With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that ...specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here, we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon complementary-determining region grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryogenic electron microscopy (cryo-EM) structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ∼50 kDa. Using an additional anti-Fab nanobody further facilitated reliable initial three-dimensional structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, is humanized, and can be conveniently expressed in
in large amounts, it may be useful not only for structural biology but also for biomedical applications.
Interleukin 17 is involved in the pathogenesis of psoriasis, a chronic debilitating disease.
To evaluate the safety/tolerability, immunogenicity, pharmacokinetics/pharmacodynamics, and efficacy of ...M1095, an anti–interleukin 17A/F nanobody, in moderate-to-severe plaque psoriasis.
This multicenter, double-blind, placebo-controlled dose escalation phase 1 study randomized 44 patients 4:1 to treatment with subcutaneous M1095 (30, 60, 120, or 240 mg) or placebo biweekly for 6 weeks, in 4 ascending dose cohorts.
The most frequent treatment-emergent adverse events with M1095 were pruritus (n = 4) and headache (n = 3); 2 patients withdrew owing to adverse events (injection site reaction and elevated liver enzyme levels). The terminal half-life of M1095 was 11 to 12 days. The area under the curve/maximum concentration was dose proportional. Of 10 M1095-treated patients positive for antidrug antibodies, 5 showed treatment-emergent antidrug antibody responses. There was no effect on M1095 exposure. Marked decreases in psoriasis inflammatory markers were observed with M1095. By day 85, 100% and 56% of patients receiving M1095, 240 mg, achieved psoriasis area and severity index 90 and 100, respectively. Improvements in static Physician's Global Assessment and affected body surface area were also seen.
Interpretation of efficacy data is limited by the small sample size.
Multiple subcutaneous doses of M1095 showed a favorable safety profile with dose-dependent improvements in psoriasis.
The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector ...functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6 Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.
Accurate prediction of the structurally diverse complementarity determining region heavy chain 3 (CDR-H3) loop structure remains a primary and long-standing challenge for antibody modeling. Here, we ...present the H3-OPT toolkit for predicting the 3D structures of monoclonal antibodies and nanobodies. H3-OPT combines the strengths of AlphaFold2 with a pre-trained protein language model and provides a 2.24 Å average RMSD Cα between predicted and experimentally determined CDR-H3 loops, thus outperforming other current computational methods in our non-redundant high-quality dataset. The model was validated by experimentally solving three structures of anti-VEGF nanobodies predicted by H3-OPT. We examined the potential applications of H3-OPT through analyzing antibody surface properties and antibody–antigen interactions. This structural prediction tool can be used to optimize antibody–antigen binding and engineer therapeutic antibodies with biophysical properties for specialized drug administration route.
Single-domain antibody (sdAb) is among the most promising vectors for developing molecular imaging tracers. Several sdAb tracers targeting human epidermal growth factor receptor 2 or programmed death ...ligand 1 have entered clinical practice. However, radiolabeled single-valent sdAbs generally have high kidney retention, limiting their therapeutic applications. Therefore, engineering strategies such as PEGylation or incorporation of renal cleavable linkers can be adapted to improve pharmacokinetics and reduce kidney retention. In this Focus on Molecular Imaging review, we try to summarize the latest developments in sdAb-derived agents and propose potential strategies that can be used to improve the theranostic value of radiolabeled sdAbs.
Significance
Vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. Currently available vaccines require cold storage and sophisticated manufacturing capacity, ...complicating their distribution, especially in less developed countries. We report a protein-based SARS-CoV-2 vaccine that directly and specifically targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (Spike
RBD
) fused to a nanobody that recognizes class II major histocompatibility complex antigens (VHH
MHCII
). Our vaccine elicits robust humoral (high-titer binding and neutralizing antibodies) and cellular immunity against SARS-CoV-2 and its variants in both young and aged mice. VHH
MHCII
-Spike
RBD
is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy, desirable attributes for logistical reasons.
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (Spike
RBD
) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHH
MHCII
). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHH
MHCII
-Spike
RBD
elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHH
MHCII
-Spike
RBD
is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.
Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320 gene. Because ...CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo‐TC (TC in complex with B12) or the interface of the human holo‐TC:TCblR complex with nanomolar affinities. We determined X‐ray crystal structures of these nanobodies bound to holo‐TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TC‐Nb4) using fluorescent light microscopy. The co‐crystal structure of holo‐TC:TCblR with another nanobody (TC‐Nb34) revealed novel features of the interface of TC and the LDLR‐A1 domain of TCblR, rationalizing the decrease in the affinity of TC‐B12 binding caused by the Δ88 mutation in CD320.
Engineered nanobodies (VHs) to the SARS-CoV-2 receptor-binding domain (RBD) were generated using phage display technology. A recombinant Wuhan RBD served as bait in phage panning to fish out ...nanobody-displaying phages from a VH/V
H phage display library. Sixteen phage-infected
clones produced nanobodies with 81.79-98.96% framework similarity to human antibodies; thus, they may be regarded as human nanobodies. Nanobodies of
clones 114 and 278 neutralized SARS-CoV-2 infectivity in a dose-dependent manner; nanobodies of clones 103 and 105 enhanced the virus's infectivity by increasing the cytopathic effect (CPE) in an infected Vero E6 monolayer. These four nanobodies also bound to recombinant Delta and Omicron RBDs and native SARS-CoV-2 spike proteins. The neutralizing VH114 epitope contains the previously reported VYAWN motif (Wuhan RBD residues 350-354). The linear epitope of neutralizing VH278 at Wuhan RBD 319RVQPTESIVRFPNITN334 is novel. In this study, for the first time, we report SARS-CoV-2 RBD-enhancing epitopes, i.e., a linear VH103 epitope at RBD residues 359NCVADVSVLYNSAPFFTFKCYG380, and the VH105 epitope, most likely conformational and formed by residues in three RBD regions that are spatially juxtaposed upon the protein folding. Data obtained in this way are useful for the rational design of subunit SARS-CoV-2 vaccines that should be devoid of enhancing epitopes. VH114 and VH278 should be tested further for clinical use against COVID-19.
Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the ...major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific V
H/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity K
ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (K
ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, the fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (K
of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND
) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL (∼0.043 nM) and 3.18 ng/mL (∼0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnostic reagents for COVID-19.
To date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics, including SARS-CoV-2 targeting antibodies, remains critical. Due to their small size (13-15 kDa), high solubility, and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multivalent formats than the conventional antibody. Here, we report a series of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.
The non-toxic immunoassay for mycotoxins is being paid more attention due to its advantages of higher safety and cost savings by using anti-idiotype antibodies to substitute toxins. In this study, ...with tenuazonic acid (TeA), a kind of highly toxic
mycotoxin as the target, an enhanced non-toxic immunoassay was developed based on the ferritin-displayed anti-idiotypic nanobody-nanoluciferase multimers. First, three specific β-type anti-idiotype nanobodies (AId-Nbs) bearing the internal image of TeA mycotoxin were selected from an immune phage display library. Then, the AId-Nb 2D with the best performance was exploited to generate a nanoluciferase (Nluc)-functionalized fusion monomer, by which a one-step non-toxic immunodetection format for TeA was established and proven to be effective. To further improve the affinity of the monomer, a ferritin display strategy was used to prepare 2D-Nluc fusion multimers. Finally, an enhanced bioluminescent enzyme immunoassay (BLEIA) was established in which the half maximal inhibitory concentration (IC
) for TeA was 6.5 ng/mL with a 10.5-fold improvement of the 2D-based enzyme-linked immunosorbent assay (ELISA). The proposed assay exhibited high selectivities and good recoveries of 80.0-95.2%. The generated AId-Nb and ferritin-displayed AId-Nb-Nluc multimers were successfully extended to the application of TeA in food samples. This study brings a new strategy for production of multivalent AId-Nbs and non-toxic immunoassays for trace toxic contaminants.
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