786 Dose selection for DuoBody®-PD-L1×4-1BB (GEN1046) using a semimechanistic pharmacokinetics/pharmacodynamics model that leverages preclinical and clinical data

BackgroundDuoBody-PD-L1×4-1BB (GEN1046) is a class-defining bispecific antibody, designed to elicit an anti-tumor immune response by simultaneous and complementary blockade of PD-L1 on tumor cells and conditional stimulation of 4-1BB on T-cells and NK cells. Optimizing target engagement for a bispecific antibody is challenging, as it involves binding with two targets, and predicting trimer levels in tumors based on affinity of individual arms and target expression. Here we describe a semimechanistic, physiologically based pharmacokinetic/pharmacodynamic (PK/PD) model that predicts a dosing regimen for DuoBody-PD-L1×4-1BB, which results in the formation of maximum levels of a therapeutically active 4-1BB-bispecific antibody-PD-L1 trimolecular complex (trimer), and optimal PD-L1 receptor occupancy (RO).MethodsAn integrated semimechanistic PK/PD model that describes the distribution of DuoBody-PD-L1×4-1BB into central and peripheral compartments and partitioning into tumor/lymph nodes was developed. The model used PK/PD data and physiological parameters from the literature for parameterizations of PD-L1 and 4-1BB expression levels and T-cell trafficking. The model incorporates dynamic binding of DuoBody-PD-L1×4-1BB to its targets to predict trimer formation and RO for PD-L1 in tumors. Model parameters were calibrated to match in vitro PD studies, such as analyses of T-cell proliferation and cytokine release, as well as clinical PK data. Sensitivity to model assumptions were assessed by varying PK/PD parameters, and assessing their impact on trimer formation and PD-L1 RO. The model was subsequently used to explore in vivo trimer levels and PD-L1 RO in tumors at various dosing regimens.ResultsThe model was able to adequately describe the PK of DuoBody-PD-L1×4-1BB in the central compartment. Simulations showed a bell-shaped response for average trimer levels in tumors that peaked at 100 mg every 3 weeks (Q3W), with doses >100 mg resulting in reduced trimer formation. Average PD-L1 receptor occupancy at the 100 mg dose was predicted to be approximately 70% over 21 days and increased at higher doses. Based on these model predictions, and available safety, anti-tumor activity, and PD data from the ongoing GCT1046-01 trial (NCT03917381), 100 mg Q3W was chosen as the expansion dose for further evaluation in Part 2 of the study.ConclusionsThis semimechanistic PK/PD model provides a novel approach for dose selection of bispecific antibodies such as DuoBody-PD-L1×4-1BB, by using preclinical and clinical PK/PD data to predict formation of optimal trimer levels and PD-L1 receptor occupancy.AcknowledgementsThe authors thank Friederike Gieseke and Zuzana Jirakova at BioNTech SE; Kalyanasundaram Subramanian at Applied Biomath LLC for their valuable contributions.Trial RegistrationWritten informed consent, in accordance with principles that originated in the Declaration of Helsinki 2013, current ICH guidelines including ICH-GCP E6(R2), applicable regulatory requirements, and sponsor policy, was provided by the patients.

Species-specific gamete recognition initiates fusion-driving trimer formation by conserved fusogen HAP2

Recognition and fusion between gametes during fertilization is an ancient process. Protein HAP2, recognized as the primordial eukaryotic gamete fusogen, is a structural homolog of viral class II fusion proteins. The mechanisms that regulate HAP2 function, and whether virus-fusion-like conformational changes are involved, however, have not been investigated. We report here that fusion between plus and minus gametes of the green alga Chlamydomonas indeed requires an obligate conformational rearrangement of HAP2 on minus gametes from a labile, prefusion form into the stable homotrimers observed in structural studies. Activation of HAP2 to undergo its fusogenic conformational change occurs only upon species-specific adhesion between the two gamete membranes. Following a molecular mechanism akin to fusion of enveloped viruses, the membrane insertion capacity of the fusion loop is required to couple formation of trimers to gamete fusion. Thus, species-specific membrane attachment is the gateway to fusion-driving HAP2 rearrangement into stable trimers.

On the specificity of protein–protein interactions in the context of disorder

With the increased focus on intrinsically disordered proteins (IDPs) and their large interactomes, the question about their specificity — or more so on their multispecificity — arise. Here we recapitulate how specificity and multispecificity are quantified and address through examples if IDPs in this respect differ from globular proteins. The conclusion is that quantitatively, globular proteins and IDPs are similar when it comes to specificity. However, compared with globular proteins, IDPs have larger interactome sizes, a phenomenon that is further enabled by their flexibility, repetitive binding motifs and propensity to adapt to different binding partners. For IDPs, this adaptability, interactome size and a higher degree of multivalency opens for new interaction mechanisms such as facilitated exchange through trimer formation and ultra-sensitivity via threshold effects and ensemble redistribution. IDPs and their interactions, thus, do not compromise the definition of specificity. Instead, it is the sheer size of their interactomes that complicates its calculation. More importantly, it is this size that challenges how we conceptually envision, interpret and speak about their specificity.

An unusual aspartic acid cluster in the reovirus attachment fiber σ1 mediates stability at low pH

ABSTRACTThe reovirus attachment protein σ1 mediates cell attachment and receptor binding and is thought to undergo conformational changes during viral disassembly. σ1 is a trimeric filamentous protein with an α-helical coil-coiled Tail, a triple β-spiral Body, and a globular Head. The Head domain features an unusual and conserved aspartic acid cluster at the trimer interface, which forms the only significant intra-trimer interactions in the Head, and must be protonated to allow trimer formation.Here we show that all domains of σ1 are remarkably thermostable across a wide range of pH, even at the low pH of the stomach. Interestingly, we determine the optimal pH for stability to be between pH 5-6, a value close to the pH of the endosome and of the jejunum. The σ1 Head is stable at acidic and neutral pH, but detrimerizes at basic pH. When Asp345 in the aspartic acid cluster is mutated to asparagine, the σ1 Head loses stability at low pH and is more prone to detrimerize. Overall, the presence of the Body stabilizes the σ1 Head.Our results confirm a role of the aspartic acid cluster as a pH-dependent molecular switch, and highlight its role in enhancing σ1 stability at low pH.

Novel Semi-Mechanistic Model Leveraging Preclinical and Clinical Data to Inform the Recommended Phase 2 Dose (RP2D) Selection for Epcoritamab (DuoBody CD3xCD20)

Introduction: Epcoritamab (DuoBody-CD3×CD20) is a subcutaneously administered bispecific antibody (bsAb) that simultaneously binds to CD3 on T cells and CD20 on malignant B cells, resulting in T-cell activation and expansion and selective T-cell-mediated killing of CD20+ cells. Target engagement (TE) and crosslinking of CD3 and CD20 (trimer formation) lead to activation and expansion of T cells, which in turn leads to tumor cell killing and is the first step driving the pharmacology of epcoritamab. Hence, the optimal clinical dose of epcoritamab can be informed by TE and trimer formation. Unlike typical monoclonal antibodies, bsAbs exhibit a hook effect in which trimer formation is impaired at high drug concentrations. Therefore, targeting complete receptor occupancy can lead to suboptimal trimer formation and clinical efficacy; instead, the aim should be to identify epcoritamab concentrations that result in maximal trimer formation. Epcoritamab is currently being investigated in an ongoing, open-label, multi-center, first-in-human trial in patients with relapsed/refractory B-cell non-Hodgkin lymphoma (NCT03625037). Endpoints include safety and identification of RP2D and pharmacokinetic/pharmacodynamic (PK/PD) analyses. Here, we present our approach for recommending the RP2D for epcoritamab based on a novel PK/PD model that predicts trimer formation. Methods: A semi-mechanistic PK/PD model was developed to quantitatively describe biodistribution of epcoritamab, trimer formation, and tumor response (Figure 1). This model makes use of preclinical data from cynomolgus monkeys, clinical PK/PD data, patient biomarker data, patient tumor characteristics, and response data. The model incorporates a minimal physiological-based PK model to predict epcoritamab concentration in tumors. The model also considers T- and B-cell dynamics, expression of CD3 and CD20 on these cells, and dynamic binding of epcoritamab to CD3 and CD20, as well as levels of trimer formation. Clinical trial simulations were performed using the PK/PD model, incorporating individual variability in key parameters, to predict the extent of trimer formation and tumor response in patients with diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Variability in model parameters was based on either interindividual variability observed in the first-in-human trial or literature values. Results: The population PK/PD model was able to describe epcoritamab concentration-time profiles across doses (range: 0.0128-60 mg). Epcoritamab exhibited slow absorption, with Tmax of 2.8 days, a terminal half-life of 8.67 days, and target-mediated disposition (TMD). The model predicted the saturation of TMD to occur at dose levels ≥48 mg, indicating engagement and saturation of CD3 and CD20 in blood. In addition, the model was able to describe the exposure-response relationship observed in the clinic. Clinical trial simulations using the PK/PD model demonstrated that a 48-mg dose can achieve optimal trimer formation and clinical response in both FL and DLBCL. An exposure-adverse event analysis showed a flat relationship between epcoritamab exposure and risk of cytokine release syndrome (CRS) in the dose range evaluated. Based on these findings, 48 mg was identified as the potential RP2D for epcoritamab. Conclusions: For bsAbs such as epcoritamab, the optimal dose is one that leads to maximum trimer formation; hence, traditional PK modeling methodologies and exposure-response analyses are not adequate to guide RP2D selection. To overcome these limitations, we have developed a novel, semi-mechanistic PK/PD model incorporating preclinical, clinical, and patient biomarker data that identified the optimal dose for epcoritamab. This PK/PD model represents a novel approach and provides a general framework that can be applied to other CD3 bsAbs. Disclosures Li: Genmab: Current Employment. Hiemstra:Genmab: Current Employment, Current equity holder in publicly-traded company. Chiu:Genmab: Current Employment. Oliveri:Genmab: Current Employment, Current equity holder in publicly-traded company. DeMarco:Genmab: Current Employment, Current equity holder in publicly-traded company. Salcedo:Genmab: Current Employment. Lihme Egerod:Genmab: Current Employment. Gupta:Genmab: Current Employment. OffLabel Disclosure: Epcoritamab is an investigational agent undergoing evaluation in patients with relapsed/refractory B-cell non-Hodgkin lymphoma.

Retracted Article: Dynamic Adsorption Self-assembly of Au nanocube (NC) for opposite charge Au nanoparticles (NPs) in real time

Using in situ liquid cell TEM imaging, the dynamic process of dimer and trimer formation were captured. The kinetic energy of NPs and the NC- Electrostatic Shielding Length (NC-ESL) are...