The field of cancer treatment has evolved significantly over the last decade with the emergence of next-generation therapeutic antibodies. Conventional treatments like chemotherapy pose significant challenges, including adverse side effects. Monoclonal antibodies have paved the way for more targeted and effective interventions. The evolution from chimeric to humanized and fully human antibodies has led to a reduction in immunogenicity and enhanced tolerance in vivo. The advent of next-generation antibodies, including bispecific antibodies, nanobodies, antibody-drug conjugates, glyco-engineered antibodies, and antibody fragments, represents a leap forward in cancer therapy. These innovations offer increased potency, adaptability, and reduced drug resistance. Challenges such as target validation, immunogenicity, and high production costs exist. However, technological advancements in antibody engineering techniques provide optimism for addressing these issues. The future promises a paradigm shift, where ongoing research will propel these powerful antibodies to the forefront, revolutionizing the fight against cancer and creating new preventive and curative treatments. This review provides an overview of three next-generation antibody-based molecules, namely bispecific antibodies, antibody-drug conjugates, and nanobodies that have shown promising results in cancer treatment. It discusses the evolution of antibodies from conventional forms to next-generation molecules, along with their applications in cancer treatment, production methods, and associated challenges. The review aims to offer researchers insights into the evolving landscape of next-generation antibody-based cancer therapeutics and their potential to revolutionize treatment strategies.

OBJECTIVE: While immunotherapy has revolutionized the oncology field, variations in therapy responsiveness limit the broad applicability of these therapies. Diagnostic imaging of immune cell, and specifically CD8+ T cell, dynamics could allow early patient stratification and result in improved therapy efficacy and safety. In this study, we report the development of a nanobody-based immunotracer for non-invasive SPECT and PET imaging of human CD8+ T-cell dynamics.
METHODS: Nanobodies targeting human CD8β were generated by llama immunizations and subsequent biopanning. The lead anti-human CD8β nanobody was characterized on binding, specificity, stability and toxicity. The lead nanobody was labeled with technetium-99m, gallium-68 and copper-64 for non-invasive imaging of human T-cell lymphomas and CD8+ T cells in human CD8 transgenic mice and non-human primates by SPECT/CT or PET/CT. Repeated imaging of CD8+ T cells in MC38 tumor-bearing mice allowed visualization of CD8+ T-cell dynamics.
RESULTS: The nanobody-based immunotracer showed high affinity and specific binding to human CD8 without unwanted immune activation. CD8+ T cells were non-invasively visualized by SPECT and PET imaging in naïve and tumor-bearing mice and in naïve non-human primates with high sensitivity. The nanobody-based immunotracer showed enhanced specificity for CD8+ T cells and/or faster in vivo pharmacokinetics compared to previous human CD8-targeting immunotracers, allowing us to follow human CD8+ T-cell dynamics already at early timepoints.
CONCLUSIONS: This study describes the development of a more specific human CD8+ T-cell-targeting immunotracer, allowing follow-up of immunotherapy responses by non-invasive imaging of human CD8+ T-cell dynamics.

Nanobodies derived from camelids and sharks offer unique advantages in therapeutic applications due to their ability to bind to epitopes that were previously inaccessible. Traditional methods of nanobody development face challenges such as ethical concerns and antigen toxicity. Our study presents a synthetic, phagedisplayed nanobody library using trinucleotide-directed mutagenesis technology, which allows precise amino acid composition in complementarity-determining regions (CDRs), with a focus on CDR3 diversity. This approach avoids common problems such as frameshift mutations and stop codon insertions associated with other synthetic antibody library construction methods. By analyzing FDA-approved nanobodies and Protein Data Bank sequences, we designed sub-libraries with different CDR3 lengths and introduced amino acid substitutions to improve solubility. The validation of our library through the successful isolation of nanobodies against targets such as PD-1, ATXN1 and STAT3 demonstrates a versatile and ethical platform for the development of high specificity and affinity nanobodies and represents a significant advance in biotechnology.

UNASSIGNED: The misfolding and aggregation of proteins are associated with various neurodegenerative diseases, such as Alzheimer\'s disease (AD). The small-molecule engineered antibodies, such as single-chain fragment variable (scFv) antibodies and nanobodies (Nbs), have gained attention in recent years due to their strong conformational specificity, ability to cross the blood-brain barrier (BBB), low immunogenicity, and enhanced proximity to active sites within aggregates.
UNASSIGNED: We have reviewed recent advances in therapies involving scFvs and Nbs that efficiently and specifically target pathological protein aggregates. Relevant publications were searched for in MEDLINE, GOOGLE SCHOLAR, Elsevier ScienceDirect and Wiley Online Library.
UNASSIGNED: We reviewed the recent and specific targeting of pathological protein aggregates by scFvs and Nbs. These engineered antibodies can inhibit the aggregation or promote the disassembly of misfolded proteins by recognizing antigenic epitopes or through conformational specificity. Additionally, we discuss strategies for improving the effective application of engineered antibodies in treating AD. These technological strategies will lay the foundation for the clinical application of small-molecule antibody drugs in developing effective treatments for neurological diseases. Through rational application strategies, small-molecule engineered antibodies are expected to have significant potential in targeted therapy for neurological disorders.

Maize lethal necrosis (MLN) is a maize disease caused by the maize chlorotic mottle virus (MCMV), a potyvirus which causes yield losses of 30-100%. The present study aimed to isolate nanobodies against the MCMV coat protein (CP) for the diagnosis of MLN. MCMV CP expressed in Escherichia coli was used for llama immunization. VHH (i.e. variable heavy domain of heavy chain) gene fragments were prepared from blood drawn from the immunized llama and used to generate a library in E. coli TG1 cells. MCMV specific nanobodies were selected by three rounds of phage display and panning against MCMV CP. The selected nanobodies were finally expressed in E. coli WK6 cells and purified. Eleven MCMV-specific nanobodies were identified and shown to detect MCMV in infected maize plants. Thus, our results show that nanobodies isolated from llama immunized with MCMV CP can distinguish infected and healthy maize plants, potentially enabling development of affordable MCMV detection protocols.

Single-domain antibodies (sdAbs) or nanobodies have received widespread attention due to their small size (~ 15 kDa) and diverse applications in bio-derived therapeutics. As many modern biotechnology breakthroughs are applied to antibody engineering and design, nanobody thermostability or melting temperature (Tm) is crucial for their successful utilization. In this study, we present TEMPRO which is a predictive modeling approach for estimating the Tm of nanobodies using computational methods. Our methodology integrates various nanobody biophysical features to include Evolutionary Scale Modeling (ESM) embeddings, NetSurfP3 structural predictions, pLDDT scores per sdAb region from AlphaFold2, and each sequence\'s physicochemical characteristics. This approach is validated with our combined dataset containing 567 unique sequences with corresponding experimental Tm values from a manually curated internal data and a recently published nanobody database, NbThermo. Our results indicate the efficacy of protein embeddings in reliably predicting the Tm of sdAbs with mean absolute error (MAE) of 4.03 °C and root mean squared error (RMSE) of 5.66 °C, thus offering a valuable tool for the optimization of nanobodies for various biomedical and therapeutic applications. Moreover, we have validated the models\' performance using experimentally determined Tms from nanobodies not found in NbThermo. This predictive model not only enhances nanobody thermostability prediction, but also provides a useful perspective of using embeddings as a tool for facilitating a broader applicability of downstream protein analyses.

Recent years have witnessed rapid progress in the discovery of therapeutic proteins and peptides for the treatment of central nervous system (CNS) diseases. However, their clinical applications have been considerably hindered by challenges such as low biomembrane permeability, poor stability, short circulation time, and the formidable blood-brain barrier (BBB). Recently, substantial improvements have been made in understanding the dynamics of the BBB and developing efficient approaches for delivering proteins and peptides to the CNS, especially by using various nanoparticles. Herein, we present an overview of the up-to-date understanding of the BBB under physiological and pathological conditions, emphasizing their effects on brain drug delivery. We summarize advanced strategies and elucidate the underlying mechanisms for delivering proteins and peptides to the brain. We highlight the developments and applications of nanocarriers in treating CNS diseases via BBB crossing. We also provide critical opinions on the limitations and obstacles of the current strategies and put forward prospects for future research.

Urine, a common source of biological markers in biomedical research and clinical diagnosis, has recently generated a new wave of interest. It has recently become a focus of study due to the presence of its content of extracellular vesicles (EVs). These uEVs have been found to reflect physiological and pathological conditions in kidney, urothelial, and prostate tissue and can illustrate further molecular processes, leading to a rapid expansion of research in this field In this work, we present the advantages of an immunoaffinity-based method for uEVs\' isolation with respect to the gold standard purification approach performed by differential ultracentrifugation [in terms of purity and antigen presence. The immunoaffinity method was made feasible by combining specific antibodies with a functionalized polymethacrylate polymer. Flow cytometry indicated a significant fluorescence shift, validating the presence of the markers (CD9, CD63, CD81) and confirming the effectiveness of the isolation method. Microscopy evaluations have shown that the morphology of the vesicles remained intact and corresponded to the expected shapes and dimensions of uEVs. The described protocol is inexpensive, fast, easy to process, has good reproducibility, and can be applied to further biological samples.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation with no cure and limited treatment options that often have systemic side effects. In this study, we developed a target-specific system to potentially treat IBD by engineering the probiotic bacterium Escherichia coli Nissle 1917 (EcN). Our modular system comprises three components: a transcription factor-based sensor (NorR) capable of detecting the inflammation biomarker nitric oxide (NO), a type 1 hemolysin secretion system, and a therapeutic cargo consisting of a library of humanized anti-TNFα nanobodies. Despite a reduction in sensitivity, our system demonstrated a concentration-dependent response to NO, successfully secreting functional nanobodies with binding affinities comparable to the commonly used drug Adalimumab, as confirmed by enzyme-linked immunosorbent assay and in vitro assays. This newly validated nanobody library expands EcN therapeutic capabilities. The adopted secretion system, also characterized for the first time in EcN, can be further adapted as a platform for screening and purifying proteins of interest. Additionally, we provided a mathematical framework to assess critical parameters in engineering probiotic systems, including the production and diffusion of relevant molecules, bacterial colonization rates, and particle interactions. This integrated approach expands the synthetic biology toolbox for EcN-based therapies, providing novel parts, circuits, and a model for tunable responses at inflammatory hotspots.

Antibody pairs-based immunoassay platforms served as essential and effective tools in the field of pathogen detection. However, the cumbersome preparation and limited detection sensitivity of antibody pairs challenge in establishment of a highly sensitive detection platform. In this study, using COVID-19 testing as a case, we utilized readily accessible nanobodies as detection antibodies and further proposed an accurate design concept with a more scientific and efficient screening strategy to obtain ultrasensitive antibody pairs. We employed nanobodies capable of binding different antigenic epitopes of the nucleocapsid (NP) or receptor-binding domain (RBD) antigens sandwich as substitutes for monoclonal antibodies (mAbs) sandwich in fast detection formats and utilized time-resolved fluorescence (TRF) microspheres as the signal probe. Consequently, we developed a multi-epitope nanobody sandwich-based fluorescence lateral flow immunoassay (FLFA) strip. Our results suggest that the NP antigen had a detection limit of 12.01pg/mL, while the RBD antigen had a limit of 6.51 pg/mL using our FLFA strip. Based on double mAb sandwiches, the values presented herein demonstrated 4 to 32-fold enhancements in sensitivity, and 32 to 256-fold enhancements compared to commercially available antigen lateral flow assay kits. Furthermore, we demonstrated the excellent characteristics of the proposed test strip, including its specificity, stability, accuracy, and repeatability, which underscores its the prospective utility. Indeed, these findings indicate that our established screening strategy along with the multi-epitope nanobody sandwich mode provides an optimized strategy in the field of pathogen detection.