There is a critical need to non-invasively assess the PD-L1 expression in tumors as a predictive biomarker for determining the efficacy of anti-PD-1/PD-L1 immunotherapies. Non-invasive imaging modality like positron emission tomography (PET) can be a powerful tool to assess the PD-L1 expression in the whole body including multiple metastases as a patient selection criterion for the anti-PD-1/PD-L1 immunotherapy. In this study, we synthesized B11-nanobody, B11-scFv and B11-diabody fragments from the full-length anti-PD-L1 B11 IgG. Out of the three antibody fragments, B11-diabody showed higher nM affinity towards PD-L1 antigen as compared to B11-scFv and B11-nanobody. All three antibody fragments were successfully radiolabeled with 64Cu, a PET radioisotope. For radiolabeling, the antibody fragments were first conjugated with p-SCN-Bn-NOTA followed by chelation with 64Cu. All three radiolabeled antibody fragments were found to be stable in mouse and human sera for up to 24 h. Additionally, all three [64Cu]Cu-NOTA-B11-antibody fragments were evaluated in PD-L1 negative and human PD-L1 expressing cancer cells and subcutaneous tumor models. Based on the results, [64Cu]Cu-NOTA-B11-diabody has potential to be used as a PET imaging probe for assessing PD-L1 expression in tumors as early as 4 h post-injection, allowing faster assessment compared to the full length IgG based PET imaging probe.

Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.

OBJECTIVE: Invasive fungal diseases, such as pulmonary aspergillosis, are common life-threatening infections in immunocompromised patients and effective treatment is often hampered by delays in timely and specific diagnosis. Fungal-specific molecular imaging ligands can provide non-invasive readouts of deep-seated fungal pathologies. In this study, the utility of antibodies and antibody fragments (Fab) targeting β-glucans in the fungal cell wall to detect Aspergillus infections was evaluated both in vitro and in preclinical mouse models.
METHODS: The binding characteristics of two commercially available β-glucan antibody clones and their respective antigen-binding Fabs were tested using biolayer interferometry (BLI) assays and immunofluorescence staining. In vivo binding of the Zirconium-89 labeled antibodies/Fabs to fungal pathogens was then evaluated using PET/CT imaging in mouse models of fungal infection, bacterial infection and sterile inflammation.
RESULTS: One of the evaluated antibodies (HA-βG-Ab) and its Fab (HA-βG-Fab) bound to β-glucans with high affinity (KD = 0.056 & 21.5 nM respectively). Binding to the fungal cell wall was validated by immunofluorescence staining and in vitro binding assays. ImmunoPET imaging with intact antibodies however showed slow clearance and high background signal as well as nonspecific accumulation in sites of infection/inflammation. Conversely, specific binding of [89Zr]Zr-DFO-HA-βG-Fab to sites of fungal infection was observed when compared to the isotype control Fab and was significantly higher in fungal infection than in bacterial infection or sterile inflammation.
CONCLUSIONS: [89Zr]Zr-DFO-HA-βG-Fab can be used to detect fungal infections in vivo. Targeting distinct components of the fungal cell wall is a viable approach to developing fungal-specific PET tracers.

Potency assays are essential for the development and quality control of biopharmaceutical drugs, but they are often a time limiting factor due to manual handling steps and consequently low analytical throughput. On the other hand, automation of potency assays can be challenging due to their complexity and the use of biological materials. ELISA (enzyme-linked immunosorbent assay) is widely used for potency determination and is a good candidate for automation as all ELISA types depend on the same basic steps: coating, blocking, sample incubation, detection, and signal measurement. Nevertheless, ELISA for relative potency measurements still require drug-specific development and assay validation thereby complicating automation efforts. To simplify potency testing by ELISA, we first developed a manual protocol generally applicable to different drugs and then adapted this protocol for automated measurements. We identified unexpected critical parameters which had to be adapted to transfer the manual ELISA to an automated liquid handling system and we demonstrated that gravimetric sample dilution is unnecessary with the automated protocol. Both manual and automated protocols were validated and compared using multiple biotherapeutics. The automated protocol showed similar or higher precision and accuracy when compared to the manual method.

Streptococcus suis serotype 2 (SS2) is an important porcine pathogen that causes diseases in both swine and human. For rapid SS2 identification, a novel latex agglutination test (LAT) based on heavy-chain variable domain antibody (VH) was developed. Firstly, the soluble 47B3 VH antibody fragment from a phage display library, in which cysteine residues were engineered at the C-terminus, was expressed in Escherichia coli. The purified protein was then gently reduced to form monomeric soluble 47B3 VH subsequently used to coat with latex beads by means of site-specific conjugation. The resulting VH-coated beads gave a good agglutination reaction with SS2. The LAT was able to distinguish S. suis serotype 2 from serotype 1/2, which shares some common sugar residues, and showed no cross-reaction with other serotypes of S. suis or other related bacteria. The detection sensitivity was found to be as high as 1.85x106 cells. The LAT was stable at 4°C for at least six months without loss of activity. To the best of our knowledge, this is the first LAT based on a VH antibody fragment that can be considered as an alternative for conventional antibody-based LAT where VHs are the most favored recombinant antibody.

Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na+ channels expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the channels and belong to the beta type scorpion toxin (β-ScTx), affecting mainly the Nav1.6 channels. A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities with other known bona fide β-ScTx. The envenomation caused by this venom in mice is treated by using commercially horse antivenom available in Mexico. The potential neutralization of the toxic components was evaluated by means of surface plasmon resonance using four antibody fragments (10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4 toxins to various degrees.

Chimeric antigen receptor-T (CAR-T) cells therapy is one of the novel immunotherapeutic approaches with significant clinical success. However, their applications are limited because of long preparation time, high cost, and interpersonal variations. Although the manufacture of universal CAR-T (U-CAR-T) cells have significantly improved, they are still not a stable and unified cell bank.
Here, we tried to further improve the convenience and flexibility of U-CAR-T cells by constructing novel modular universal CAR-T (MU-CAR-T) cells. For this purpose, we initially screened healthy donors and cultured their T cells to obtain a higher proportion of stem cell-like memory T (TSCM) cells, which exhibit robust self-renewal capacity, sustainability and cytotoxicity. To reduce the alloreactivity, the T cells were further edited by double knockout of the T cell receptor (TCR) and class I human leukocyte antigen (HLA-I) genes utilizing the CRISPR/Cas9 system. The well-growing and genetically stable universal cells carrying the CAR-moiety were then stored as a stable and unified cell bank. Subsequently, the SDcatcher/GVoptiTag system, which generate an isopeptide bond, was used to covalently connect the purified scFvs of antibody targeting different antigens to the recovered CAR-T cells.
The resulting CAR-T cells can perform different functions by specifically targeting various cells, such as the eradication of human immunodeficiency virus type 1 (HIV-1)-latenly-infected cells or elimination of T lymphoma cells, with similar efficiency as the traditional CAR-T cells did.
Taken together, our strategy allows the production of CAR-T cells more modularization, and makes the quality control and pharmaceutic manufacture of CAR-T cells more feasible.

The polishing step in the downstream processing of therapeutic antibodies removes residual impurities from Protein A eluates. Among the various classes of impurities, antibody fragments are especially challenging to remove due to the broad biomolecular diversity generated by a multitude of fragmentation patterns. The current approach to fragment removal relies on ion exchange or mixed-mode adsorbents operated in bind-and-gradient-elution mode. However, fragments that bear strong similarity to the intact product or whose biophysical features deviate from the ensemble average can elude these adsorbents, and the lack of a chromatographic technology enabling robust antibody polishing is recognized as a major gap in downstream bioprocessing. Responding to this challenge, this study introduces size-exclusion mixed-mode (SEMM) silica resins as a novel chromatographic adsorbent for the capture of antibody fragments irrespective of their biomolecular features. The pore diameter of the silica beads features a narrow distribution and is selected to exclude monomeric antibodies, while allowing their fragments to access the pores where they are captured by the mixed-mode ligands. The static and dynamic binding capacity of the adsorbent ranged respectively between 30-45 and 25-33 gs of antibody fragments per liter of resin. Selected SEMM-silica resins also demonstrated the ability to capture antibody aggregates, which adsorb on the outer layer of the beads. Optimization of the SEMM-silica design and operation conditions - namely, pore size (10 nm) and ligand composition (quaternary amine and alkyl chain) as well as the linear velocity (100 cm/h), ionic strength (5.7 mS/cm), and pH (7) of the mobile phase - afforded a significant reduction of both fragments and aggregates, resulting into a final antibody yield up to 80% and monomeric purity above 97%.

The development of highly potent antibodies and antibody fragments as binding agents holds significant implications in fields such as biosensing and biotherapeutics. Their binding strength is intricately linked to the arrangement and composition of residues at the binding interface. Computational techniques offer a robust means to predict the three-dimensional structure of these complexes and to assess the affinity changes resulting from mutations. Given the interdependence of structure and affinity prediction, our objective here is to disentangle their roles. We aim to evaluate independently six side-chain reconstruction methods and ten binding affinity estimation techniques. This evaluation was pivotal in predicting affinity alterations due to single mutations, a key step in computational affinity maturation protocols. Our analysis focuses on a data set comprising 27 distinct antibody/hen egg white lysozyme complexes, each with crystal structures and experimentally determined binding affinities. Using six different side-chain reconstruction methods, we transformed each structure into its corresponding mutant via in silico single-point mutations. Subsequently, these structures undergo minimization and molecular dynamics simulation. We therefore estimate ΔΔG values based on the original crystal structure, its energy-minimized form, and the ensuing molecular dynamics trajectories. Our research underscores the critical importance of selecting reliable side-chain reconstruction methods and conducting thorough molecular dynamics simulations to accurately predict the impact of mutations. In summary, our study demonstrates that the integration of conformational sampling and scoring is a potent approach to precisely characterizing mutation processes in single-point mutagenesis protocols and crucial for computational antibody design.

Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.