Search

Background: HER2-targeted therapies have substantially improved outcomes for patients with HER2-positive breast and gastric cancers. Several other cancers exhibit HER2 expression and/or amplification of its gene (ERBB2), suggesting that HER2-targeted agents may have broader therapeutic utility. Zanidatamab is a humanized, novel bispecific antibody directed against two non-overlapping domains of HER2. The aim of this Phase 1 dose-escalation and expansion study (NCT02892123) was to evaluate the safety and efficacy of zanidatamab across a range of solid tumors. Parallel to drug development, there has been rapid advancements in NGS technologies including the Guardant360 assay that can specifically sequence ctDNA and detect amplifications of the ERBB2 gene, which can lead to overexpression of HER2. FISH, the current gold standard for HER2 amplification detection, is a tissue-based assay that assesses the raw ERBB2 copy number as well as ratio of ERBB2 to a centromeric protein of chromosome 17 where the ERBB2 gene resides. We evaluated concordance of the FISH and Guardant360 assays to detect ERBB2 amplification in plasma samples. Unlike gene copy number in tissue analysis, the observed plasma copy number (pCN) is also a function of the tumor burden and rate of tumor shedding of ctDNA into the bloodstream. Methods: HER2 status was determined from a fresh tumor biopsy or in archival FFPE tissue samples by IHC and FISH according to ASCO-CAP guidelines from the Phase 1 study with zanidatamab in multiple cancer types (cholangiocarcinoma [21], colorectal carcinoma [27], all other [87]). Plasma samples were collected prior to the first cycle of zanidatamab and on-treatment for testing with Guardant360, 74 gene ctDNA NGS-based assays. Results: A concordance of 82% was observed in ERBB2/HER2 amplifications between the Guardant360 and FISH assays. An exploratory adjustment method based on tumor DNA shedding was developed by Guardant using the maximum mutant allele fraction (maxVAF) as a surrogate for tumor content. Majority of patients experienced a decrease in HER2 pCN post treatment, with 9 PD patients having the least and 21 PR patients the largest changes in ctDNA fraction (maxVAF). Conclusion: These results indicate that ERBB2 amplification detected by the Guardant360 assay could be used as a surrogate for FISH analysis in lieu of invasive surgical procedures. Citation Format: Diana Shpektor, Daryanaz Dargahi, Antonios Samiotakis, Sara Wienke, Ali Livernois, Arielle Yablonovitch, Geethika Yalamanchili, Elaina Gartner, Funda Meric-Bernstam. ERBB2 amplification detected in ctDNA as a surrogate for tumor tissue FISH analysis of HER2 status in a phase 1 study with zanidatamab for the treatment of locally advanced or metastatic HER2 expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT278.

In this study, we describe the discovery of antibodies against a MAGE-A4 peptide-major histocompatibility complex (pMHC). These antibodies will form the basis for the tumor-binding arm of T-cell engagers (TCEs) against this target. Bispecific CD3 TCEs have the potential to transform cancer treatment by redirecting T cells to tumor targets, but technological barriers have limited their development for solid tumors. Targets for TCEs have generally been limited to surface-expressed proteins, however, access to intracellular proteins that are mutated and/or differentially expressed in cancer cells would expand the target pool. Peptides of these intracellular proteins presented on MHC class I (MHC-I) provide opportunities for TCE development. Technologies powering discovery of rare antibodies that are ultra-specific, high-affinity pMHC binders are needed to expand this promising class of tumor targets. We have developed a technology platform for the discovery of optimal TCEs, including a diverse panel of CD3-binding antibodies and an antibody discovery and development engine that includes multispecific engineering capabilities, powered by OrthoMabTM. We are applying this platform to develop TCEs against MAGE-A4, an intracellular tumor target expressed by many solid tumors, but not by healthy tissues. Using proprietary immunization technologies, we triggered robust, diverse antibody responses against a complex of a human MAGE-A4 peptide presented on MHC-I. We used high-throughput microfluidic technology to screen single B cells using a multiplexed bead-binding assay to identify antibodies specific to the target, but not closely-related pMHCs. We then expressed and purified antibodies for downstream validation and characterization. Antibody specificity was initially validated using a panel of related pMHC complexes, and developability properties were assessed, including hydrophobicity, self-association, polyspecificity, stability, and aggregation. With complex data integration and analysis, we identified a panel of diverse and developable antibodies that bind with high affinity to a human MAGE-A4 peptide sequence of 10 amino acids presented on MHC-I (HLA:02*01). Strategic selection and pairing of these target-binding antibodies with our large and diverse panel of fully human CD3-binders will power the discovery of ultra-specific MAGE-A4 TCEs with optimal potency and cytokine release. Citation Format: Davide Tortora, Peter Bergqvist, Grace P. Leung, Elena Vigano, Antonios Samiotakis, Harveer Dhupar, Wei Wei, Shirley R. Zhi, Yukiko Sato, Allison Goodman, Cindy-Lee Crichlow, Melissa Cid, Jessica Fernandes Scortecci, Ping Xiang, Ahn Lee, Vivian Li, Stephanie Masterman, Sherie Duncan, Aaron Yamniuk, Kush Dalal, Tim Jacobs, Raffi Tonikian, Bryan C. Barnhart. Breaking barriers to access intracellular targets with T-cell engagers: Discovery of diverse, developable, and ultra-specific antibodies against a MAGE-A4 pMHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1891.

Previous experiments with two single-domain proteins showed that macromolecular crowding can stabilize dramatically toward heat perturbation and modulate native-state structure and shape. To assess the generality of this, we here tested the effects of the synthetic crowding agents on cytochrome c, a small single-domain protein. Using far-UV circular dichroism (CD), we discovered that there is no effect on cytochrome c's secondary structure upon addition of Ficoll or dextran (0-400 mg/mL, pH 7). Thermal experiments revealed stabilizing effects (5-10 degrees C) of Ficoll 70 and dextran 70; this effect was enhanced by the presence of low levels of guanidine hydrochloride (GuHCl) that destabilize the protein. When using a smaller dextran, dextran 40, the thermal effects were larger (10-20 degrees C). In silico analysis, using structure-based (Go-like) interactions for cytochrome c, is in excellent agreement with the in vitro thermodynamic data and also agrees with scaled particle theory. Simulations of a range of crowder size and shape demonstrated that the smaller the crowder the larger the favorable effect on cytochrome c's folded-state stability. Together with previous data, we conclude that protein size, stability, conformational malleability, and folding routes, as well as crowder size and shape, are key factors that modulate the net effect of macromolecular crowding on proteins.

How the crowded environment inside cells affects folding, stability and structures of proteins is a vital question, since most proteins are made and function inside cells. Here we describe how crowded conditions can be created in vitro and in silico and how we have used this to probe effects on protein properties. We have found that folded forms of proteins become more compact in the presence of macromolecular crowding agents; if the protein is aspherical, the shape also changes (extent dictated by native-state stability and chemical conditions). It was also discovered that the shape of the macromolecular crowding agent modulates the folding mechanism of a protein; in addition, the extent of asphericity of the protein itself is an important factor in defining its folding speed.

Proteins fold and function in the crowded environment of the cell's interior. In the recent years it has been well established that the so-called “macromolecular crowding” effect enhances the folding stability of proteins by destabilizing their unfolded states for selected proteins. On the other hand, chemical and thermal denaturation is often used in experiments as a tool to destabilize a protein by populating the unfolded states when probing its folding landscape and thermodynamic properties. However, little is known about the complicated effects of these synergistic perturbations acting on the kinetic properties of proteins, particularly when large structural fluctuations, such as protein folding, have been involved. In this study, we have first investigated the folding mechanism of Trp-cage dependent on urea concentration by coarse-grained molecular simulations where the impact of urea is implemented into an energy function of the side chain and/or backbone interactions derived from the all-atomistic molecular dynamics simulations with urea through a Boltzmann inversion method. In urea solution, the folding rates of a model mini protein Trp-cage decrease and the folded state slightly swells due to a lack of contact formation between side chains at the terminal regions. In addition, the equilibrium m-values of Trp-cage from the computer simulations are in agreement with experimental measurements. We have further investigated the combined effects of urea denaturation and macromolecular crowding on Trp-cage's folding mechanism where crowding agents are modeled as hard-spheres. The enhancement of folding rates of Trp-cage is most pronounced by macromolecular crowding effect when the extended conformations of Trp-cast dominate at high urea concentration. Our study makes quantitatively testable predictions on protein folding dynamics in a complex environment involving both chemical denaturation and macromolecular crowding effects.

Chemical and thermal denaturation methods have been widely used to investigate folding processes of proteins in vitro. However, a molecular understanding of the relationship between these two perturbation methods is lacking. Here, we combined computational and experimental approaches to investigate denaturing effects on three structurally different proteins. We derived a linear relationship between thermal denaturation at temperature T(b) and chemical denaturation at another temperature T(u) using the stability change of a protein (ΔG). For this, we related the dependence of ΔG on temperature, in the Gibbs-Helmholtz equation, to that of ΔG on urea concentration in the linear extrapolation method, assuming that there is a temperature pair from the urea (T(u)) and the aqueous (T(b)) ensembles that produces the same protein structures. We tested this relationship on apoazurin, cytochrome c, and apoflavodoxin using coarse-grained molecular simulations. We found a linear correlation between the temperature for a particular structural ensemble in the absence of urea, T(b), and the temperature of the same structural ensemble at a specific urea concentration, T(u). The in silico results agreed with in vitro far-UV circular dichroism data on apoazurin and cytochrome c. We conclude that chemical and thermal unfolding processes correlate in terms of thermodynamics and structural ensembles at most conditions; however, deviations were found at high concentrations of denaturant.

We developed a multiscale approach (MultiSCAAL) that integrates the potential of mean force (PMF) obtained from all-atomistic molecular dynamics simulations with a knowledge-based energy function for coarse-grained molecular simulations in better exploring the energy landscape of a small protein under chemical interference such as chemical denaturation. An excessive amount of water molecules in all-atomistic molecular dynamics simulations often negatively impacts the sampling efficiency of some advanced sampling techniques such as the replica exchange method and it makes the investigation of chemical interferences on protein dynamics difficult. Thus, there is a need to develop an effective strategy that focuses on sampling structural changes in protein conformations rather than solvent molecule fluctuations. In this work, we address this issue by devising a multiscale simulation scheme (MultiSCAAL) that bridges the gap between all-atomistic molecular dynamics simulation and coarse-grained molecular simulation. The two key features of this scheme are the Boltzmann inversion and a protein atomistic reconstruction method we previously developed (SCAAL). Using MultiSCAAL, we were able to enhance the sampling efficiency of proteins solvated by explicit water molecules. Our method has been tested on the folding energy landscape of a small protein Trp-cage with explicit solvent under 8M urea using both the all-atomistic replica exchange molecular dynamics (AA-REMD) and MultiSCAAL. We compared computational analyses on ensemble conformations of Trp-cage with its available experimental NOE distances. The analysis demonstrated that conformations explored by MultiSCAAL better agree with the ones probed in the experiments because it can effectively capture the changes in side chain orientations that can flip out of the hydrophobic pocket in the presence of urea and water molecules., Comment: This article has been accepted by the Journal of Chemical Physics (2010)

Topological frustrated proteins can give rise to complex folding pathways and transition state profiles. To better understand such systems, we combine experimental and computational methods to study Desulfovibrio desulfuricans apoflavodoxin by producing several point mutation variants. By equilibrium unfolding experiments, we first revealed how different secondary-structure elements contribute to overall protein resistance towards heat and urea. Next using stopped-flow mixing coupled to far-UV circular dichroism (CD), we probed how individual residues affect the amount of structure formed in the experimentally-detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of the growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central β-strand 3 (i.e. at β-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in the folding nucleigrowth correlates with the in vitro burst phase CD amplitude. In addition, Φ-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the β-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central β-sheet directs initial misfolding while proper alignment on both sides of β-strand 3 is necessary for productive folding.

Flavodoxin adopts the common repeat beta/alpha topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, we probed how individual residues affect the amount of structure formed in the experimentally detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central beta-strand 3 (i.e., at beta-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, phi-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the beta-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central beta-sheet directs initial misfolding, while proper alignment on both sides of beta-strand 3 is necessary for productive folding.

How the crowded environment inside cells affects the structures of proteins with aspherical shapes is a vital question because many proteins and protein–protein complexes in vivo adopt anisotropic shapes. Here we address this question by combining computational and experimental studies of a football-shaped protein (i.e., Borrelia burgdorferi VlsE) in crowded, cell-like conditions. The results show that macromolecular crowding affects protein-folding dynamics as well as overall protein shape. In crowded milieus, distinct conformational changes in VlsE are accompanied by secondary structure alterations that lead to exposure of a hidden antigenic region. Our work demonstrates the malleability of “native” proteins and implies that crowding-induced shape changes may be important for protein function and malfunction in vivo .

Chemical and thermal denaturation has been widely used in order to investigate the folding processes of proteins in vitro. However, a comprehensive understanding of relationship between chemical and thermal perturbations of proteins at molecular level has been challenging. Here, we have used combined computational and experimental approaches to investigate the denaturing effect on structurally different, unrelated proteins. We have identified a linear relationship between thermal denaturation and chemical denaturation through the stability of protein folding (ΔG) by relating the dependence of ΔG on temperature in the Gibbs-Helmholtz equation and that of ΔG on urea concentration in the linear extrapolation method. We have tested this relationship on apoazurin, cytochrome c and apoflavodoxin by examining their structural similarities of the ensembles perturbed by chemical versus thermal means using coarse-grained molecular simulations at a broad range of urea concentration and temperature. We have found that the temperature of a system in the absence of urea at Tb is linearly proportional to the temperature of the same system under low urea concentration at Tu at a slope of one. The slope of this linear correspondence deviates from one at high urea concentration. In addition, the folding route of apoflavodoxin that involves topological frustration is influenced by the presence of urea. This in silico finding has agreed with in vitro far UV circular dichorism data for apoazurin and cytochrome c. Thus, chemical and thermal unfolding processes correlate in terms of thermodynamics and structural ensembles at most conditions, but deviations occur at high concentrations of denaturant.

Proteins fold and function in the crowded environment of the cell's interior. In the recent years it has been well established that the so-called "macromolecular crowding" effect enhances the folding stability of proteins by destabilizing their unfolded states for selected proteins. On the other hand, chemical and thermal denaturation is often used in experiments as a tool to destabilize a protein by populating the unfolded states when probing its folding landscape and thermodynamic properties. However, little is known about the complicated effects of these synergistic perturbations acting on the kinetic properties of proteins, particularly when large structural fluctuations, such as protein folding, have been involved. In this study, we have first investigated the folding mechanism of Trp-cage dependent on urea concentration by coarse-grained molecular simulations where the impact of urea is implemented into an energy function of the side chain and/or backbone interactions derived from the all-atomistic molecular dynamics simulations with urea through a Boltzmann inversion method. In urea solution, the folding rates of a model miniprotein Trp-cage decrease and the folded state slightly swells due to a lack of contact formation between side chains at the terminal regions. In addition, the equilibrium m-values of Trp-cage from the computer simulations are in agreement with experimental measurements. We have further investigated the combined effects of urea denaturation and macromolecular crowding on Trp-cage's folding mechanism where crowding agents are modeled as hard-spheres. The enhancement of folding rates of Trp-cage is most pronounced by macromolecular crowding effect when the extended conformations of Trp-cast dominate at high urea concentration. Our study makes quantitatively testable predictions on protein folding dynamics in a complex environment involving both chemical denaturation and macromolecular crowding effects.

We use computer simulation to investigate the effects of synthetic crowders on cytochrome c, a small single-domain protein with a cofactor heme. The folding energy landscape of cytochrome was computed in the presence of crowders with various sizes and shapes by using a coarse-grained protein model and the structure-based (Go-like) interactions. Our results demonstrated that given the same volume fraction of crowders, the stability of a folded protein inversely increases with the radius of crowders. In addition, a crowder with an anisotropic geometry imposes a greater stabilizing effect on the folded protein than isotropic crowders. This is in agreement with the predictions by the scaled particle theory. In addition, the distribution of contact formation between heme and cytochrome c protein was found to be varied by different types of crowders, demonstrating that the geometry of crowders may be one of the key factors for tuning heme-protein contact formation under cell-like conditions. Prospects of mixed crowders will be presented.

In the quest to develop multiscale molecular simulation methods for complex protein dynamics that fuse high-resolution and low-resolution protein representations, it is important to investigate the required information of reconstruction of all-atomistic proteins from low resolution ones with manageable uncertainly. In this paper, we introduce a robust, accurate, and fast reconstruction method (SCAAL) that produces reliable all-atomistic protein structure by taking few beads from a coarse-grained model with at least one side chain bead and one Cα bead in the backbone (Side chain-Cα Model, SCM) into accounts. Our algorithm (SCAAL) is compared with SCWRL3.0 and it excels in robustness and is more accurate in the reconstruction of large amino acids. In addition, we further test SCAAL in the reconstruction of a complete protein folding trajectory from SCM coarse-grained models. We show that the efficiency, accuracy, and robustness of SCAAL as leverage for multi-scale simulations are excellent in terms of low root mean square deviations that lie within 1A resolution.

We developed a multiscale approach (MultiSCAAL) that integrates the potential of mean force (PMF) obtained from all-atomistic molecular dynamics simulations with a knowledge-based energy function for coarse-grained molecular simulation in better exploring the energy landscape of a small protein under chemical interference such as chemical denaturation. The two key features of this scheme are the Boltzmann inversion and a protein atomistic reconstruction method we previously developed (SCAAL). Using MultiSCAAL, we were able to enhance the sampling efficiency of proteins solvated by explicit water molecules. Our method has been tested on the folding energy landscape of a small protein Trp-cage with explicit solvent under 8M urea using both the all-atomistic replica exchange molecular dynamics (AA-REMD) and MultiSCAAL. We compared computational analyses on ensemble conformations of Trp-cage with its available experimental NOE distances. The analysis demonstrated that conformations explored by MultiSCAAL better agree with the ones probed in the experiments because it can effectively capture the changes in side chain orientations that can flip out of the hydrophobic pocket in the presence of urea and water molecules. In this regard, MultiSCAAL is a promising and effective sampling scheme for investigating chemical interference which presents a great challenge when modeling protein interactions in vivo.