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Monoclonal antibodies are being used to treat a remarkable breadth of human disorders. Nevertheless, there are several key challenges at the earliest stages of antibody drug development that need to be addressed using simple and widely accessible methods, especially related to generating antibodies against membrane proteins and identifying antibody candidates with drug-like biophysical properties (high solubility and low viscosity). Here we highlight key bionanotechnologies for preparing functional and stable membrane proteins in diverse types of lipoparticles that are being used to improve antibody discovery and engineering efforts. We also highlight key bionanotechnologies for high-throughput and ultra-dilute screening of antibody biophysical properties during antibody discovery and optimization that are being used for identifying antibodies with superior combinations of in vitro (formulation) and in vivo (half-life) properties.

Neutralizing monoclonal antibodies and nanobodies have shown promising results as potential therapeutic agents for COVID-19. Identifying such antibodies and nanobodies requires evaluating the neutralization activity of a large number of lead molecules via biological assays, such as the virus neutralization test (VNT). These assays are typically time-consuming and demanding on lab facilities. Here, we present a rapid and quantitative assay that evaluates the neutralizing efficacy of an antibody or nanobody within 1.5 hours, does not require BSL-2 facilities, and consumes only 8 μL of low concentration (ng/mL) sample for each assay run. We tested the human angiotensin-converting enzyme 2 (ACE2) binding inhibition efficacy of seven antibodies and eight nanobodies and verified that the IC(50) values of our assay are comparable with those from SARS-CoV-2 pseudovirus neutralization tests. We also found that our assay could evaluate the neutralizing efficacy against three widespread SARS-CoV-2 variants. We observed increased affinity of these variants for ACE2, including the Beta and Gamma variants. Finally, we demonstrated that our assay enables rapid identification of an immune-evasive mutation of the SARS-CoV-2 spike protein utilizing a set of nanobodies with known binding epitopes.

Agonist antibodies that activate cellular signaling have emerged as promising therapeutics for treating myriad pathologies. Unfortunately, the discovery of rare antibodies with the desired agonist functions is a major bottleneck during drug development. Nevertheless, there has been important recent progress in discovering and optimizing agonist antibodies against a variety of therapeutic targets that are activated by diverse signaling mechanisms. Herein, we review emerging high-throughput experimental and computational methods for agonist antibody discovery as well as rational molecular engineering methods for optimizing their agonist activity.

SARS-CoV-2 variants with enhanced transmissibility represent a serious threat to global health. Here we report machine learning models that can predict the impact of receptor-binding domain (RBD) mutations on receptor (ACE2) affinity, which is linked to infectivity, and escape from human serum antibodies, which is linked to viral neutralization. Importantly, the models predict many of the known impacts of RBD mutations in current and former Variants of Concern on receptor affinity and antibody escape as well as novel sets of mutations that strongly modulate both properties. Moreover, these models reveal key opposing impacts of RBD mutations on transmissibility, as many sets of RBD mutations predicted to increase antibody escape are also predicted to reduce receptor affinity and vice versa. These models, when used in concert, capture the complex impacts of SARS-CoV-2 mutations on properties linked to transmissibility and are expected to improve the development of next-generation vaccines and biotherapeutics.

The COVID‐19 pandemic continues to be a severe threat to human health, especially due to current and emerging SARS‐CoV‐2 variants with potential to escape humoral immunity developed after vaccination or infection. The development of broadly neutralizing antibodies that engage evolutionarily conserved epitopes on coronavirus spike proteins represents a promising strategy to improve therapy and prophylaxis against SARS‐CoV‐2 and variants thereof. Herein, a facile multivalent engineering approach is employed to achieve large synergistic improvements in the neutralizing activity of a SARS‐CoV‐2 cross‐reactive nanobody (VHH‐72) initially generated against SARS‐CoV. This synergy is epitope specific and is not observed for a second high‐affinity nanobody against a non‐conserved epitope in the receptor‐binding domain. Importantly, a hexavalent VHH‐72 nanobody retains binding to spike proteins from multiple highly transmissible SARS‐CoV‐2 variants (B.1.1.7 and B.1.351) and potently neutralizes them. Multivalent VHH‐72 nanobodies also display drug‐like biophysical properties, including high stability, high solubility, and low levels of non‐specific binding. The unique neutralizing and biophysical properties of VHH‐72 multivalent nanobodies make them attractive as therapeutics against SARS‐CoV‐2 variants., This work reports a multivalent engineering strategy to synergistically increase the neutralization potency of nanobodies against SARS‐CoV‐2. An engineered hexavalent nanobody directed against an evolutionarily conserved epitope potently neutralizes SARS‐CoV‐2 and two key variants of concern (B.1.1.7 and B.1.351). This facile approach significantly improves the neutralization breadth and potency of promising nanobodies for potential use in therapeutic applications.

We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in three modes: a constitutive mode that enables consistent and high levels of expression; a tightly repressed mode in which there is very little background expression; and an inducible mode in which concentrations of two signals (arabinose and autoinducer-2) determine the net amplification of the gene(s)-of-interest. We demonstrate the utility of the circuit for the controlled expression of human granulocyte macrophage colony stimulating factor in an engineered probiotic E. coli. This dual-input genetic controller is the first homologous AI-2 quorum sensing circuit that has the ability to be operated in three different modes. We believe it has the potential for wide-ranging biotechnological applications due its versatile features.

There is widespread interest in facile methods for generating potent neutralizing antibodies, nanobodies, and other affinity proteins against SARS-CoV-2 and related viruses to address current and future pandemics. While isolating antibodies from animals and humans are proven approaches, these methods are limited to the affinities, specificities, and functional activities of antibodies generated by the immune system. Here we report a surprisingly simple directed evolution method for generating nanobodies with high affinities and neutralization activities against SARS-CoV-2. We demonstrate that complementarity-determining region swapping between low-affinity lead nanobodies, which we discovered unintentionally but find is simple to implement systematically, results in matured nanobodies with unusually large increases in affinity. Importantly, the matured nanobodies potently neutralize both SARS-CoV-2 pseudovirus and live virus, and possess drug-like biophysical properties. We expect that our methods will improve in vitro nanobody discovery and accelerate the generation of potent neutralizing nanobodies against diverse coronaviruses., Graphical abstract, Zupancic et al. report potent neutralizing nanobodies against SARS-CoV-2. They demonstrate an approach that involves swapping the complementarity-determining regions of low-affinity clones to generate matured nanobodies with large increases in affinity and neutralization activity.

Expression of the receptor tyrosine kinase HER3 is negatively correlated with survival in ovarian cancer, and HER3 overexpression is associated with cancer progression and therapeutic resistance. Thus, improvements in HER3-targeted therapy could lead to significant clinical impact for ovarian cancer patients. Previous work from our group established multivalency as a potential strategy to improve the therapeutic efficacy of HER3-targeted ligands, including affibodies. Others have established HER3 affibodies as viable and potentially superior alternatives to monoclonal antibodies for cancer therapy. Here, bivalent HER3 affibodies were engineered for optimized production, specificity, and function as evaluated in an ovarian cancer xenograft model. Enhanced inhibition of HER3-mediated signaling and increased HER3 downregulation associated with multivalency could be achieved with a simplified construct, potentially increasing translational potential. Additionally, functional effects of affibodies due to multivalency were found to be specific to HER3 targeting, suggesting a unique molecular mechanism. Further, HER3 affibodies demonstrated efficacy in ovarian cancer xenograft mouse models, both as single agents and in combination with carboplatin. Overall, these results reinforce the potential of HER3-targeted affibodies for cancer therapy and establish treatment of ovarian cancer as an application where multivalent HER3 ligands may be useful. Further, this work introduces the potential of HER3 affibodies to be utilized as part of clinically relevant combination therapies (e.g., with carboplatin).

Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA via sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy.

For therapies targeting diseases of the gastrointestinal tract, we and others envision probiotic bacteria that synthesize and excrete biotherapeutics at disease sites. Toward this goal, we have engineered commensal E. coli that selectively synthesize and secrete a model biotherapeutic in the presence of nitric oxide (NO), an intestinal biomarker for Crohn's disease (CD). This is accomplished by co‐expressing the pore forming protein TolAIII with the biologic, granulocyte macrophage‐colony stimulating factor (GM‐CSF). We have additionally engineered these bacteria to accumulate at sites of elevated NO by engineering their motility circuits and controlling pseudotaxis. Importantly, because we have focused on in vitro test beds, motility and biotherapeutics production are spatiotemporally characterized. Together, the targeted recognition, synthesis, and biomolecule delivery comprises a “smart” probiotics platform that may have utility in the treatment of CD. Further, this platform could be modified to accommodate other pursuits by swapping the promoter and therapeutic gene to reflect other disease biomarkers and treatments, respectively.

Extracellular vesicles (EVs)—comprising a heterogeneous population of cell-derived lipid vesicles including exosomes, microvesicles, and others—have recently emerged as both mediators of intercellular information transfer in numerous biological systems and vehicles for drug delivery. In both roles, EVs have immense potential to impact tissue engineering and regenerative medicine applications. For example, the therapeutic effects of several progenitor and stem cell-based therapies have been attributed primarily to EVs secreted by these cells, and EVs have been recently reported to play direct roles in injury-induced tissue regeneration processes in multiple physiological systems. In addition, EVs have been utilized for targeted drug delivery in regenerative applications and possess unique potential to be harnessed as patient-derived drug delivery vehicles for personalized medicine. This review discusses EVs in the context of tissue repair and regeneration, including their utilization as drug carriers and their crucial role in cell-based therapies. Furthermore, the article highlights the growing need for bioengineers to understand, consider, and ultimately design and specifically control the activity of EVs to maximize the efficacy of tissue engineering and regenerative therapies.

The receptor tyrosine kinase HER3 has emerged as a therapeutic target in ovarian, prostate, breast, lung, and other cancers due to its ability to potently activate the PI3K/Akt pathway, especially via dimerization with HER2, as well as for its role in mediating drug resistance. Enhanced efficacy of HER3-targeted therapeutics would therefore benefit a wide range of patients. This study evaluated the potential of multivalent presentation, through protein engineering, to enhance the effectiveness of HER3-targeted affibodies as alternatives to monoclonal antibody therapeutics. Assessment of multivalent affibodies on a variety of cancer cell lines revealed their broad ability to improve inhibition of Neuregulin (NRG)-induced HER3 and Akt phosphorylation compared to monovalent analogues. Engineered multivalency also promoted enhanced cancer cell growth inhibition by affibodies as single agents and as part of combination therapy approaches. Mechanistic investigations revealed that engineered multivalency enhanced affibody-mediated HER3 downregulation in multiple cancer cell types. Overall, these results highlight the promise of engineered multivalency as a general strategy for enhanced efficacy of HER3-targeted therapeutics against a variety of cancers.

The reaction p(e,e'pi+)n was measured at the Mainz Microtron MAMI at an invariant mass of W=1125 MeV and four-momentum transfers of Q^2=0.117, 0.195 and 0.273 (GeV/c)^2. For each value of Q^2, a Rosenbluth separation of the transverse and longitudinal cross sections was performed. An effective Lagrangian model was used to extract the `axial mass' from experimental data. We find a value of M_A=(1.077+-0.039) GeV which is (0.051+-0.044) GeV larger than the axial mass known from neutrino scattering experiments. This is consistent with recent calculations in chiral perturbation theory.

We have measured the absolute unpolarized cross sections for photon electro-production off the proton ep → epγ with the Three-Spectrometer-Setup at MAMI at a momentum transfer q=600 MeV/c and a virtual photon polarization ɛ=0.62. The momentum q ′ of the outgoing real photon range from 33 to 111 MeV/c. We extracted two combinations of the recently introduced generalized polarizabilities [1,2].

The neutron magnetic form factor Gmn has been determined via a measurement of the ratio of cross sections D(e,e′n) and D(e,e′p). The absolute detection efficiency of the neutron detector was measured with high accuracy using tagged neutrons produced from H(n,p)n elastic scattering by means of a high intensity neutron beam. This approach minimizes the model dependence and improves upon the weakest points of previous experiments. Data in the range q2=0.2–0.8 (GeV/c)2 with uncertainties of

The reaction $p({e,e}^{\ensuremath{'}}p){\ensuremath{\pi}}^{0}$ has been studied very close to threshold $\ensuremath{\Delta}W\ensuremath{\le}4\mathrm{MeV}$ and at 4-momentum transfer ${Q}^{2}\phantom{\rule{0ex}{0ex}}=\phantom{\rule{0ex}{0ex}}0.1(\mathrm{GeV}/c{)}^{2}$. The scattered electron and the kinematically focused recoil proton were detected in coincidence using the 3-magnetic spectrometer setup at the Mainz Microtron MAMI. The transverse and longitudinal as well as the longitudinal-transverse and the transverse-transverse interference structure functions have been determined. From the data the $s$-wave multipoles ${E}_{0+}$ and ${L}_{0+}$ have been extracted. The experimental results are compared to recent calculations in chiral perturbation theory.

Two-proton correlations were studied in close to 4 π geometry using the reaction 12 C (e,e′pp) . The beam energy was 705 MeV, the energy transfer 225 MeV, and the three-momentum transfer 412 MeV/c. The electrons were observed in a large-acceptance magnetic spectrometer in coincidence with protons observed in a BGO crystal ball. Missing energy and momentum, relative momentum and angular distributions were derived and compared with a factorized two-nucleon emission model. Soft-core correlation functions are favoured by the data.

A set-up of three high-resolution magnetic spectrometers, for simplicity named A, B and C, has been built as the central facility for the precise determination of double and triple coincidence cross sections of hadron knock-out and meson production through the scattering of electrons at the Mainz microtron MAMI. The spectrometers A and C with point-to-point optics in the dispersive plane and parallel-to-point optics in the non-dispersive plane have a solid angle of 28 msr and a momentum acceptance of 20 and 25%, respectively. They each consist of a quadrupole, a sextupole and two dipole magnets, reaching maximum momenta of 735 and 550 MeV/c, respectively. The spectrometer B has a solid angle of 5.6 msr and a somewhat reduced momentum acceptance of 15%, but it reaches a maximum momentum larger than that of the MAMI electron beam (855 MeV/c). It consists of a single-clamshell dipole magnet with point-to-point optics in both planes. Each spectrometer is equipped with a position-sensitive detector system consisting of four planes of vertical drift chambers, two planes of plastic scintillators and a threshold gas Cherenkov detector. The operational experiences demonstrate that all three spectrometers exceed the specifications. Selected results of double (e, e′ x ) and triple (e, e′ x 1 x 2 ) coincidence experiments, x 1 and x 2 standing for charged hadrons, are presented, which demonstrate the performance of the whole set-up.

The differential coincident pion electroproduction cross section in the 3 He(e,e π + ) 3 H reaction has been measured with high resolution with the three-spectrometer set-up at the Mainz Microtron (MAMI) electron accelerator. Measurements were performed at the four incident energies E 0 = 855, 675, 600, and 555 MeV at fixed four-momentum transfer Q 2 = 0.045 GeV 2 , with the pions detected in parallel kinematics. This enables a separation of the measured cross section into the transverse and longitudinal structure functions by means of the Rosenbluth method. The experimental data are compared to model calculations, in which the elementary pion production amplitude includes the standard Born-amplitudes and also delta and higher resonance terms. Three-body Fadeev wave functions are used and the final state interaction of the outgoing pion is taken into account. The calculation describes the data only after medium modifications of the delta and of the pion are included.

The reactione+p →> e+π ++n at c.m. energyW=1125MeV and momentum transfer Q2=0.117GeV2/c2 has been measured. The transverse and longitudinal structure functions have been separated by varying the polarization of the virtual photon (Rosenbluth plot) with a 3 to 4% error. In addition the longitudinal-transverse interference term has been determined measuring the right-left asymmetry with an accuracy of 3%. The experimental data are compared to model calculations, and the sensitivity of the results to the axial and pion formfactors is discussed.

Forward-angle coincident electroproduction cross sections of charged pions from ${}^{3}\mathrm{He}$ have been measured at electron energies ${E}_{0}\phantom{\rule{0ex}{0ex}}=\phantom{\rule{0ex}{0ex}}855$, 675, 600, and 555 MeV. The overall features of the data for energy transfers of $\ensuremath{\omega}\phantom{\rule{0ex}{0ex}}=\phantom{\rule{0ex}{0ex}}370$ to 430 MeV with pions detected along the momentum transfer axis are reproduced in terms of a microscopic model, including pole terms, final state rescattering and produced and preformed $\ensuremath{\Delta}$ resonances. Separation of the cross section into its longitudinal and transverse parts was performed at ${Q}^{2}\phantom{\rule{0ex}{0ex}}=\phantom{\rule{0ex}{0ex}}0.045\phantom{\rule{0ex}{0ex}}(\mathrm{GeV}/c{)}^{2}$. The longitudinal part of the cross section in the ${\ensuremath{\pi}}^{+}$ channel does not contradict with the assumption of a preformed ${\ensuremath{\Delta}}^{++}$.

An intensity profile monitor has been integrated into the beam diagnostic system of the Mainz microtron MAMI. The monitor consists of two arrays of five 50 μm thick tungsten wires each, stretched from a common center with angles of 18° to each other. The arrays are moved through the electron beam in the horizontal and vertical direction, respectively. For each wire, the beam profile is recorded by measuring the secondary electron emission current which is proportional to the projected beam intensity. The intensity distribution is reconstructed from the ten beam profiles by using a two-dimensional Fourier transformation similar to the method of computer tomography. The data available in digital form are used for an on-line graphical output or as input for a computer-controlled beam handling system.

We have investigated that part of the proton momentum distribution of 16O that is linked through the (e, e′p) reaction to low-lying discrete states in 15N. The MAMI facility with its new generation of 100%-duty-cycle electron accelerator has allowed to cover in this experiment the previously not accessible momentum range of 265

A method for the separation of structure functions in (e, e′ p) experiments is proposed, which is an extension of the traditional Rosenbluth-type techniques of [1,2]. In our approach, we use a very flexible Ansatz to describe the structure functions within an extended kinematical regionG and determine its free parameters with a x2 minimization. The procedure is tested by pseudo data (12C(e, e′p)11Bg.s.) in the quasi-free region.

We have measured the first complete angular correlations of {alpha}-particle emission from the {sup 16}O isoscalar giant quadrupole resonance (GQ{sub 0}R) following excitation by inelastic electron scattering. Analysis of these determines the GQ{sub 0}R strength distribution and resolves a discrepancy between previous results from photonuclear excitation and inelastic {alpha} scattering.

The interference response function f{sub LT}(R{sub LT}) of the {sup 2}H(e,e{sup '}p)n reaction has been determined at squared four-momentum transfer Q{sup 2}=0.33 (GeV/c){sup 2} and for missing momenta up to p{sub m}=0.29 GeV/c. The results have been compared to calculations that reproduce f{sub LT} quite well but overestimate the cross sections by 10-20% for missing momenta between 0.1 GeV/c and 0.2 GeV/c.

Oral application of atropine was used to test if a modulation of cholinergic neurotransmission changed motor excitability. Healthy volunteers received either 1 or 2 mg atropine. Paired transcranial magnetic stimulation was used to study intracortical inhibition and intracortical facilitation before, 1 h and 24 h after ingestion of atropine. In addition, the silent period, motor threshold, F wave and motor response amplitudes were measured. The 1 mg dose of atropine induced a loss of intracortical inhibition, the 2 mg dose produced an intracortical disinhibition and enhanced intracortical facilitation. These changes returned to baseline after 24 h. Other electrophysiological parameters remained unchanged. Thus, an antagonist of pre- and postsynaptic muscarinic receptors increased excitability in the human motor cortex in a dose-dependent manner, indicating an influence of the cholinergic system on motor cortex excitation.

We measured the absolute cross sections for photon electro-production off the proton, ep to ep gamma , with the high resolution spectrometers at MAMI at momentum transfer q=600 MeV/c and photon polarization epsilon =0.62. We covered the momentum range for the outgoing real photon q'=33/111 MeV/c. From the extracted virtual Compton scattering amplitude we deduce values for two structure functions related to the generalized polarizabilities of the proton.

Virtual Compton scattering, i.e. the exclusive reaction γ ∗ p → γ′p′ with γ ∗ denoting a virtual photon, provides new insights on the internal structure of the proton. Below π 0 production threshold, this experiment measures the generalized polarizabilities of the proton as defined by Guichon et al [1], [2] and Drechsel et al [3]. These new electromagnetic observables, functions of Q 2 , enlarge the concept of electric (α0 and magnetic (β) polarizabilities in Real Compton Scattering ( Q 2 =0) [4]. The first VCS experiment [5] of this kind was measured at the three spectrometer facility at the Mainz Microtron MAMI for Q 2 =0.33 GeV 2 and we present in this paper the preliminary results.

The virtual Compton scattering (VCS) is the electron scattering on a proton which radiates a real photon before being detected. The new observables, called Generalized Polarizabilities (GP), extracted from this VCS at threshold can be understood as the deformation of the charge and current distributions of the proton [1]. These GP are functions of the mass of the virtual photon Q2. In real Compton scattering (Q2 = 0), some polarizabilities of the nucleon are already measured [2]. With the VCS, we will generalize these observables by measuring them at different values of Q2.

The12C(e, e'p)11B g.s reaction has been measured in parallel and anti-parallel kinematics over ap m range of −120