotherapy provides a window into the relationship between NfL levels and future non-relapsing progression.FundingF. Hoffmann-La Roche Ltd.

Ocrelizumab (OCREVUS®) is a humanized anti-CD20 monoclonal antibody approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). Here, we discuss the strategic and technical considerations needed to develop a robust antibody-dependent cellular cytotoxicity (ADCC)-based neutralizing antibody (NAb) assay to detect anti-ocrelizumab NAb in patients enrolled in the ocrelizumab registered clinical trials. The NAb detection assay consisted of a two-tier assay that included a screening assay and a confirmation assay. In the screening assay, patient samples were analyzed in the presence of ocrelizumab. Samples that tested positive in the screening assay were subsequently analyzed in the confirmatory assay where another anti-CD20 mAb, obinutuzumab, was replaced by ocrelizumab, to verify NAb specificity. Both assays utilized MEC-2 cells, a chronic B cell leukemia cell line, pre-labeled with calcein AM as the target cells, and natural killer (NK) cells engineered to stably express Fc gamma receptor IIIa_ F158 as effector cells. Both cell lines were prepared to be thaw-and-use cells. The NAb assay measures fluorescence from the calcein AM released into the assay media upon the lysis of target cells by ADCC in the presence of ocrelizumab or obinutuzumab. Our validated NAb assay showed a relative sensitivity of 743 ng/mL and can detect 1500 ng/mL of a surrogate positive control antibody in the presence of 1500 ng/mL ocrelizumab. This ADCC assay is the first reported NAb assay that directly measures target cell lysis by using thaw-and-use target and effector cells simultaneously.

analyses, supporting it as a standard-ofcare therapy for relapsed MM pts.

Introduction This phase 1 trial assessed the safety, pharmacokinetics, and preliminary antitumor activity of RO7297089, an anti-BCMA/CD16a bispecific antibody. Methods RO7297089 was administered weekly by intravenous infusion to patients with relapsed/refractory multiple myeloma. The starting dose was 60 mg in this dose-escalation study utilizing a modified continual reassessment method with overdose control model. Results Overall, 27 patients were treated at doses between 60 and 1850 mg. The maximally administered dose was 1850 mg due to excipients in the formulation that did not allow for higher doses to be used. The maximum tolerated dose was not reached. The most common adverse events irrespective of grade and relationship to the drug were anemia, infusion-related reaction, and thrombocytopenia. Most common treatment-related grade ≥ 3 toxicities were ALT/AST increase and reduced lymphocyte count. Pharmacokinetic studies suggested non-linear pharmacokinetics and target-mediated drug disposition, with a trend of approaching linear pharmacokinetics at doses of 1080 mg and higher. Partial response was observed in two patients (7%), minimal response in two patients (7%), and stable disease in 14 patients (52%). Conclusions RO7297089 was well tolerated at doses up to 1850 mg, and the efficacy data supported activity of RO7297089 in multiple myeloma. Combination with other agents may further enhance its potential as an innate immune cell engager in multiple myeloma. Trial Registration ClinicalTrials.gov: NCT04434469; Registered June 16, 2020; https://www.clinicaltrials.gov/ct2/show/NCT04434469 .

AbstractObjectiveTo develop an age‐adjustment model for neurofilament light chain (NfL), an emerging injury marker in patients with a range of neurologic conditions including multiple sclerosis (MS).MethodsSerum and plasma samples were collected from a healthy donor (HD) cohort of 118 individuals aged 24 to 66 years, 90 patients with relapsing MS (RMS) and 22 patients with progressive MS (PMS). Serum and plasma samples were assessed for NfL using the SIMOA assay (Quanterix NfL Advantage Kit™). A log‐linear model was used to evaluate the relationship between NfL and age and to calculate age‐adjusted NfL levels.ResultsHigher serum and plasma NfL levels were significantly associated with increasing HD age. Log‐transformation of blood NfL levels reduced heteroscedasticity and skewness. A log‐linear model enabled adjustment for age‐related increase in serum and plasma NfL levels (2.3% [95% CI, 1.6–2.9] and 2.6% [95% CI, 1.3–3.3] per year, respectively). Following age adjustment, NfL did not show significant association with HD sex or ethnicity. While unadjusted serum NfL levels were elevated in patients with PMS (mean age 56 years) compared with those with RMS (mean age 37 years), age‐adjusted NfL levels did not differ.InterpretationA log‐linear, age adjustment model was developed to enable comparison of NfL levels across populations with different ages. While additional data and evidence are needed for patient‐level adoption, this could be a valuable tool for interpreting NfL levels across a range of patient groups with neurologic conditions.

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089 induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

RO7297089, an anti-B-cell maturation antigen (BCMA)/CD16A bispecific tetravalent antibody, is being developed as a multiple myeloma (MM) therapeutic. This study characterized nonclinical pharmacokinetics (PK), pharmacodynamics (PD), soluble BCMA (sBCMA), and soluble CD16 (sCD16) changes following administration of RO7297089 to support clinical trials. Unbound and total RO7297089 concentrations were measured in cynomolgus monkeys. RO7297089 exhibited a bi-phasic systemic concentration-time profile, similar to a typical human immunoglobulin 1 antibody. Target engagement by RO7297089 led to a robust increase (~100-fold) in total systemic sBCMA levels and relatively mild increase (~2-fold) in total sCD16 levels. To describe the relationship of nonclinical PK/PD data, we developed a target-mediated drug disposition (TMDD) model that includes the systemic target engagement of membrane BCMA (mBCMA), sBCMA, membrane CD16 (mCD16), and sCD16. We then used this model to simulate the PK/PD relationship of RO7297089 in MM patients by translating relevant PK parameters and target levels, based on the literature and newly generated data such as baseline sCD16A levels. Our model suggested that the impact of TMDD on RO7297089 exposure may be more significant in MM patients due to significantly higher expression levels of both mBCMA and sBCMA compared to healthy cynomolgus monkeys. Based on model simulations, we propose more frequent dosing of RO7297089 compared to regular monthly frequency in the clinic at the beginning of treatment to ensure sustained target engagement. This study demonstrates a translational research strategy for collecting relevant nonclinical data, establishing a TMDD model, and using simulations from this model to inform clinical dose regimens.

Background: RO7297089 is a bispecific tetravalent antibody targeting BCMA (Genentech licensed from Affimed GMBH), which is highly expressed on MM cells and CD16a, which is expressed on innate immune cells including natural killer (NK) cells, macrophages, and monocyte subsets. Here we report findings from a Phase I dose-escalation study of RO7297089 in patients with RRMM (GO41582, Study registration NCT04434469).

Abstract RO7297089 is a bispecific antibody (IgG-scFv) targeting Bcell maturation antigen (BCMA) and CD16a (FcγRIIIA) that is being developed for the treatment of multiple myeloma (MM). BCMA is exclusively expressed on plasmablasts and differentiated plasma cells (PCs), and is overexpressed on malignant PCs in MM patients. CD16a is expressed on natural killer (NK) cells, monocytes, mast cells, and macrophages. Herein, we characterized the mode of action and safety profile of RO7297089 in vitro and in vivo. RO7297089 showed potent cell killing when using BCMA+ MM tumor cell lines as target cells and human peripheral blood mononuclear cells, NK cells or macrophages as effector cells. Minimal increases in TNFα (2x) and IFNγ (4x), but not other cytokines/chemokines, were observed compared to the vehicle control treatment only in the presence of the BCMA+ MM tumor cell line up to the concentration tested. This suggests that, unlike T-cell engagers, the risk of cytokine release syndrome in patients receiving RO7297089 is low. Cynomolgus monkey is the only relevant nonclinical species for RO7297089 as it showed binding to both recombinant CD16 and BCMA with comparable affinity to human antigens. Following five weekly intravenous administrations to monkeys at 0, 15, and 50 mg/kg, RO7297089 was well tolerated. In line with the mechanism of action, there were no test article-related cytokine increases or adverse findings observed in both dose levels. Systemic exposure of RO7297089 was approximately dose proportional from 15 to 50 mg/kg. Anti-drug antibodies (ADA) were observed in some animals at both dose levels, and ADA-related decreases in concentrations were observed at only 15 mg/kg. To evaluate in vivo activity, RO7297089-related effects on total plasma sBCMA and PCs were assessed. Elevations of sBCMA levels (100x) were observed post dose at both dose levels, and these effects returned to predose levels in animals that did not maintain concentrations at 15 mg/kg, suggesting that RO7297089 bound to and stabilized circulating cynomolgus sBCMA. Time- and dose-dependent reductions in serum IgM levels were observed at both dose levels. Changes in PC numbers were not detected by immunophenotyping; however, gene expression analysis of PC markers was included and demonstrated clear reductions in mRNA expression levels of PC markers including BCMA and J-chain in blood at both dose levels, suggesting reductions in BCMA+ cells. Collectively, these studies suggest that RO7297089 selectively kills BCMA+ cells by engaging CD16a-positive immune cells and has a favorable safety profile. Citation Format: Satoko Kakiuchi-Kiyota, Melissa M. Schutten, Adeyemi O. Adedeji, Hao Cai, Robert Hendricks, Luna Liu, Sivan Cohen, Aaron M. Fullerton, Nicholas Corr, Lanlan Yu, Denise de Almeida Nagata, Shelly Zhong, Michael Dillon, Christoph Spiess, Steve R. Leong, Bing Zheng, Susanne Wingert, Uwe Reusch, Stefan Knackmuss, Thorsten Ross, Andrew Polson, Ayse M. Ovacik. Preclinical pharmacology and safety of RO7297089, a novel anti-BCMA/CD16a bispecific antibody for the treatment of multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4556.

To provide reference ranges of cerebrospinal fluid (CSF), serum, and plasma neurofilament light (NfL) levels from a healthy donor cohort using the Quanterix NfL Advantage Kit™