Receptor Saturation Research Articles

P-207: A multi-center, phase 1b study to assess the safety, Pharmacokinetics and efficacy of subcutaneous Isatuximab plus Pomalidomide and Dexamethasone, in patients with Relapsed/Refractory Multiple Myeloma

<h3>Background</h3> Intravenous (IV) isatuximab (Isa) + pomalidomide and dexamethasone (Pd) is an approved regimen for the treatment of adults with relapsed/refractory multiple myeloma (RRMM). Subcutaneous (SC) delivery would optimize convenience of administration. <h3>Methods</h3> This multicenter, open-label, Phase 1b study evaluated the safety, pharmacokinetics (PK), and efficacy of SC vs IV Isa + Pd in patients (pts) with RRMM who had received ≥2 lines including lenalidomide and a proteasome inhibitor. Pts were randomized 2:1 to Cohorts 1a (SC–1000 mg) or 1b (IV–10 mg/kg) and, after evaluation of Isa SC safety, PK and CD38 Receptor Occupancy (RO), randomized to Cohorts 2a (SC–1400 mg) or 2b (IV). SC was delivered through a syringe pump. Primary endpoints assessed safety including dose-limiting toxicity (DLT), injection site reactions (ISR), and PK parameters. Key secondary endpoints were overall response rate (ORR), progression-free survival (PFS), and CD38 RO. <h3>Results</h3> 34 pts were randomized and treated: 12 pts Isa IV 10 mg/kg + Pd, 12 pts Isa SC1000 + Pd, 10 pts Isa SC1400 + Pd. On March 31, 2021, 7 pts (58%) IV, 4 pts (33%) SC1000, and 7 pts (70%) SC1400 remained on study treatment. At study entry, International Staging System (ISS) stage II–III was 58% in IV, 33% in SC1000 and 60% in SC1400 pts. Due to sequential accrual, the median follow-up was longer in IV (15.1 months [mos]) and SC1000 (14.8 mos) cohorts than SC1400 (8.8 mos). Infusion reactions were infrequent (≤10% across cohorts, all Grade (G) 2), and only at first injection. Local tolerability of SC injection was very good with only a single episode of G2 ISR in SC1400 cohort. Similar occurrence of ≥G3 treatment emergent adverse events (TEAE), treatment-related TEAE, and neutropenia occurred across cohorts. One DLT was reported in each SC cohort: G4 neutropenia (SC1000) and G3 pulmonary infection (SC1400). No maximum tolerated dose was identified. ORR, ≥VGPR, and complete response were 67%, 33%, and 17% in the IV cohort; 67%, 42%, and 25% in the SC1000 cohort and 80%, 40%, and 20% in the SC1400 cohort, respectively. 8 mos PFS-free rate was 73% in the IV and SC1000 cohorts and 89% in the SC1400 cohort. Mean Ctrough after the 4th weekly administration was higher in the SC cohorts (339 µg/mL for SC1000, 338 µg/mL for SC1400) vs 235 µg/mL for IV cohort. High CD38 receptor saturation by Isa on bone marrow plasma cells was reached for both SC doses; mean CD38 RO was 76% in IV, 80% in SC1000, and 81% in SC1400. <h3>Conclusions</h3> The safety of Isa SC at 1000 mg and 1400 mg + Pd was consistent with the known safety profile associated with IV administration, with no new safety signals identified. Local tolerability of SC Isa was very good. Efficacy results were comparable with the Phase 3 ICARIA study. Higher Ctrough at 4 weeks (PK best predictor of Isa efficacy) was achieved following SC administration compared with IV. Isa SC administration appears to be a promising and convenient option. Funded by Sanofi.

Effect of using different co-ligands during 99mTc-labeling of J18 peptide on SK-MES-1 cell binding and tumor targeting.

Objective(s):Lung cancer is the main cause of cancer death, and its incidence is increasing worldwide. The goal of this study is to evaluate in vitro and in vivo tumor targeting behavior of [99mTc]Tc -HYNIC-(Ser)3-J18 in lung carcinoma (SK-MES-1)-bearing mice.Materials and Methods:The J18 (RSLWSDFYASASRGP) peptide was conjugated with hydrazinonicotinamide (HYNIC) via three serine amino acids as a linker at the peptide’s N-terminal and then labeled with technetium-99m using tricine and tricine/EDDA as the co-ligands. The radiolabeled peptides were assessed for in vitro receptor binding, specific binding, and saturation affinity. In vivo biodistribution studies were also performed for 99mTc-peptide 1 (tricine co-ligand) and 99mTc-peptide 2 (tricine/EDDA coligands) in nude mice bearing SK-MES-1 xenograft tumors. Results: In vitro studies showed high specific binding for 99mTc-peptide 1 in SKMES-1 cells compared with 99mTc-peptide 2 (11.5 vs. 4.5). The KD values for 99mTc-peptide 1 and 99mTc-peptide 2 were reported to be 3.1±0.3 nM and 3.46 ± 0.8 nM, respectively. The biodistribution study also showed high significant tumor to muscle ratios of 5.1 and 6.18 for 99mTc-peptide 1 at 1 and 2 hr after injection, respectively, while these ratios were 3.81 and 5.18 for peptide 2, respectively. Conclusion:Overall, 99mTc-labeled J18 peptide in the presence of tricine as co-ligand has better in vitro and in vivo tumor targeting properties in SK-MES-1 cells than tricine/EDDA co-ligands. These findings show that the 99mTc-labeled J18 peptide is a good candidate for lung carcinoma targeting.

Melatonin binds with high affinity and specificity to beta-amyloid: LC-MS provides insight into Alzheimer's disease treatment.

To study the potential relationship between melatonin and beta‐amyloid (Abeta), we established a liquid chromatography–mass spectrometry (LC‐MS) method to quantitatively analyze melatonin, deuterated isotopes (melatonin‐D4), and internal standard 6‐iodo‐2‐(4'‐dimethylamino‐) phenyl‐imidazo(1,2) pyridine (IMPY) under positive (+) mode. The gradient elution was set to 6 min, and the corresponding peak time of melatonin and its isotope melatonin‐D4 was 3.14 min, while the peak time for the internal standard IMPY was 3.24 min. Next, we established and optimized the molecule receptor saturation binding assay based on LC‐MS to determine the melatonin affinity for beta‐amyloid (Abeta). Melatonin showed a high and specific binding for Abeta. The corresponding equilibrium dissociation constant (Kd) of melatonin with Abeta 1‐40 and Abeta 1‐42 was 814.37 ± 36.62 and 628.33 ± 13.57 nmol·L−1; besides, the Kd of melatonin with mixed plaques (1‐40 and 1‐42) was 461.13 ± 45.37 nmol·L−1. The results may suggest the potential mechanism of action of MT on Abeta and provide a theoretical basis for the improvement of MT treatment of Alzheimer's disease.

Neuroblastoma xenograft models demonstrate the therapeutic potential of 177Lu-octreotate

BackgroundNeuroblastoma (NB) is one of the most frequently diagnosed tumors in infants. NB is a neuroendocrine tumor type with various characteristics and features, and with diverse outcome. The most malignant NBs have a 5-year survival rate of only 40–50%, indicating the need for novel and improved treatment options. 177Lu-octreotate is routinely administered for treatment of neuroendocrine tumors overexpressing somatostatin receptors (SSTR). The aim of this study was to examine the biodistribution of 177Lu-octreotate in mice bearing aggressive human NB cell lines, in order to evaluate the potential usefulness of 177Lu-octreotate for treatment of NB.MethodsBALB/c nude mice bearing CLB-BAR, CLB-GE or IMR-32 tumor xenografts (n = 5–7/group) were i.v. injected with 0.15 MBq, 1.5 MBq or 15 MBq 177Lu-octreotate and sacrificed 1 h, 24 h, 48 h and 168 h after administration. The radioactivity concentration was determined for collected tissue samples, tumor-to-normal-tissue activity concentration ratios (T/N) and mean absorbed dose for each tissue were calculated. Immunohistochemical (IHC) staining for SSTR1–5, and Ki67 were carried out for tumor xenografts from the three cell lines.ResultsHigh 177Lu concentration levels and T/N values were observed in all NB tumors, with the highest for CLB-GE tumor xenografts (72%IA/g 24 h p.i.; 1.5 MBq 177Lu-octreotate). The mean absorbed dose to the tumor was 6.8 Gy, 54 Gy and 29 Gy for CLB-BAR, CLB-GE and IMR-32, respectively, p.i. of 15 MBq 177Lu-octreotate. Receptor saturation was clearly observed in CLB-BAR, resulting in higher concentration levels in the tumor when lower activity levels where administered. IHC staining demonstrated highest expression of SSTR2 in CLB-GE, followed by CLB-BAR and IMR-32.ConclusionT/N values for all three human NB tumor xenograft types investigated were high relative to previously investigated neuroendocrine tumor types. The results indicate a clear potential of 177Lu-octreotate as a therapeutic alternative for metastatic NB.

Evaluation of atezolizumab immunogenicity: Clinical pharmacology (part 1).

Baseline patient characteristics and prognostic factors are important considerations in oncology when evaluating the impact of immunogenicity on pharmacokinetics (PK) and efficacy. Here, we assessed the impact of anti‐drug antibodies (ADA) on the PK of the immune checkpoint inhibitor atezolizumab (an anti–PD‐L1 monoclonal antibody). We evaluated data from ≈ 4500 patients from 12 clinical trials across different tumor types, treatment settings, and dosing regimens. In our dataset, ~ 30% of patients (range, 13–54%) developed treatment‐emergent ADA, and in vitro neutralizing antibodies (NAb) were seen in ~ 50% of ADA‐positive (+) patients. Pooled time course data showed a trend toward lower atezolizumab exposure in ADA+ patients, which was more pronounced in ADA+/NAb+ patients. However, the atezolizumab concentration distributions overlapped, and drug concentrations exceeded 6 µg/ml, the target concentration required for receptor saturation, in greater than 95% of patients. Patients had sufficient exposure regardless of ADA status. The dose selected to allow for dosing over effects from ADA resulted in a flat exposure‐response relationship. Analysis of study results by ADA titer showed that exposure and overall survival were not affected in a clinically meaningful way. High tumor burden, low albumin, and high CRP at baseline showed the greatest association with ADA development but not with subsequent NAb development. These imbalanced factors at baseline can confound analysis of ADA impact. ADA increases atezolizumab clearance minimally (9%), and its impact on exposure based on the totality of the clinical pharmacology assessment does not appear to be clinically meaningful.

Erythroferrone and hepcidin as mediators between erythropoiesis and iron metabolism during allogeneic hematopoietic stem cell transplant.

Hematopoietic cell transplantation (HCT) brings important alterations in erythropoiesis and iron metabolism. Hepcidin, which regulates iron metabolism, increases in iron overload or inflammation and decreases with iron deficiency or activated erythropoiesis. Erythroferrone (ERFE) is the erythroid regulator of hepcidin. We investigated erythropoiesis and iron metabolism after allogeneic HCT in 70 patients randomized between erythropoietin (EPO) treatment or no EPO, by serially measuring hepcidin, ERFE, CRP (inflammation), soluble transferrin receptor (sTfR, erythropoiesis), serum iron and transferrin saturation (Tsat; iron for erythropoiesis) and ferritin (iron stores). We identified biological and clinical factors associated with serum hepcidin and ERFE levels. Serum ERFE correlated overall with sTfR and reticulocytes and inversely with hepcidin. Erythroferrone paralleled sTfR levels, dropping during conditioning and recovering with engraftment. Inversely, hepcidin peaked after conditioning and decreased during engraftment. Erythroferrone and hepcidin were not significantly different with or without EPO. Multivariate analyses showed that the major determinant of ERFE was erythropoiesis (sTfR, reticulocytes or serum Epo). Pretransplant hepcidin was associated with previous RBC transfusions and ferritin. After transplantation, the major determinants of hepcidin were iron status (ferritin at all time points and Tsat at day 56) and erythropoiesis (sTfR or reticulocytes or ERFE), while the impact of inflammation was less clear and clinical parameters had no detectable influence. Hepcidin remained significantly higher in patients with high compared to low pretransplant ferritin. After allogeneic HCT with or without EPO therapy, significant alterations of hepcidin occur between pretransplant and day 180, in correlation with iron status and inversely with erythroid ERFE.

Role of AMPA receptor desensitization in short term depression - lessons from retinogeniculate synapses.

Repetitive synapse activity induces various forms of short-term plasticity. The role of presynaptic mechanisms such as residual Ca2+ and vesicle depletion in short-term facilitation and short-term depression is well established. On the other hand, the contribution of postsynaptic mechanisms such as receptor desensitization and saturation to short-term plasticity is less well known and often ignored. In this review, I will describe short-term plasticity in retinogeniculate synapses of relay neurons of the dorsal lateral geniculate nucleus (dLGN) to exemplify the synaptic properties that facilitate the contribution of AMPA receptor desensitization to short-term plasticity. These include high vesicle release probability, glutamate spillover and, importantly, slow recovery from desensitization of AMPA receptors. The latter is strongly regulated by the interaction of AMPA receptors with auxiliary proteins such as CKAMP44. Finally, I discuss the relevance of short-term plasticity in retinogeniculate synapses for the processing of visual information by LGN relay neurons.

P306 MORF-057, an oral selective α4β7 integrin inhibitor for Inflammatory Bowel Disease, leads to specific target engagement in a single and multiple ascending dose study in healthy subjects

Abstract Background MORF-057 is an orally administered small molecule designed to inhibit the α 4β 7 receptor, addressing an unmet medical need in inflammatory bowel disease (IBD) patients, and avoiding the need for periodic therapeutic infusions and the risk for infusion-related reactions. This study evaluated single (SAD) and multiple ascending doses (MAD) of MORF-057 in healthy volunteers. Methods This was a randomized, double-blind, placebo-controlled, single and multiple doses, phase 1 study of the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of MORF-057 conducted in a Phase I Unit in the USA. Subjects were randomized 3:1 to MORF-057 or placebo capsules once daily in the SAD cohorts: 25, 50, 100, 150, and 400 mg; or twice daily (BID) in the MAD cohorts: 100 and 200 mg total daily doses (dosed as 50 and 100 mg BID, respectively). In the MAD cohorts, trough PK samples were obtained premorning or pre-evening dose following administration of 50 or 100 mg BID, respectively. Blood samples to assess receptor occupancy (RO) of α 4β 7 and α 4β 1 integrins were obtained prior to the first dose and 12 hours after treatment. Results To date, 51 healthy subjects were dosed; 1 subject withdrew consent on Day 2 for personal reasons. Eleven non-serious adverse events (AEs) were reported; AEs were mild and did not result in discontinuation. Two reversible mild AEs in the 200 mg MAD cohort were possibly related to MORF-057 (macular / maculopapular rash). No safety signals were identified to date. Following dosing, MORF-057 was rapidly absorbed and systemic exposure increased approximately dose-proportionally (Fig.1). Mean α 4β 7 RO was found to be greater than 95% after each of the three highest single doses (Fig.2). In multiple dosing, mean α 4β 7 RO was greater than 90% at the lower dose, while the highest dose tested resulted in α 4β 7 RO saturation at steady state. α 4β 1 RO was below the limit of quantitation with mean trough values estimated to be &amp;lt;10% at any of the dose levels. Conclusion Current results demonstrated that single and multiple ascending doses of MORF-057 were well tolerated, with only mild, non-serious AEs reported that did not result in study drug discontinuation. MORF-057 demonstrated a favorable PK profile where target engagement was confirmed and resulted in α4β7 receptor saturation in many subjects receiving higher doses. These results support further investigation of MORF-057 and provide a basis for dose selection phase 2 studies in patients with IBD. (NCT04580745)

Low activity [11C]raclopride kinetic modeling in the mouse brain using the spatiotemporal kernel method

Depending on the molar activity of the tracer, the maximal allowable injected activity in mouse brain PET studies can be extremely low in order to avoid receptor saturation. Therefore, a high level of noise can be present in the image. We investigate several dynamic PET reconstruction methods in reduced counts, or equivalently in reduced injected activity, data exemplified in [11C]raclopride BP ND and quantification using the simplified reference tissue model (SRTM). We compared independent frame reconstruction (IFR), post-reconstruction HYPR denoising (IFR + HYPR), direct reconstruction using the SRTM model (DIR-SRTM), and the spatial (KERS) and spatiotemporal kernel reconstruction (KERST). Additionally, HYPR denoising of the frames used as features for the calculation of the spatial kernel matrix, was investigated (KERS-HYPR and KERST-HYPR). In vivo data of 11 mice, was used to generate list-mode data for five reduced count levels corresponding to reductions by a factor 4, 8, 12, 16 and 32 (equivalently 2.07, 1.04, 0.691, 0.518, and 0.260 MBq). Correlation of regional BP ND and values (reduced versus full counts reconstructions) was high (r > 0.94) for all methods, with KERS-HYPR and KERST-HYPR reaching the highest correlation (r > 0.96). Among methods with regularization, DIR-SRTM showed the largest variability in BP ND (Bland–Altman SD from 3.0% to 12%), while IFR showed it for (5.1%–14.6%). KERST and KERST-HYPR were the only methods with Bland–Altman bias and SD below 5% for noise level up to a reduction factor of 16. At the voxel level, BP ND and correlation was gradually decreased with increasing noise, with the largest correlation (BP ND r > 0.88, r > 0.62) for KERS-HYPR and KERST-HYPR. The spatial and the spatiotemporal kernel methods performed similarly, while using only temporal regularization with direct reconstruction showed more variability. Although R1 values present noise, using the spatiotemporal kernel reconstruction, accurate estimates of binding potential could be obtained with mouse injected activities as low as 0.26–0.518 MBq. This is desirable in order to maintain the tracer kinetics principle in mouse studies.

NVG-111, a novel ROR1xCD3 bispecific antibody for non-Hodgkin lymphoma.

7549 Background: Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is a type I transmembrane protein is highly expressed on an array of haematological and solid tumours. NVG-111 is a humanised, tandem scFv ROR1xCD3 bispecific antibody previously shown to elicit potent killing of tumour cells in vitro and in vivo by engaging a membrane-proximal epitope in the Wnt5a-binding Frizzled domain of ROR1 and redirecting T cell activity. The in vitro potency and pharmacodynamic responses to NVG-111 were assessed to support progression to a first-in-human study. Methods: The potency of NVG-111 in vitro was determined by evaluating the concentration response for cytotoxicity, T cell activation, and cytokine release in co-cultured Jeko-1 and unstimulated human T cells. Comparative data were generated for the marketed CD19xCD3 bispecific antibody, blinatumomab. Potency data for NVG-111 were used together with allometric scaling from murine PK studies to inform planned clinical doses. Results: NVG-111 demonstrated T cell-dependent cytotoxicity, T cell activation and levels of cytokine release similar in potency to blinatumomab. Cytotoxic responses of both NVG-111 and blinatumomab were more potent than T cell activation and cytokine release. Dose response curves for NVG-111 showed a decrease in activity beyond the concentration of maximal response (ie “hook effect”). We hypothesise this is due to receptor saturation, inhibiting synapse formation. NVG-111 has progressed to a Phase 1/2 first-in-human study in patients with debulked, relapsed/refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), the drug given as add-on to ≥2nd line therapy with a Bruton’s tyrosine kinase inhibitor, or venetoclax. Phase 1 includes escalating doses of 0.3 to 360 µg/day via continuous infusion over 3 cycles (each 21 days on, 7 days off) to establish safety, PK, pharmacodynamics (PD) and recommended phase 2 dose (RP2D). Predicted exposure at 0.3 µg/day is ̃EC20 for cytotoxicity in vitro and below the lowest EC10 for cytokine release. PD biomarkers in the study include systemic cytokines. Phase 2 will study efficacy and safety of the RP2D in CLL and MCL, with primary endpoint complete response rate; other efficacy endpoints include minimal residual disease and progression free survival. Conclusions: NVG-111 shows potent T-cell mediated lymphoma cell cytotoxicity in vitro at concentrations well below those associated with extensive cytokine release. NVG-111 is in an ongoing Phase 1/2 study and may present a novel option for adoptive immunotherapy in patients with non-Hodgkin lymphoma and potentially other cancers. Clinical trial information: 2020-000820-20. [Table: see text]

Pharmacokinetics and Exposure-Response Relationship of Teprotumumab, an Insulin-Like Growth Factor-1 Receptor-Blocking Antibody, in Thyroid Eye Disease.

Background and ObjectiveThyroid eye disease (TED) is characterized by inflammation/expansion of orbital tissues, proptosis, and diplopia. Teprotumumab is the first US Food and Drug Administration-approved therapy for TED, administered as an initial intravenous infusion of 10 mg/kg followed by 20 mg/kg every 3 weeks for an additional seven infusions. The objective of this article is to discuss the pharmacokinetics and exposure-response profile for teprotumumab in patients with TED.MethodsA population pharmacokinetic analysis was performed to characterize pharmacokinetics and select dosing in patients with TED. Exposure-response was evaluated for efficacy (proptosis response, clinical activity score categorical response, and diplopia response) and safety (hyperglycemia, muscle spasms, and hearing impairment) parameters.ResultsTeprotumumab pharmacokinetics was linear in patients with TED, with low systemic clearance (0.334 L/day), low volume of distribution (3.9 and 4.2 L for the central and peripheral compartment, respectively), and a long elimination half-life (19.9 days). The approved dosing regimen provided > 20 µg/mL for > 90% insulin-like growth factor 1 receptor saturation throughout the dosing interval. Model-predicted mean (± standard deviation) steady-state area under the concentration-time curve, peak, and trough concentrations in patients with TED were 131 (± 30.9) mg∙h/mL, 643 (± 130) µg/mL, and 157 (± 50.6) µg/mL, respectively. Female patients had a 15% higher steady-state peak concentration but a similar steady-state area under the concentration-time curve vs male patients. No other covariates affected teprotumumab pharmacokinetics. No meaningful correlations between teprotumumab exposures and efficacy or safety parameters were observed.ConclusionsTeprotumumab pharmacokinetics was well characterized in patients with TED, and generally consistent with other IgG1 antibodies. Efficacy was consistent across the exposure range with a well-tolerated safety profile supporting the current dose regimen for patients with TED.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40262-021-01003-3.

An Evaluation of Dose and Sourcedependent Activity of Ethanolic Extractof Trigonellafoenum-graecum againstDiabetes Using Alloxan-Induced RatModel

Metabolic disorder diabetes results from an alteration of the secretion or action of insulin. Trigonella foenum-graecum is a traditionally used specimen since ancient times. We aimed to investigate the hypoglycemic potential of ethanolic extract of T. foenum-graecum seed powder solution both in a dose and source-dependent manner as well as to fathom out its safety profile so that this plant can be used to ameliorate diabetes. Diabetes was induced in the rat model via intraperitoneal injection of alloxan (150 mg/kg). Ethanolic extract of T. foenum-graecum was administered to rats’ belonged to different groups. Blood glucose levels were assessed periodically and the safety profiles were evaluated through assessment of SGOT, SGPT, creatinine, and lipid profiles after sacrificing the animals. It has been evidenced that T. foenum-graecum possesses anti-diabetic activity. Furthermore, the extract is capable of reversing the disturbed pathological state towards a healthy status. Besides, these therapeutic consequences possess dose-dependent potentiality (p<0.05), further a noteworthy source dependent (p<0.01) response were experienced. It may confer that the inconsistency associated with the remedial impacts between 2 same doses belonged to two distinct sources are due to accuracy of lab-based preparation, geographic area of cultivation, and also the season of collection. Apart from that, the visual and statistical inspections have evidence that the medium and the high dose are imparting almost indistinguishable therapeutic effects. Presumably, the reason lies beneath the receptor saturation issue.

An Assessment of Source and dosedependent Diabetes Ameliorating activity of Ethanolic Extract of Nigella sativa on Alloxan-Induced Diabetic Rat Model

Metabolic disorder diabetes results from an alteration of the secretion or action of insulin. Nigella sativa is a traditionally used specimen since ancient times. We aimed to investigate the hypoglycemic potential of ethanolic extract of Nigella sativa seed powder solution both in a dose and source-dependent manner as well as to fathom out its safety profile so that this plant can be used to ameliorate diabetes. Diabetes was induced in the rat model via intraperitoneal injection of alloxan (150 mg/kg). Ethanolic extract of T. Nigella sativa was administered to rats’ belonged to different groups. Blood glucose levels were assessed periodically and the safety profiles were evaluated through assessment of SGOT, SGPT, creatinine, and lipid profiles after sacrificing the animals. It has been evidenced that Nigella sativa possesses anti-diabetic activity. Furthermore, the extract is capable of reversing the disturbed pathological state towards a healthy status. Besides, these therapeutic consequences possess dose-dependent potentiality (p>0.05), further a noteworthy source dependent (p> 0.01) response were experienced. It may confer that the inconsistency associated with the remedial impacts between 2 same doses belonged to two distinct sources are due to accuracy of lab-based preparation, geographic area of cultivation, and also the season of collection. Apart from that, the visual and statistical inspections have evidence that the medium and the high dose are imparting almost indistinguishable therapeutic effects. Presumably, the reason lies beneath the receptor saturation issue.

Combinatorial nanodot stripe assay to systematically study cell haptotaxis

Haptotaxis is critical to cell guidance and development and has been studied in vitro using either gradients or stripe assays that present a binary choice between full and zero coverage of a protein cue. However, stripes offer only a choice between extremes, while for gradients, cell receptor saturation, migration history, and directional persistence confound the interpretation of cellular responses. Here, we introduce nanodot stripe assays (NSAs) formed by adjacent stripes of nanodot arrays with different surface coverage. Twenty-one pairwise combinations were designed using 0, 1, 3, 10, 30, 44 and 100% stripes and were patterned with 200 × 200, 400 × 400 or 800 × 800 nm2 nanodots. We studied the migration choices of C2C12 myoblasts that express neogenin on NSAs (and three-step gradients) of netrin-1. The reference surface between the nanodots was backfilled with a mixture of polyethylene glycol and poly-d-lysine to minimize nonspecific cell response. Unexpectedly, cell response was independent of nanodot size. Relative to a 0% stripe, cells increasingly chose the high-density stripe with up to ~90% of cells on stripes with 10% coverage and higher. Cell preference for higher vs. lower netrin-1 coverage was observed only for coverage ratios >2.3, with cell preference plateauing at ~80% for ratios ≥4. The combinatorial NSA enables quantitative studies of cell haptotaxis over the full range of surface coverages and ratios and provides a means to elucidate haptotactic mechanisms.

Establishment of an In Vivo Mouse Model to Study and Overcome Infusion Related Reactions Associated with Fcgriib Antibody Administration

Introduction Administration of therapeutic monoclonal antibodies (mAb) is often accompanied by infusion related reactions (IRR), of ill-determined mechanism. During the clinical development of BI-1206 (NCT02933320 and NCT03571568), a fully human mAb that binds specifically to hFcγRIIB (hCD32B), frequent IRR were observed, independent of sustained FcγRIIB blockade. At 100mg/patient, BI-1206 showed transient receptor saturation for up to 72h, whereas IRR most often resolved within 24h. Administration of BI-1206 was aslo associated with transient thrombocytopenia but not increased bleeding, and most episodes resolved within a week. Elevated transaminases (i.e. alanine transaminase (ALT) and aspartate transaminase (AST)) and a transient cytokine release was also observed alongside thrombocytopenia. Methods Six to eight-week-old C57BL/6 mice were injected either through intravenous (i.v.), intra-peritoneal (i.p.) or subcutaneous (s.c.) routes with a surrogate mouse anti-FcγRIIB (AT130-2 IgG2a) in doses ranging from 1-400μg/mouse. Mice were monitored post injection for changes in behavior and macroscopic symptoms such as isolation, mobility, and fur condition. Serum concentration of AT130-2 were quantified using an in-house ELISA. Platelet counts were analysed in fresh blood using a Vetscan (Triolab). Transaminases were analysed by IDEXX BioResearch. Cytokines were analysed with V-plex Proinflammatory Panel 1 Mouse kit (MesoScale Discovery), including interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL, 4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO and tumor necrosis factor (TNF)-α. Results Following administration of 200μg AT130-2 i.v., rapid onset of IRR was seen within 5-7 minutes. Blood sampling of these mice indicated reduced blood pressure. The mice recovered 10-15 minutes post IRR onset and no macroscopic symptoms were observed by 1h. Tolerability was dose-dependent and showed a clear pattern of increasing IRR with regards route of administration in the order s.c.&amp;lt;i.p.&amp;lt;i.v. When comparing PK and receptor occupancy with onset, severity and duration of IRR, there was a clear correlation between high dose and rapid increase in serum concentration, rather than FcγRIIB saturation. A decrease in platelet count was seen at the same time as IRRs after both i.v. and i.p. administration. The decrease was transient and normalised within 8h post injection. AST, ALT, IL-5, IL-6, IL-10 KC/GRO, TNF-α showed a transient increase, all peaking 1-3 post injection, except for IL-5. IL-5 showed a delayed peak 3-8h post injection. To investigate whether premedication with corticosteroids could inhibit the IRR and associated toxicities, mice were premedicated with betamethasone or dexamethasone 16-24h s.c. and 1h i.v. pre injection of AT130-2. Development of IRR and platelet reduction was completely inhibited with premedication. Also, the increase in liver transaminases and cytokine release was significantly reduced. A single dose of premedication at 1h pre injection did not inhibit the IRR nor prevent the decrease in platelet count, Whilst a single dose premedication 16-24h pre injection reduced the IRR and platelet decrease, it not completely block the changes, indicating that two doses of corticosteroids are optimal. Conclusions Here we demonstrate anin vivomodel that recapitulates the tolerability profile seen with BI-1206 in human cancer subjects, including IRR, decreased platelet count, elevated transaminases and transient cytokine release. In the mouse model, there was a correlation between IRR with high dose and rapid increase in serum concentration, rather than FcγRIIB saturation following administration of anti-FcγRIIB without IRR. This pre-medication regimen has been implemented in clinical trials and shown to improve tolerability to BI-1206. Disclosures Karlsson: BioInvent International AB:Current Employment.Mårtensson:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Borggren:BioInvent International AB:Current Employment.Kovacek:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Roos:BioInvent International AB:Current Employment.Blidberg:BioInvent International AB:Current Employment.Oldham:BioInvent International AB:Research Funding.Jerkeman:Roche:Research Funding;Abbvie:Research Funding;Janssen:Research Funding;Gilead:Research Funding;Celgene:Research Funding.Dyer:BioInvent International AB:Research Funding.Davies:Roche, Celgene, Kite Pharma, Acerta, Karyopharma, Regeneron, Incyte:Consultancy;Roche, Acerta Pharma, AstraZeneca, Celgene, Gilead, ADC Therapeutics, Gilead:Research Funding;Celegene, Roche, Kite Pharma, Celegene:Honoraria;Roche:Other: TRAVEL, ACCOMMODATIONS, EXPENSES.Córdoba:Roche:Honoraria, Other: travel and accommodation;Abbvie:Honoraria, Other: travel and accommodation;Janssen:Honoraria, Other: travel and accommodation;Takeda Farmacéutica España S.A.:Speakers Bureau;Gilead:Honoraria, Other: travel and accommodation.Abrisqueta:AbbVie:Consultancy, Honoraria, Speakers Bureau;Janssen:Consultancy, Honoraria, Speakers Bureau;Celgene:Consultancy, Honoraria;Roche:Consultancy, Honoraria, Speakers Bureau.Beers:BioInvent International AB:Research Funding.Teige:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.McAllister:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Lim:BioInvent International AB:Patents &amp; Royalties, Research Funding.Cragg:BioInvent International AB:Patents &amp; Royalties, Research Funding.Frendéus:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.

17-BI-1206-02 Phase 1/2a Clinical Trial of BI-1206, a Monoclonal Antibody to Fcgriib, in Combination with Rituximab in Subjects with Indolent B-Cell Non-Hodgkin Lymphoma That Has Relapsed or Is Refractory to Rituximab

Introduction BI-1206 is a fully-human IgG1 monoclonal antibody that exquisitely recognizes and blocks FcγRIIB. BI-1206 enhances the activity of anti-CD20 antibodies such as rituximab by preventing interaction with FcγRIIB and may thus overcome resistance to those treatments. BI-1206 is currently in clinical investigation in combination with rituximab for the treatment of indolent NHL. This report presents a preliminary pharmacokinetic evaluation of the data generated in the trial. Methods The safety and tolerability profile of BI-1206 in combination with rituximab is currently investigated in the Phase 1/2a clinical trial 17-BI-1206-02. The study population includes patients with follicular lymphoma (FL), marginal zone lymphoma (MZL), and mantle cell lymphoma (MCL) who have relapsed or are refractory to rituximab. BI-1206 and rituximab are administered as i.v. infusions once per week for four weeks. In Phase 1a, a 3+3 study design is used, with escalating doses of BI-1206 and a fixed dose of rituximab (375 mg/m2), with the aim of selecting the RP2D of BI-1206 for an expansion cohort in Phase 2a. Patients showing clinical benefit, are eligible for continued maintenance therapy with dosing of BI-1206 and rituximab every 8 weeks. The assessment of the pharmacokinetics (PK) of BI-1206 included non-compartmental analysis (NCA), and the assessment of the pharmacodynamics (PD) included receptor occupancy (RO%). PK modelling was conducted to further characterize the PK behavior and to provide predictions of upcoming dose levels. In addition, the effect of BI-1206 on the PK of rituximab was investigated by comparing PK parameters of rituximab to literature values of rituximab monotherapy. Results Up to 100 mg BI-1206 has been administered in combination with rituximab (375 mg/m2). Increasing doses of BI-1206 from 30 mg to 70 or 100 mg gave rise to a supra-proportional increase in Cmax as well as an increase in the half-life of BI-1206. A trend of accumulation after consecutive doses was also seen. When comparing the serum-concentrations against the associated RO% of FcγRIIB there is a trend that higher doses almost fully saturate the receptors immediately after dosing and for up to 72 hours. It is likely that increasing the dose further, will give rise to full receptor saturation, which should be maintained for an extended period. Clinical response, assessed by reduction of tumour size, has been observed at the 70 mg cohort. This at a dose which typically does not saturate receptors for the entire dose interval. It may therefor be speculated that doses which enable full RO% over the entire dosing interval, may show additional clinical benefit for patients. PK modelling showed that there was a significant contribution of a non-linear component on the elimination of BI-1206, which may be attributed to receptor binding. A two-compartment model with linear (non-saturating) and non-linear (saturating) elimination best describes the data. Using the model for predictions of upcoming doses revealed that doses close to the ones already administered may be sufficient for full receptor saturation during the entire dosing interval. Finally, the Cmax after one dose of rituximab was in the same range as previously reported values, indicating no substantial effect on the PK of rituximab. Consequently, at the current dose levels, there is no apparent need for dose-adjustments for rituximab. Conclusions This report presents preliminary data of the clinical trial 17-BI-1206-02, where BI-1206 is combined with rituximab. The presented data is encouraging, both in terms of first clinical response against tumors, as well as showing signs of overcoming target-mediated drug disposition, which may allow weekly or even less frequent dosing at clinically relevant dose levels. Disclosures Jerkeman: Roche:Research Funding;Celgene:Research Funding;Gilead:Research Funding;Abbvie:Research Funding;Janssen:Research Funding.McAllister:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Roos:BioInvent International AB:Current Employment.Lindell Andersson:BioInvent International AB:Current Employment.Karlsson:BioInvent International AB:Current Employment.Borggren:BioInvent International AB:Current Employment.Abrisqueta:Celgene:Consultancy, Honoraria;Janssen:Consultancy, Honoraria, Speakers Bureau;AbbVie:Consultancy, Honoraria, Speakers Bureau;Roche:Consultancy, Honoraria, Speakers Bureau.Teige:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.Frendéus:BioInvent International AB:Current Employment, Current equity holder in publicly-traded company.

Glutamate, d-(-)-2-Amino-5-Phosphonopentanoic Acid, and N-Methyl-d-Aspartate Do Not Directly Modulate Glycine Receptors.

Replication studies play an essential role in corroborating research findings and ensuring that subsequent experimental works are interpreted correctly. A previously published paper indicated that the neurotransmitter glutamate, along with the compounds N-methyl-d-aspartate (NMDA) and d-(-)-2-amino-5-phosphonopentanoic acid (AP5), acts as positive allosteric modulators of inhibitory glycine receptors. The paper further suggested that this form of modulation would play a role in setting the spinal inhibitory tone and influencing sensory signaling, as spillover of glutamate onto nearby glycinergic synapses would permit rapid crosstalk between excitatory and inhibitory synapses. Here, we attempted to replicate this finding in primary cultured spinal cord neurons, spinal cord slice, and Xenopus laevis oocytes expressing recombinant human glycine receptors. Despite extensive efforts, we were unable to reproduce the finding that glutamate, AP5, and NMDA positively modulate glycine receptor currents. We paid careful attention to critical aspects of the original study design and took into account receptor saturation and protocol deviations such as animal species. Finally, we explored possible explanations for the experimental discrepancy. We found that solution contamination with a high-affinity modulator such as zinc is most likely to account for the error, and we suggest methods for preventing this kind of misinterpretation in future studies aimed at characterizing high-affinity modulators of the glycine receptor. SIGNIFICANCE STATEMENT: A previous study indicates that glutamate spillover onto inhibitory synapses can directly interact with glycine receptors to enhance inhibitory signalling. This finding has important implications for baseline spinal transmission and may play a role when chronic pain develops. However, we failed to replicate the results and did not observe glutamate, d-(-)-2-amino-5-phosphonopentanoic acid, or N-methyl-d-aspartate modulation of native or recombinant glycine receptors. We ruled out various sources for the discrepancy and found that the most likely cause is solution contamination.

Natalizumab in Multiple Sclerosis Treatment: From Biological Effects to Immune Monitoring.

Multiple sclerosis is a chronic demyelinating disease of the central nervous system (CNS) with an autoimmune component. Among the recent disease-modifying treatments available, Natalizumab, a monoclonal antibody directed against the alpha chain of the VLA-4 integrin (CD49d), is a potent inhibitor of cell migration toward the tissues including CNS. It potently reduces relapses and active brain lesions in the relapsing remitting form of the disease. However, it has also been associated with a severe infectious complication, the progressive multifocal leukoencephalitis (PML). Using the standard protocol with an injection every 4 weeks it has been shown by a close monitoring of the drug that trough levels soon reach a plateau with an almost saturation of the target cell receptor as well as a down modulation of this receptor. In this review, mechanisms of action involved in therapeutic efficacy as well as in PML risk will be discussed. Furthermore the interest of a biological monitoring that may be helpful to rapidly adapt treatment is presented. Indeed, development of anti-NAT antibodies, although sometimes unapparent, can be detected indirectly by normalization of CD49d expression on circulating mononuclear cells and might require to switch to another drug. On the other hand a stable modulation of CD49d expression might be useful to follow the circulating NAT levels and apply an extended interval dose scheme that could contribute to limiting the risk of PML.

Abstract 4464: Pharmacodynamic and predictive biomarkers of clinical responses to PD-1 inhibitor budigalimab from the first-in-human clinical trial

Abstract Background: Programmed cell death 1 (PD-1) is a receptor upregulated on activated lymphocytes that mediates a dominant-negative checkpoint signal limiting antigen receptor-driven cellular activation. PD-1 immune checkpoint inhibitors have shown durable clinical responses in patients with non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC). Budigalimab (formerly ABBV-181) is a blocking humanized recombinant anti-PD-1 monoclonal antibody, currently under evaluation in multiple studies such as phase 1 clinical trial (NCT03000257) in patients with solid tumors including HNSCC and NSCLC. Multiple clinical biomarker assessments have been incorporated into this study to accommodate pharmacodynamic (PD) and predictive analyses. Methods: Patients with previously treated, advanced HNSCC (n=41) or NSCLC (n=40) that were PD-1 inhibitor naïve received budigalimab IV at 250 mg every two weeks or 500 mg every four weeks to progression, with responses assessed every 8 weeks. Archival tumor FFPE specimens from patients with HNSCC (n=37) and NSCLC (N=33) were evaluated for CD8 infiltration and tumor PD-L1 expression by Dako 28-8 IHC assay, with remaining samples processed for RNA sequencing and whole-exome sequencing. Serum (for soluble biomarkers) and whole blood (for flow cytometric analysis of T cell biomarkers) were taken at baseline and selected on-treatment time points. Univariate analysis of biomarkers associated with the best overall response (BOR) and progression-free survival was conducted. Results: As of July 2019, responses per response evaluation criteria in solid tumors (RECIST) v1.1 or iRECIST were observed at the expected rates for a PD-1 inhibitor (BOR of partial or complete response of 15% in HNSCC and 19% in NSCLC), with responses in PD-L1+ and PD-L1- tumors in both groups. Budigalimab dosing was associated with rapid complete PD-1 receptor saturation, transient decreases in circulating lymphocytes, upregulation of proliferation (Ki67) biomarkers in T cells, and upregulation of multiple soluble biomarkers, including IFNγ-induced chemokines. Differential neoantigen burden and lymphocyte-associated gene expression in tumors were observed in responders versus non-responders. Preliminary univariate analysis of biomarkers found that baseline tumor size, serum IL-6, and T cell counts were significantly associated with outcomes. Conclusions: Our biomarker analysis of budigalimab-treated HNSCC and NSCLC patients has identified early PD biomarkers consistent with PD-1 inhibitor activity as well as pre-treatment tumor and peripheral biomarkers associated with observed clinical responses. These findings confirm that budigalimab is a biologically active PD-1 inhibitor. Citation Format: Stacie Lambert, Chun Zhang, Stefan Englert, Claire Guo, Tolga Turan, Catherine Tribouley, David Masica, Robert T. McLaughlin, Gregory S. Vosganian, Daniel E. Afar. Pharmacodynamic and predictive biomarkers of clinical responses to PD-1 inhibitor budigalimab from the first-in-human clinical trial [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 4464.

Abstract 5602: BT-001, an oncolytic vaccinia virus armed with a Treg-depletion-optimized recombinant human anti-CTLA4 antibody and GM-CSF to target the tumor microenvironment

Abstract Oncolytic virotherapy is a clinically validated class of immune modulatory drugs with activity in solid cancer. The mode of action of armed oncolytic Vaccinia viruses (oVV) includes cell death induction following selective replication in tumor cells, local inflammation and priming of adaptive immune responses, and intra-tumoral delivery of therapeutic modalities e.g. antibodies, cytokines, enzymes and engineered receptor ligands. BioInvent and Transgene are collaborating in the development of a next generation armed oncolytic viruses, bringing respectively their n-CoDeR F.I.R.S.T antibody (mAb) discovery and Invir.IO oVV platforms. The first product, named BT-001, consists of the Copenhagen oVV strain deleted in J2R and I4L viral genes allowing restricted replication in proliferating cells, and the human IgG1 4-E03 recognizing human CTLA4. 4-E03 shows improved Treg-depleting activity compared with ipilimumab. BT-001 also encodes GM-CSF, the cytokine expressed in clinically advanced products like T-Vec (Imlygic) or Pexa-Vec. A surrogate mAb with similar pronounced Treg-depleting properties to 4-E03 was vectorized into the same oVV (mBT-1) allowing for functional and mechanistic in vivo studies. Since virus replication and expression of recombinant transgenes are tightly linked, the armed oVV is an interesting design for intra-tumoral delivery of therapeutic proteins exhibiting toxicological limitation (e.g. anti-CTLA4) and/or pharmacokinetic issues (e.g. GM-CSF). Oncoviral tumor-localized delivery of anti-CTLA4 may also allow better tolerated and more effective combination therapy with approved antibodies targeting the PD-1/PDL1 axis. Our studies demonstrate that 4-E03 and GM-CSF were expressed at high level as functional molecules after infection by BT-001 of several human tumoral cell lines in vitro. Moreover, following intratumoral administration in immune competent and immune deficient mice transplanted with mouse or human tumors, transgene expression was sustained at levels associated with receptor saturation for days to weeks. In contrast, and supporting the tumor-selective nature of oVV, blood concentrations of anti-CTLA4 mAb were lower compared to those observed following I.V. administration of therapeutic doses (3 mg/kg) of mAb. The in vivo anti-tumor activity of mBT-1 was assessed in multiple syngeneic mouse tumor models including CT26, EMT6, A20, and C38. mBT-1 conferred cures in the majority of challenged mice irrespective of tumor origin. The excellent anti-tumoral profile depends on anti-CTLA4 expression and could be boosted by co-administration of anti-PD-1 mAb. A clinical batch of BT-001 has been produced and toxicological evaluation is ongoing. Transgene and BioInvent are preparing CTA and IND filings for a multicentre clinical trial targeting injectable superficial tumors. Citation Format: Jean-Baptiste Marchand, Monika Semmrich, Laetitia Fend, Matilda Rehn, Nathalie Silvestre, Ingrid Teige, Johann Foloppe, Linda Mårtensson, Eric Quéméneur, Björn Frendeus. BT-001, an oncolytic vaccinia virus armed with a Treg-depletion-optimized recombinant human anti-CTLA4 antibody and GM-CSF to target the tumor microenvironment [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 5602.