A Novel Tetravalent Bispecific Immune Cell Engager Activates Natural Killer Cells to Kill Cancer Cells without Mediating Fratricide.

We previously reported the structure, affinity, and anticancer activity of a bivalent bispecific natural killer cell engager (BiKE) composed of one anti-CD16a VHH and one anti-HER2 VHH fused via a linker. In this study, we explored the engineering of a tetravalent BiKE by fusing two anti-CD16a and two anti-HER2 VHHs in tandem, using bivalent BiKE as a template. The tetravalent BiKE was genetically engineered, and its tertiary structure was predicted using in silico modeling. The antigen binding and affinity of the tetravalent BiKE were assessed using ELISA, flow cytometry, and biolayer interferometry. The ability of the BiKEs to kill cancer cells was evaluated through classical and residual antibody-dependent cellular cytotoxicity (ADCC) assays. Additionally, we investigated the potential for NK cell fratricide via CD16a-CD16a crosslinking. Our results revealed that the tetravalent BiKE exhibited at least 100-fold higher affinity toward its target antigens compared to its bivalent counterpart. The residual ADCC assay indicated that the tetravalent BiKE was more effective in killing cancer cells than the bivalent BiKE, attributable to its lower Koff value, which prolonged its binding to NK cell surfaces. Fratricide assays demonstrated that neither the bivalent nor the tetravalent BiKE mediated fratricide. Notably, our findings showed that daratumumab-induced NK fratricide was restricted to CD38-CD38 crosslinking and was not related to ADCC via CD16a-CD38 crosslinking. This study is the first in the literature to show the successful engineering of a tetravalent immune cell engager composed of tandem VHH units, which achieves high affinity and anticancer activity without mediating fratricide.

A high affinity and specificity anti-HER2 single-domain antibody (VHH) that targets trastuzumab's epitope with versatile biochemical, biological, and medical applications.

In the past decade, various single-domain antibodies from llamas, also known as VHH or nanobody, have been discovered with applications in tumor imaging and cancer therapy. However, the potential application of anti-HER2 VHHs as a diagnostic tool suitable for ELISA, flow cytometry, cell imaging, bispecific antibody engineering, and immunohistochemistry has not been fully elucidated. To investigate this potential, HER2 antigen was expressed in HEK293 F cells, purified, and used to immunize llama. Using phage display, anti-HER2 VHHs with high affinity and specificity were isolated, sequenced, and constructed with a Histag and c-Myc tag. The constructed anti-HER2 VHHs were then expressed in E. coli, purified, and evaluated for their use in ELISA, flow cytometry, cell imaging, and immunohistochemistry. The affinities of the anti-HER2 VHHs toward the HER2 antigen were determined using biolayer interferometry. Furthermore, the binding sites of the anti-HER2 VHHs were evaluated by epitope mapping and in silico modeling and docking. Here, we report the sequence of an anti-HER2 VHH with high affinity (sub-nanomolar), specificity, and selectivity. This VHH binds to the same epitope as trastuzumab and can be utilized to generate bispecific antibodies or used as a diagnostic tool to differentiate HER2+ from HER2- antigens on plates, cells, and tissues. This discovery has broad applications in biochemical, biological, and medical sciences.

Evidence for Plant-Conserved Region Mediated Trimeric CESAs in Plant Cellulose Synthase Complexes.

Higher plants synthesize cellulose using membrane-bound, six-lobed cellulose synthase complexes, each lobe containing trimeric cellulose synthases (CESAs). Although molecular biology reports support heteromeric trimers composed of different isoforms, a homomeric trimer was reported for in vitro studies of the catalytic domain of CESA1 of Arabidopsis (AtCESA1CatD) and confirmed in cryoEM structures of full-length CESA8 and CESA7 of poplar and cotton, respectively. In both structures, a small portion of the plant-conserved region (P-CR) forms the only contacts between catalytic domains of the monomers. We report inter-subunit lysine-crosslinks that localize to the small P-CR, negative-stain EM structure, and modeling data for homotrimers of AtCESA1CatD. Molecular dynamics simulations for AtCESA1CatD trimers based on the CESA8 cryoEM structure were stable and dependent upon a small set of residue contacts. The results suggest that homomeric CESA trimers may be important for the synthesis of primary and secondary cell walls and identify key residues for future mutagenic studies.

Bispecific killer cell engager with high affinity and specificity toward CD16a on NK cells for cancer immunotherapy.

Introduction: The Fc region of monoclonal antibodies (mAbs) interacts with the CD16a receptor on natural killer (NK) cells with "low affinity" and "low selectivity". This low affinity/selectivity interaction results in not only suboptimal anticancer activity but also induction of adverse effects. CD16a on NK cells binds to the antibody-coated cells, leading to antibody-dependent cell-mediated cytotoxicity (ADCC). Recent clinical data have shown that the increased binding affinity between mAb Fc region and CD16a receptor is responsible for significantly improved therapeutic outcomes. Therefore, the objective of this study was to develop a bispecific killer cell engager (BiKE) with high affinity and specificity/selectivity toward CD16a receptor for NK cell-based cancer immunotherapy. Methods: To engineer BiKE, a llama was immunized, then high binding anti-CD16a and anti-HER2 VHH clones were isolated using phage display. ELISA, flow cytometry, and biolayer interferometry (BLI) data showed that the isolated anti-CD16a VHH has high affinity (sub-nanomolar) toward CD16a antigen without cross-reactivity with CD16b-NA1 on neutrophils or CD32b on B cells. Similarly, the data showed that the isolated anti-HER2 VHH has high affinity/specificity toward HER2 antigen. Using a semi-flexible linker, anti-HER2 VHH was recombinantly fused with anti-CD16a VHH to create BiKE:HER2/CD16a. Then, the ability of BiKE:HER2/CD16a to activate NK cells to release cytokines and kill HER2+ cancer cells was measured. As effector cells, both high-affinity haNK92 (CD16+, V176) and low-affinity laNK92 (CD16+, F176) cells were used. Results and discussion: The data showed that the engineered BiKE:HER2/CD16a activates haNK92 and laNK92 cells to release cytokines much greater than best-in-class mAbs in the clinic. The cytotoxicity data also showed that the developed BiKE induces higher ADCC to both ovarian and breast cancer cells in comparison to Trazimera™ (trastuzumab). According to the BLI data, BiKE:HER2/CD16 recognizes a different epitope on CD16a antigen than IgG-based mAbs; thus, it provides the opportunity for not only monotherapy but also combination therapy with other antibody drugs such as checkpoint inhibitors and antibody-drug conjugates. Taken together, the data demonstrate the creation of a novel BiKE with high affinity and specificity toward CD16a on NK cells with the potential to elicit a superior therapeutic response in patients with HER2+ cancer than existing anti-HER2 mAbs.

The structure of a potassium-selective ion channel reveals a hydrophobic gate regulating ion permeation.

Protein dynamics are essential to function. One example of this is the various gating mechanisms within ion channels, which are transmembrane proteins that act as gateways into the cell. Typical ion channels switch between an open and closed state via a conformational transition which is often triggered by an external stimulus, such as ligand binding or pH and voltage differences. The atomic resolution structure of a potassium-selective ion channel named NaK2K has allowed us to observe that a hydro-phobic residue at the bottom of the selectivity filter, Phe92, appears in dual conformations. One of the two conformations of Phe92 restricts the diameter of the exit pore around the selectivity filter, limiting ion flow through the channel, while the other conformation of Phe92 provides a larger-diameter exit pore from the selectivity filter. Thus, it can be concluded that Phe92 acts as a hydro-phobic gate, regulating the flow of ions through the selectivity filter.

BraggNet: integrating Bragg peaks using neural networks.

Neutron crystallography offers enormous potential to complement structures from X-ray crystallography by clarifying the positions of low-Z elements, namely hydrogen. Macromolecular neutron crystallography, however, remains limited, in part owing to the challenge of integrating peak shapes from pulsed-source experiments. To advance existing software, this article demonstrates the use of machine learning to refine peak locations, predict peak shapes and yield more accurate integrated intensities when applied to whole data sets from a protein crystal. The artificial neural network, based on the U-Net architecture commonly used for image segmentation, is trained using about 100 000 simulated training peaks derived from strong peaks. After 100 training epochs (a round of training over the whole data set broken into smaller batches), training converges and achieves a Dice coefficient of around 65%, in contrast to just 15% for negative control data sets. Integrating whole peak sets using the neural network yields improved intensity statistics compared with other integration methods, including k-nearest neighbours. These results demonstrate, for the first time, that neural networks can learn peak shapes and be used to integrate Bragg peaks. It is expected that integration using neural networks can be further developed to increase the quality of neutron, electron and X-ray crystallography data.

Crystallization of a potassium ion channel and X-ray and neutron data collection.

The mechanism by which potassium ions are transported through ion channels is currently being investigated by several groups using many different techniques. Clarification of the location of water molecules during transport is central to understanding how these integral membrane proteins function. Neutrons have a unique sensitivity to both hydrogen and potassium, rendering neutron crystallography capable of distinguishing waters from K+ ions. Here, the collection of a complete neutron data set from a potassium ion channel to a resolution of 3.55 Šusing the Macromolecular Neutron Diffractometer (MaNDi) is reported. A room-temperature X-ray data set was also collected from the same crystal to a resolution of 2.50 Å. Upon further refinement, these results will help to further clarify the ion/water population within the selectivity filter of potassium ion channels.

Anomalous X-ray diffraction studies of ion transport in K+ channels.

Potassium ion channels utilize a highly selective filter to rapidly transport K+ ions across cellular membranes. This selectivity filter is composed of four binding sites which display almost equal electron density in crystal structures with high potassium ion concentrations. This electron density can be interpreted to reflect a superposition of alternating potassium ion and water occupied states or as adjacent potassium ions. Here, we use single wavelength anomalous dispersion (SAD) X-ray diffraction data collected near the potassium absorption edge to show experimentally that all ion binding sites within the selectivity filter are fully occupied by K+ ions. These data support the hypothesis that potassium ion transport occurs by direct Coulomb knock-on, and provide an example of solving the phase problem by K-SAD.

Temperature-Induced Replacement of Phosphate Proton with Metal Ion Captured in Neutron Structures of A-DNA.

Nucleic acids can fold into well-defined 3D structures that help determine their function. Knowing precise nucleic acid structures can also be used for the design of nucleic acid-based therapeutics. However, locations of hydrogen atoms, which are key players of nucleic acid function, are normally not determined with X-ray crystallography. Accurate determination of hydrogen atom positions can provide indispensable information on protonation states, hydrogen bonding, and water architecture in nucleic acids. Here, we used neutron crystallography in combination with X-ray diffraction to obtain joint X-ray/neutron structures at both room and cryo temperatures of a self-complementary A-DNA oligonucleotide d[GTGG(CSe)CAC]2 containing 2'-SeCH3 modification on Cyt5 (CSe) at pH 5.6. We directly observed protonation of a backbone phosphate oxygen of Ade7 at room temperature. The proton is replaced with hydrated Mg2+ upon cooling the crystal to 100 K, indicating that metal binding is favored at low temperature, whereas proton binding is dominant at room temperature.

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A ß-Lactamase with a Monobactam Substrate.

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum ß-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M ß-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum ß-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A ß-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.

Comparative Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the Plant Cellulose Synthesis Complex.

A six-lobed membrane spanning cellulose synthesis complex (CSC) containing multiple cellulose synthase (CESA) glycosyltransferases mediates cellulose microfibril formation. The number of CESAs in the CSC has been debated for decades in light of changing estimates of the diameter of the smallest microfibril formed from the ß-1,4 glucan chains synthesized by one CSC. We obtained more direct evidence through generating improved transmission electron microscopy (TEM) images and image averages of the rosette-type CSC, revealing the frequent triangularity and average cross-sectional area in the plasma membrane of its individual lobes. Trimeric oligomers of two alternative CESA computational models corresponded well with individual lobe geometry. A six-fold assembly of the trimeric computational oligomer had the lowest potential energy per monomer and was consistent with rosette CSC morphology. Negative stain TEM and image averaging showed the triangularity of a recombinant CESA cytosolic domain, consistent with previous modeling of its trimeric nature from small angle scattering (SAXS) data. Six trimeric SAXS models nearly filled the space below an average FF-TEM image of the rosette CSC. In summary, the multifaceted data support a rosette CSC with 18 CESAs that mediates the synthesis of a fundamental microfibril composed of 18 glucan chains.

Neutron diffraction analysis of Pseudomonas aeruginosa peptidyl-tRNA hydrolase 1.

Perdeuterated peptidyl-tRNA hydrolase 1 from Pseudomonas aeruginosa was crystallized for structural analysis using neutron diffraction. Crystals of perdeuterated protein were grown to 0.15 mm(3) in size using batch crystallization in 22.5% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol with a 20-molar excess of trilysine as an additive. Neutron diffraction data were collected from a crystal at room temperature using the MaNDi single-crystal diffractometer at Oak Ridge National Laboratory.

The structure of Toho1 ß-lactamase in complex with penicillin reveals the role of Tyr105 in substrate recognition.

The role of the conserved residue Tyr105 in class A ß-lactamases has been the subject of investigation using both structural studies and saturation mutagenesis. Both have shown that while it does not need to be strictly conserved for activity, it is important for substrate recognition. With this in mind we determined the crystal structure of Toho1 ß-lactamase at 15 K to 1.10 Å resolution in complex with penicillin. As expected a ring-opened penicillin molecule bound to Ser70 the catalytic nucleophile, can clearly be seen in electron density in the active site. In addition to the trapped penicillin, however, are two additional intact ring-closed penicillin molecules, captured by the enzyme through noncovalent interactions at the edge of the active site.

A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers.

A cellulose synthesis complex with a "rosette" shape is responsible for synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. This work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. The conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. This study strongly supports the "hexamer of trimers" model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product.

Exploring the Mechanism of ß-Lactam Ring Protonation in the Class A ß-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography.

The catalytic mechanism of class A ß-lactamases is often debated due in part to the large number of amino acids that interact with bound ß-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type ß-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 ß-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzyme's native machinery.