S309-CAR-NK cells bind the Omicron variants in vitro and reduce SARS-CoV-2 viral loads in humanized ACE2-NSG mice.

Recent progress on chimeric antigen receptor (CAR)-NK cells has shown promising results in treating CD19-positive lymphoid tumors with minimal toxicities [including graft versus host disease (GvHD) and cytokine release syndrome (CRS) in clinical trials. Nevertheless, the use of CAR-NK cells in combating viral infections has not yet been fully explored. Previous studies have shown that CAR-NK cells expressing S309 single-chain fragment variable (scFv), hereinafter S309-CAR-NK cells, can bind to SARS-CoV-2 wildtype pseudotyped virus (PV) and effectively kill cells expressing wild-type spike protein in vitro. In this study, we further demonstrate that the S309-CAR-NK cells can bind to different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants in vitro. We also show that S309-CAR-NK cells reduce virus loads in the NOD/SCID gamma (NSG) mice expressing the human angiotensin-converting enzyme 2 (hACE2) receptor challenged with SARS-CoV-2 wild-type (strain USA/WA1/2020). Our study demonstrates the potential use of S309-CAR-NK cells for inhibiting infection by SARS-CoV-2 and for the potential treatment of COVID-19 patients unresponsive to otherwise currently available therapeutics. IMPORTANCE: Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

Smoothened stimulation by membrane sterols drives Hedgehog pathway activity.

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3-5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results-combined with signalling studies and molecular dynamics simulations-delineate the structural basis for PTCH1-SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.

Development of an effective single-chain variable fragment recognizing a novel epitope in the hepatitis C virus E2 protein that restricts virus entry into hepatocytes.

Previously, we reported a neutralizing monoclonal antibody, A8A11, raised against a novel conserved epitope within the hepatitis C virus (HCV) E2 protein, that could significantly reduce HCV replication. Here, we report the nucleotide sequence of A8A11 and demonstrate the efficacy of a single-chain variable fragment (scFv) protein that mimics the antibody, inhibits the binding of an HCV virus-like particle to hepatocytes, and reduces viral RNA replication in a cell culture system. More importantly, scFv A8A11 was found to effectively restrict the increase of viral RNA levels in the serum of HCV-infected chimeric mice harbouring human hepatocytes. These results suggest a promising approach to neutralizing-antibody-based therapeutic interventions against HCV infection.

Development of alkaline phosphatase-linked single-chain variable fragment fusion proteins for one-step immunodetection of deoxynivalenol in cereals.

Deoxynivalenol (DON) is a mycotoxin that widely distributes in various foods and seriously threatens food safety. To minimize the consumers' dietary exposure to DON, there is an urgent demand for developing rapid and sensitive detection methods for DON in food. In this study, a bifunctional single-chain variable fragment (scFv) linked alkaline phosphatase (ALP) fusion protein was developed for rapid and sensitive detection of deoxynivalenol (DON). The scFv gene was chemically synthesized and cloned into the expression vector pET25b containing the ALP gene by homologous recombination. The prokaryotic expression, purification, and activity analysis of fusion proteins (scFv-ALP and ALP-scFv) were well characterized and performed. The interactions between scFv and DON were investigated by computer-assisted simulation, which included hydrogen bonds, hydrophobic interactions, and van der Waals forces. The scFv-ALP which showed better bifunctional activity was selected for developing a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for DON in cereals. The dc-ELISA takes 90 min for one test and exhibits a half inhibitory concentration (IC50) of 11.72 ng/mL, of which the IC50 was 3.08-fold lower than that of the scFv-based dc-ELISA. The developed method showed high selectivity for DON, and good accuracy was obtained from the spike experiments. Furthermore, the detection results of actual cereal samples analyzed by the method correlated well with that determined by high-performance liquid chromatography (R2=0.97165). These results indicated that the scFv-ALP is a promising bifunctional probe for developing the one-step colorimetric immunoassay, providing a new strategy for rapid and sensitive detection of DON in cereals.

Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice.

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.

Neutralizing anti-diphtheria toxin scFv produced by phage display.

BACKGROUND: Diphtheria can be prevented by vaccination, but some epidemics occur in several places, and diphtheria's threat is considerable. Administration of diphtheria antitoxin (DAT) produced from hyperimmunized animals is the most common treatment. Recombinant human antibody fragments such as single-chain variable fragments (scFv) produced by phage display library may introduce an interesting approach to overcome the limitations of the traditional antibody therapy. In the present study, B cells of immunized volunteers were used to construct a human single-chain fragment (HuscFv) library. MATERIALS AND METHODS: The library was constructed with the maximum combination of heavy and light chains. As an antigen, Diphtheria toxoid (DTd) was used in four-round phage bio-panning to select phage clones that display DTd bound HuscFv from the library. After panning, individual scFv clones were selected. Clones that were able to detect DTd in an initial screening assay were transferred to Escherichia coli HB2151 to express the scFvs and purification was followed by Ni metal ion affinity chromatography. Toxin neutralization test was performed on Vero cells. The reactivity of the soluble scFv with diphtheria toxin were done and affinity calculation based on Beatty method was calculated. RESULTS: The size of the constructed scFv library was calculated to be 1.3 × 106 members. Following four rounds of selection, 40 antibody clones were isolated which showed positive reactivity with DTd in an ELISA assay. Five clones were able to neutralize DTd in Vero cell assay. These neutralizing clones were used for soluble expression and purification of scFv fragments. Some of these soluble scFv fragments show neutralizing activity ranging from 0.6 to 1.2 µg against twofold cytotoxic dose of diphtheria toxin. The affinity constant of the selected scFv antibody was determined almost 107 M-1. CONCLUSION: This study describes the prosperous construction and isolation of scFv from the immune library, which specifically neutralizes diphtheria toxin. The HuscFv produced in this study can be a potential candidate to substitute the animal antibody for treating diphtheria and detecting toxins.

Anti-Idiotypic VHHs and VHH-CAR-T Cells to Tackle Multiple Myeloma: Different Applications Call for Different Antigen-Binding Moieties.

CAR-T cell therapy is at the forefront of next-generation multiple myeloma (MM) management, with two B-cell maturation antigen (BCMA)-targeted products recently approved. However, these products are incapable of breaking the infamous pattern of patient relapse. Two contributing factors are the use of BCMA as a target molecule and the artificial scFv format that is responsible for antigen recognition. Tackling both points of improvement in the present study, we used previously characterized VHHs that specifically target the idiotype of murine 5T33 MM cells. This idiotype represents one of the most promising yet challenging MM target antigens, as it is highly cancer- but also patient-specific. These VHHs were incorporated into VHH-based CAR modules, the format of which has advantages compared to scFv-based CARs. This allowed a side-by-side comparison of the influence of the targeting domain on T cell activation. Surprisingly, VHHs previously selected as lead compounds for targeted MM radiotherapy are not the best (CAR-) T cell activators. Moreover, the majority of the evaluated VHHs are incapable of inducing any T cell activation. As such, we highlight the importance of specific VHH selection, depending on its intended use, and thereby raise an important shortcoming of current common CAR development approaches.

Structure-guided affinity maturation of a single-chain variable fragment antibody against the Fu-bc epitope of the dengue virus envelope protein.

Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (KD) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (KD = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy.

Single-Chain Variable Fragments of Broadly Neutralizing Antibodies Prevent HIV Cell-Cell Transmission.

Broadly neutralizing antibodies (bNAbs) are able to prevent HIV infection following passive administration. Single-chain variable fragments (scFv) may have advantages over IgG as their smaller size permits improved diffusion into mucosal tissues. We have previously shown that scFv of bNAbs retain significant breadth and potency against cell-free viral transmission in a TZM-bl assay. However, scFv have not been tested for their ability to block cell-cell transmission, a model in which full-sized bNAbs lose potency. We tested four scFv (CAP256.25, PGT121, 3BNC117, and 10E8v4) compared to IgG, in free-virus and cell-cell neutralization assays in A3.01 cells, against a panel of seven heterologous viruses. We show that free-virus neutralization titers in the TZM-bl and A3.01 assays were not significantly different and confirm that scFv show a 1- to 32-fold reduction in activity in the cell-free model, compared to IgG. However, whereas IgG shows 3.4- to 19-fold geometric mean potency loss in cell-cell neutralization compared to free-virus transmission, scFv had more comparable activity in the two assays, with only a 1.3- to 2.3-fold reduction. Geometric mean 50% inhibitory concentration (IC50) of scFv for cell-cell transmission ranged from 0.65 µg/mL (10E8v4) to 2.3 µg/mL (3BNC117), with IgG and scFv neutralization showing similar potency against cell-associated transmission. Therefore, despite the reduced activity of scFv in cell-free assays, their retention of activity in the cell-cell format may make scFv useful for the prevention of both modes of transmission in HIV prevention studies. IMPORTANCE Broadly neutralizing antibodies (bNAbs) are a major focus for passive immunization against HIV, with the recently concluded HVTN Antibody Mediated Protection trial providing proof of concept. Most studies focus on cell-free HIV; however, cell-associated virus may play a significant role in HIV infection, pathogenesis, and latency. Single-chain variable fragments (scFv) of antibodies may have increased tissue penetration and reduced immunogenicity. We previously demonstrated that scFv of four HIV-directed bNAbs (CAP256.25, PGT121, 3BNC117, and 10E8v4) retain significant potency and breadth against cell-free HIV. As some bNAbs have been shown to lose potency against cell-associated virus, we investigated the ability of bNAb scFv to neutralize this mode of transmission. We demonstrate that unlike IgG, scFv of bNAbs are able to neutralize cell-free and cell-associated virus with similar potency. These scFv, which show functional activity in the therapeutic range, may therefore be suitable for further development as passive immunity for HIV prevention.

CD70-specific CAR T cells have potent activity against acute myeloid leukemia without HSC toxicity.

The prognosis of patients with acute myeloid leukemia (AML) remains dismal, highlighting the need for novel innovative treatment strategies. The application of chimeric antigen receptor (CAR) T-cell therapy to patients with AML has been limited, in particular by the lack of a tumor-specific target antigen. CD70 is a promising antigen to target AML, as it is expressed on most leukemic blasts, whereas little or no expression is detectable in normal bone marrow samples. To target CD70 on AML cells, we generated a panel of CD70-CAR T cells that contained a common single-chain variable fragment (scFv) for antigen detection, but differed in size and flexibility of the extracellular spacer and in the transmembrane and the costimulatory domains. These CD70scFv CAR T cells were compared with a CAR construct that contained human CD27, the ligand of CD70 fused to the CD3ζ chain (CD27z). The structural composition of the CAR strongly influenced expression levels, viability, expansion, and cytotoxic capacities of CD70scFv-based CAR T cells, but CD27z-CAR T cells demonstrated superior proliferation and antitumor activity in vitro and in vivo, compared with all CD70scFv-CAR T cells. Although CD70-CAR T cells recognized activated virus-specific T cells (VSTs) that expressed CD70, they did not prevent colony formation by normal hematopoietic stem cells. Thus, CD70-targeted immunotherapy is a promising new treatment strategy for patients with CD70-positive AML that does not affect normal hematopoiesis but will require monitoring of virus-specific T-cell responses.

A phase 1 study of a novel fully human BCMA-targeting CAR (CT103A) in patients with relapsed/refractory multiple myeloma.

B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapies have shown efficacy in relapsed/refractory multiple myeloma (RRMM). Because the non-human originated antigen-targeting domain may limit clinical efficacy, we developed a fully human BCMA-specific CAR, CT103A, and report its safety and efficacy in a phase 1 trial. Eighteen consecutive patients with RRMM, including 4 with prior murine BCMA CAR exposures, were enrolled. CT103A was administered at 1, 3, and 6 × 106 CAR-positive T cells/kg in the dose-escalation phase, and 1 × 106 CAR-positive T cells/kg in the expansion cohort. The overall response rate was 100%, with 72.2% of the patients achieving complete response or stringent complete response. For the 4 murine BCMA CAR-exposed patients, 3 achieved stringent complete response, and 1 achieved a very good partial response. At 1 year, the progression-free survival rate was 58.3% for all cohorts and 79.1% for the patients without extramedullary myeloma. Hematologic toxicities were the most common adverse events; 70.6% of the patients experienced grade 1 or 2 cytokine release syndromes. No immune effector cell-associated neurotoxicity syndrome was observed. To the cutoff date, CAR transgenes were detectable in 77.8% of the patients. The median CAR transgene persistence was 307.5 days. Only 1 patient was positive for the anti-drug antibody. Altogether, CT103A is safe and highly active in patients with RRMM and can be developed as a promising therapy for RRMM. Patients who relapsed from prior murine BCMA CAR T-cell therapy may still benefit from CT103A. This trial was registered at http://www.chictr.org.cn as #ChiCTR1800018137.

Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2.

Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml-1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.

Targeting insulin-like growth factor-1 receptor (IGF1R) for brain delivery of biologics.

The blood-brain barrier (BBB) prevents the majority of drugs from crossing into the brain and reaching neurons. To overcome this challenge, safe and non-invasive technologies targeting receptor-mediated pathways have been developed. In this study, three single-domain antibodies (sdAbs; IGF1R3, IGF1R4, and IGF1R5) targeting the extracellular domain of the human insulin-like growth factor-1 receptor (IGF1R), generated by llama immunization, showed enhanced transmigration across the rat BBB model (SV-ARBEC) in vitro. The rate of brain uptake of these sdAbs fused to mouse Fc (sdAb-mFc) in vivo was estimated using the fluorescent in situ brain perfusion (ISBP) technique followed by optical brain imaging and distribution volume evaluation. Compared to the brains perfused with the negative control A20.1-mFc, the brains perfused with anti-IGF1R sdAbs showed a significant increase of the total fluorescence intensity (~2-fold, p < .01) and the distribution volume (~4-fold, p < .01). The concentration curve for IGF1R4-mFc demonstrated a linear accumulation plateauing at approximately 400 µg (~1 µM), suggesting a saturable mechanism of transport. Capillary depletion and mass spectrometry analyses of brain parenchyma post-ISBP confirmed the IGF1R4-mFc brain uptake with ~25% of the total amount being accumulated in the parenchymal fraction in contrast to undetectable levels of A20.1-mFc after a 5-min perfusion protocol. Systemic administration of IGF1R4-mFc fused with the non-BBB crossing analgesic peptide galanin (2 and 5 mg/kg) induced a dose-dependent suppression of thermal hyperalgesia in the Hargreaves pain model. In conclusion, novel anti-IGF1R sdAbs showed receptor-mediated brain uptake with pharmacologically effective parenchymal delivery of non-permeable neuroactive peptides.

Structure of PINK1 in complex with its substrate ubiquitin.

Autosomal-recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular the genes encoding the E3 ubiquitin ligase Parkin (PARK2, also known as PRKN) and its upstream protein kinase PINK1 (also known as PARK6). PINK1 phosphorylates both ubiquitin and the ubiquitin-like domain of Parkin on structurally protected Ser65 residues, triggering mitophagy. Here we report a crystal structure of a nanobody-stabilized complex containing Pediculus humanus corporis (Ph)PINK1 bound to ubiquitin in the 'C-terminally retracted' (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the N lobe of PINK1 binds ubiquitin via a unique insertion. The flexible Ser65 loop in the Ub-CR conformation contacts the activation segment, facilitating placement of Ser65 in a phosphate-accepting position. The structure also explains how autophosphorylation in the N lobe stabilizes structurally and functionally important insertions, and reveals the molecular basis of AR-JP-causing mutations, some of which disrupt ubiquitin binding.

A conformation-specific antibody against oligomeric ß-amyloid restores neuronal integrity in a mouse model of Alzheimer's disease.

Conformationally distinct aggregates of the amyloid ß (Aß) peptide accumulate in brains of patients with Alzheimer's disease (AD), but the roles of the different aggregates in disease progression are not clear. We previously isolated two single-chain variable domain antibody fragments (scFvs), C6T and A4, that selectively bind different toxic conformational variants of oligomeric Aß. Here, we utilize these scFvs to localize the presence of these Aß variants in human AD brain and to demonstrate their potential as therapeutic agents for treating AD. Both A4 and C6T label oligomeric Aß in extracellular amyloid plaques, whereas C6T also labels intracellular oligomeric Aß in human AD brain tissue and in an AD mouse model. For therapeutic studies, the A4 and C6T scFvs were expressed in the AD mice by viral infection of liver cells. The scFvs were administered at 2 months of age, and mice sacrificed at 9 months. The scFvs contained a peptide tag to facilitate transport across the blood brain barrier. While treatment with C6T only slightly decreased Aß deposits and plaque-associated inflammation, it restored neuronal integrity to WT levels, significantly promoted growth of new neurons, and impressively rescued survival rates to WT levels. Treatment with A4 on the other hand significantly decreased Aß deposits but did not significantly decrease neuroinflammation or promote neuronal integrity, neurogenesis, or survival rate. These results suggest that the specific Aß conformation targeted in therapeutic applications greatly affects the outcome, and the location of the targeted Aß variants may also play a critical factor.

Blood-brain barrier transport using a high affinity, brain-selective VNAR antibody targeting transferrin receptor 1.

Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.

Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the ß2-adrenergic receptor (ß2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the ß2AR stabilizes a 'closed' receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR­G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.

Engineering and Functional Evaluation of Neutralizing Antibody Fragments Against Congenital Toxoplasmosis.

Maternal-fetal transmission of Toxoplasma gondii tachyzoites acquired during pregnancy has potentially dramatic consequences for the fetus. Current reference-standard treatments are not specific to the parasite and can induce severe side effects. In order to provide treatments with a higher specificity against toxoplasmosis, we developed antibody fragments-single-chain fragment variable (scFv) and scFv fused with mouse immunoglobulin G2a crystallizable fragment (scFv-Fc)-directed against the major surface protein SAG1. After validating their capacity to inhibit T. gondii proliferation in vitro, the antibody fragments' biological activity was assessed in vivo using a congenital toxoplasmosis mouse model. Dams were treated by systemic administration of antibody fragments and with prevention of maternal-fetal transmission being used as the parameter of efficacy. We observed that both antibody fragments prevented T. gondii dissemination and protected neonates, with the scFv-Fc format having better efficacy. These data provide a proof of concept for the use of antibody fragments as effective and specific treatment against congenital toxoplasmosis and provide promising leads.

Fusion of apoptosis-related protein Cytochrome c with anti-HER-2 single-chain antibody targets the suppression of HER-2+ breast cancer.

Cancer treatment has gradually developed from toxic chemotherapy to targeted therapy with fewer side effects. Approximately 30% of breast cancer patients overexpress human epidermal growth factor receptor 2 (HER-2). Previous studies have successfully produced single-chain antibodies (scFv) targeting HER-2+ breast cancer; however, scFv have poor stability, easy aggregation and a shorter half-life, which have no significant effect on targeting therapy. Moreover, scFv has been considered as a drug delivery platform that can kill target cells by effector molecules. However, the functional killing domains of immunotoxins are mainly derived from plant or bacterial toxins, which have a large molecular weight, low tissue permeability and severe side effects. To address these concerns, we designed several apoptotic immune molecules to replace exogenous toxins using endogenous apoptosis-related protein DNA fragmentation factor 40 (DFF40) and tandem-repeat Cytochrome c base on caspase-3 responsive peptide (DEVD). Our results suggest that DFF40 or Cytc fusion scFv specifically targets HER-2 overexpressing breast cancer cells (SK-BR-3 and BT-474) rather than HER-2 negative cells (MDA-MB-231 and MCF-7). Following cellular internalization, apoptosis-related proteins inhibited tumour activity by initiating endogenous apoptosis pathways, which significantly reduced immunogenicity and toxic side effects. Therefore, we suggest that immunoapoptotic molecules may become potential drugs for targeted immunotherapy of breast cancer.

Generation and validation of recombinant antibodies to study human aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have noncanonical functions, and several of the aaRSs have been linked to autoimmune diseases, cancer, and neurological disorders. Deciphering these roles has been challenging because of a lack of tools to enable their study. To help solve this problem, we have generated recombinant high-affinity antibodies for a collection of thirteen cytoplasmic and one mitochondrial aaRSs. Selected domains of these proteins were produced recombinantly in Escherichia coli and used as antigens in phage display selections using a synthetic human single-chain fragment variable library. All targets yielded large sets of antibody candidates that were validated through a panel of binding assays against the purified antigen. Furthermore, the top-performing binders were tested in immunoprecipitation followed by MS for their ability to capture the endogenous protein from mammalian cell lysates. For antibodies targeting individual members of the multi-tRNA synthetase complex, we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a subset of binders for each target was also confirmed using immunofluorescence. The sequences of these proteins have been deposited in publicly available databases and repositories. We anticipate that this open source resource, in the form of high-quality recombinant proteins and antibodies, will accelerate and empower future research of the role of aaRSs in health and disease.