Domain Antibody Research Articles

Development and evaluation of a single domain antibody targeting folate receptor alpha for radioligand therapy

BackgroundFolate receptor alpha (FRα) overexpression is seen in many cancers. Radioligand therapy (RLT) has emerged as a promising tool to target FRα and has been investigated previously, but further progression was limited due to high kidney retention and, subsequently, toxicity. To circumvent this, we present here the development of a [131I]I-GMIB-conjugated anti-human FRα (hFRα) single-domain antibody (sdAb), with intrinsically fast renal clearance and concomitant low kidney retention. We report the hit-to-lead development of an anti-hFRα sdAb. We evaluated its potential in vitro and assessed its targeting ability using SPECT imaging in hFRα-knockin and tumour-bearing mice. The toxicity and therapeutic efficacy of the [131I]I-GMIB-sdAb were investigated in mouse models.ResultsThe lead anti-hFRα sdAb 2BD42 was developed with picomolar affinities, low koff, and radiolabelled using [131I]I with yields of > 41% and purity > 99%. [131I]I-GMIB-2BD42 retained tumour uptake (> 5%IA/g at 1 h p.i. and > 1.5%IA/g at 24 h p.i.) and fast kidney clearance (< 1%IA/g at 24 h p.i.) in athymic and hFRα-knock-in mice. Athymic mice bearing hFRα-positive xenografts treated with [131I]I-GMIB-2BD42 showed prolonged survival without toxicity compared to animals that received the vehicle solution or radioactive control.ConclusionThe therapeutic lead radiopharmaceutical [131I]I-GMIB-2BD42 showed fast pharmacokinetics with specific retention in hFRα + tumours. In addition, we report therapeutic efficacy with no signs of toxicity. In this study, we successfully designed a new drug for RLT, overcoming previous limitations, such as high kidney retention, which could aid in revitalising FRα-targeted radiotherapy.Graphical

Enzymatic, structural, and biophysical characterization of a single-domain antibody (VHH) selectively and tightly inhibiting neutrophil elastase and exhibiting favorable developability properties.

Human neutrophil elastase (hNE), a serine protease released by neutrophils during inflammation, plays a major role in the pathophysiology of several conditions especially in inflammatory lung diseases. Its inhibition constitutes, therefore, a promising therapeutic strategy to combat these diseases. In this work, we characterized the in vitro properties of a VHH (i.e., the antigen binding domain of camelid heavy chain-only antibodies), referred to as NbE201. This VHH is able to inhibit tightly, selectively and competitively both human and murine elastases with the inhibition constants (Ki) of 4.1 ± 0.9 nM and 36.8 ± 3.9 nM, respectively. The IC50 for the inhibition of the hydrolysis of elastin is in the same range to that of alpha-1 antitrypsin (i.e., the main endogenous inhibitor of hNE also used in the clinic) and 14 times better than that of Sivelestat (i.e., the 2nd clinically approved hNE inhibitor). The X-ray crystal structure of the NbE201-hNE complex reveals that the Complementarity Determining Regions CDR1 and CDR3 of the VHH bind into the substrate binding pocket of hNE and prevent the access to small or macromolecular substrates. They do not, however, bind deep enough into the pocket to be hydrolyzed. NbE201 is highly stable towards oxidation, deamidation, and chemical or thermal denaturation. NbE201 is therefore likely to tolerate manufacturing processes during drug development. These results highlight the high potential of NbE201 as a (pre)clinical tool to diagnose and treat diseases associated with excessive hNE activity, and for fundamental research to better understand the role of hNE in these conditions.

Using Machine Learning for Personalized Prediction of Longitudinal COVID-19 Vaccine Responses in Transplant Recipients

The COVID-19 pandemic has underscored the importance of vaccines, especially for immunocompromised populations like solid organ transplant (SOT) recipients, who often have weaker immune responses. The purpose of this study was to compare deep learning architectures for predicting SARS-CoV-2 vaccine responses 12 months post-vaccination in this high-risk group. Utilizing data from 303 SOT recipients from a Canadian multicenter cohort, models were developed to forecast anti-receptor binding domain (RBD) antibody levels. The study compared traditional machine learning models—logistic regression, epsilon-support vector regression, random forest regressor, and gradient boosting regressor—and deep learning architectures, including long short-term memory (LSTM), recurrent neural networks, and a novel model, routed LSTM. This new model combines capsule networks with LSTM to reduce the need for large datasets. Demographic, clinical, and transplant-specific data, along with longitudinal antibody measurements, were incorporated into the models. The routed LSTM performed best, achieving a mean square error (MSE) of 0.02±0.02 and a Pearson correlation coefficient (PCC) of 0.79±0.24, outperforming all other models. Key factors influencing vaccine response included age, immunosuppression, breakthrough infection, BMI, sex, and transplant type. These findings suggest that AI could be a valuable tool in tailoring vaccine strategies, improving health outcomes for vulnerable transplant recipients.

Selection of LRP1 ligand phage-displayed single domain antibody that transmigrates BBB

Effective drug delivery to the central nervous system (CNS) remains a challenge due to the blood–brain barrier (BBB). Macromolecules such as proteins and peptides are unable to cross BBB and have poor therapeutic efficacy due to little or no drug distribution. A promising alternative is the conjugation of a drug to a shuttle molecule that can reach the CNS via receptor-mediated transcytosis (RMT). Several receptors have been described for RMT, such as low-density lipoprotein receptor-related protein 1 (LRP1). We used phage display technology combined with an in vitro BBB model to identify LRP1 ligands. A single domain antibody (dAb) library was used to enrich for species that selectively bind to immobilised LRP1 ligand. We obtained a novel nanobody, dAb D11, that selectively binds to LRP1 receptor and mediates in vitro internalisation of phage particles in brain endothelial cells, with a dissociation constant Kd of 183.1 ± 85.8 nM. The high permeability of D11 was demonstrated by an in vivo biodistribution assay in mice. We discovered D11, the first LRP1 binding dAb with BBB permeability. Our findings will contribute to the development of RMT-based drugs for the treatment of CNS diseases.

High expression of variable domain of heavy-chain antibodies in Expi293F cells with optimized signal peptide and codons

The variable domain of heavy-chain antibody (VHH) has been developed widely in drug therapy, diagnosis, and research. Escherichia coli is the most popular expression system for VHH production, whereas low bioactivity occurs sometimes. Mammalian cells are one of the most ideal hosts for VHH expression at present. To improve the yield of VHH in Expi293F cells, we optimized the signal peptide (SP) and codons of VHH. Firstly, the fusion protein VHH1-Fc was used to screen SPs. The SP IFN-α2 showed the highest secretion as quantified by enzyme-linked immunosorbent assay (ELISA). Subsequently, codon optimization by improving GC3 and GC content doubled the yield of VHH1 and kept its binding activity to Senecavirus A (SVA). Finally, the mean yields of other 5 VHHs that fused with SP IFN-α2 and codon-optimized were over 191.6 mg/L, and these VHHs had high recovery and high purity in the culture supernatant. This study confirms that SP IFN-α2 and codon optimization could produce VHHs in Expi293F cells efficiently, which provides a reference for the large-scale production of VHHs.

Automated radiofluorination of HER2 single domain antibody: the road towards the clinical translation of [18F]FB-HER2 sdAb

BackgroundWith the next generation of Human Epidermal Growth Factor Receptor 2 (HER2) -targeting therapies, such as antibody–drug conjugates, showing benefit in “HER2 low” and even “HER2 ultralow” patients, the need for novel methods to quantify HER2 expression accurately becomes even more important for clinical decision making. A HER2 PET/CT imaging assessment could evaluate HER2 positive disease locations while improving patient care, reducing the need for invasive biopsies. A single-domain antibody (sdAb)-based PET tracer could combine the high specificity of sdAbs with short-lived radionuclides such as fluorine-18 (18F) and gallium-68 (68Ga). SdAb-based PET tracers have clinically been used via a 68Ga-chelator approach. However, the distribution of 68Ga-labelled pharmaceuticals to peripheral PET centres is more challenging to organize due to the short half-life of 68Ga, most certainly when the available activity is limited by a generator. Cyclotron produced 68Ga has removed this limitation. Distribution of 18F-labelled pharmaceuticals remains less challenging due to its slightly longer half-life, and radiofluorination of sdAbs via N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) has shown to be a promising strategy for developing sdAb-based PET tracers. Although [18F]SFB automation has been reported, automating protein conjugation proves challenging. Herein we report the fully automated, cartridge-based production of [18F]FB-HER2 sdAb on a single synthesis module.Results[18F]FB-HER2 sdAb (> 6 GBq) was obtained after a fully automated production (95 min), with a RCP > 95%, apparent molar activity > 20 GBq/µmol and decay-corrected radiochemical yield (RCY d.c.) of 14 ± 2% (n = 4). Further upscaling amounted to production batches of 16 GBq with an apparent molar activity > 40 GBq/µmol and RCY d.c. of 8 ± 1% (n = 4). Ex vivo biodistribution and PET imaging showed specific HER2-positive tumour targeting and low kidney retention.ConclusionThe [18F]FB-HER2 sdAb tracer was produced with clinically relevant activities using a fully automated production method. The automated production method was designed to ease the translation to the clinic and has the potential to be used not only in mono-centre but also multi-centre clinical trials with one central production site. [18F]FB-HER2 sdAb showed a favourable biodistribution pattern and could be a valuable alternative to 68Ga-labelled sdAb-based PET tracers in the clinic.

Disease-associated B cells and immune endotypes shape adaptive immune responses to SARS-CoV-2 mRNA vaccination in human SLE.

Severe acute respiratory syndrome coronavirus 2 mRNA vaccination has reduced effectiveness in certain immunocompromised individuals. However, the cellular mechanisms underlying these defects, as well as the contribution of disease-induced cellular abnormalities, remain largely unexplored. In this study, we conducted a comprehensive serological and cellular analysis of patients with autoimmune systemic lupus erythematosus (SLE) who received the Wuhan-Hu-1 monovalent mRNA coronavirus disease 2019 vaccine. Our findings revealed that patients with SLE exhibited reduced avidity of anti-receptor-binding domain antibodies, leading to decreased neutralization potency and breadth. We also observed a sustained anti-spike response in IgD-CD27- 'double-negative (DN)' DN2/DN3 B cell populations persisting during memory responses and with greater representation in the SLE cohort. Additionally, patients with SLE displayed compromised anti-spike T cell immunity. Notably, low vaccine efficacy strongly correlated with higher values of a newly developed extrafollicular B and T cell score, supporting the importance of distinct B cell endotypes. Finally, we found that anti-BAFF blockade through belimumab treatment was associated with poor vaccine immunogenicity due to inhibition of naive B cell priming and an unexpected impact on circulating T follicular helper cells.

STEM-17. THE UROKINASE RECEPTOR AS A NOVEL IMMUNOTHERAPEUTIC TARGET IN BRAIN CANCERS

Abstract Glioblastoma (GBM) is the common malignant brain tumor in adults, accounting for approximately 15% of all CNS tumors, and 48.6% of malignant brain tumors, with a median survival of approximately 15 months. GBM is characterized by extensive inter- and intra-tumoral heterogeneity and an extremely immunosuppressive tumor microenvironment. Following Standard-of-Care surgical resection and chemoradiotherapy, patients inevitably relapse, at which point few therapeutic avenues exist, owing in part due to a lack of clinically relevant targets. Data from our target identification pipeline shows the urokinase plasminogen activator receptor (uPAR) is significantly upregulated at recurrence on putative GBM brain tumor initiating cells (BTICs), which are believed to drive de novo tumor formation, recurrence, and therapeutic resistance. uPAR plays an important role in the plasminogen activation system, and in the context of cancer, has been implicated in numerous pro-tumorigenic processes such as invasion, proliferation, and therapy resistance. We used CRISPR-Cas9 to genetically delete uPAR from our in-house patient-derived GBM cell lines, and found that knockout of uPAR in recurrent GBM cells significantly reduces various functional characteristics of BTICs in vitro. In our patient-derived mouse model of GBM, we found that knockout of uPAR significantly increases survival, and decreases tumor burden. Further, we generated novel anti-uPAR single domain antibodies (sdAbs) which specifically and efficiently bind uPAR at low nanomolar concentrations in vitro. We developed anti-uPAR CAR Ts using the binder sequence of the sdAbs, which showed potent cytotoxicity in vitro, and drastically reduced tumor burden and extended survival in vivo. Further, we identified uPAR as being highly expressed on brain metastases from multiple origins (lung-to-brain, melanoma-to-brain, etc.), and demonstrate that anti-uPAR CAR Ts effectively kill brain metastases in vitro, and significantly extend survival using in vivo brain metastasis models. From this work we believe uPAR to be a clinically relevant target in both recurrent GBM and brain metastases, and investigation into therapeutic strategies targeting uPAR-positive brain cancers should be explored further.

EXTH-24. THERAPEUTIC TARGETING OF MISLOCALIZED NUCLEAR ENVELOPE PROTEINS INMYC-DRIVEN GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common, malignant pediatric brain tumour comprised of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). A subset of Group 3 MB tumors harbors focal amplifications of the MYC oncogene (MYC-G3MB) and are particularly prone to tumour recurrence due to their highly proliferative nature linked to its embryonic stem cell-of-origin and programming. We discovered that LBR, an inner nuclear transmembrane protein, while present in all cells at the nuclear envelope (NE), is aberrantly presented to the cell surface in MYC-G3MBs. Transcriptomic profiling of publicly available datasets identified significant NE protein enrichment in MYC-G3MB compared to normal tissue and high expression during fetal development that dampens postnatally and in adult samples. Immunohistochemistry analysis of LBR on patient tumors was able to significantly prognosticate overall and progression free survival. Flow cytometry analysis identified LBR cell surface expression in MYC-G3MBs with low to no expression in human neural stem cells and other MB subgroups. High resolution microscopy of endogenously tagged LBR revealed cell surface presentation to be linked to ER-like vesicles that directly trafficked to the cell surface in MYC-G3MBs. Transcriptomic analysis of LBR cell surface positive and negative cells identified LBR to be linked to mitotic processes and mislocalization was significantly enriched following exposure to chemotherapy and radiation in vitro and in vivo. Therefore, we demonstrate that LBR is an ideal therapeutic target for patients facing treatment-refractory MB and have generated single domain antibodies (sdAb) to develop LBR-CAR T cells. Validation of LBR sdAbs demonstrate on-target binding and comparable affinity and avidity to commercial LBR antibodies. There is an undeniable paucity of targets for MB patients stemming from the lack of neoantigens and genomic aberrations in pediatric tumors. The preclinical development and validation of novel immunotherapeutic targets such as LBR provides alternative therapies for patients otherwise facing palliation.

Effect of framework and complementarity determining region H1 charge on the human VH-B1a domain expression, folding, stability, and solubility

Many difficulties related to using antibodies in diagnostic and therapeutic applications can sometimes be circumvented by using smaller, less complex single domain antibodies based on variable heavy chain (VH) domain constructs such as camelid VHH domains. However, VH domains have their own limitations, including an increased tendency to aggregate. VH domains often contain hydrophobic residues within their complementarity-determining regions (CDRs) that facilitate binding to target antigens but can also mediate VH domain aggregation, which is a concern for therapeutic applications since this can trigger immune responses. In this study, we engineered the human VH-1Ba domain to improve its stability and solubility by introducing charged amino acids in the VH domain framework region and CDRH1. We followed two strategies to improve the stability and solubility of VH domains. First, we introduced positive and negatively charged amino acids in the framework region of an autonomous human VH domain (VH–B1a) and observed the effect of framework net charge on VH domain refolding, stability, and solubility. Introducing positive charge into the VH-B1a framework increased its thermostability but slightly lowered its refolding ability and solubility. We were not able to obtain correctly folded negatively charged (-VH) VH domains. Second, we introduced a series of positive and negatively charged amino acids in the CDRH1 loop of near-neutral (VH–B1a) and positively charged (+VH) VH domains, and observed their effect on expression, refolding, stability, and solubility. For both the VH-B1a and +VH domains, we observed a decrease in melting temperature (Tm) and room temperature solubility as more negative or positive charged amino acids were added to the CDRH1. The VH-B1a domain had higher room temperature solubility for negative and slightly positive CDRH1 net charges. The + VH domain had higher Tms for all CDRH1 net charges and was better able to tolerate the adverse effects of adding positive charge to CDRH1. We observed a similar response in refolding and solubility of VH-B1a and +VH in response to changes in CDRH1 net charge after temperature-induced denaturation for negative and neutral CDRH1s. We observed a positional effect with a single Lys (31K) and double Lys (31, 32 KK) substitutions in CDRH1, which promoted VH-B1a aggregation and was partially overcome by the +VH domain. In summary, this study provides information for designing VH domains with improved biophysical properties and a +VH domain that will be useful for applications where positive surface charge and CDRH1 are desirable.

Neutralizing VHH Antibodies Targeting the Spike Protein of PEDV.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that infect pigs' intestinal epithelial cells, causing high morbidity and mortality. Due to the rapid mutation of PEDV, vaccine efficacy is uncertain, prompting exploration of alternative treatments. Nanobodies, also known as variable heavy chain domains of heavy chain-only antibodies (VHHs), offer significant potential in biomedical applications due to their small size and high specificity. In this study, yeast two-hybrid technology was employed to screen for eight specific VHH sequences targeting the PEDV S protein from a synthetically constructed nanobody yeast library. The VHH genes were then cloned into expression plasmids for recombinant protein production, and the resulting VHHs (termed PEDV S-VHHs) were purified. Indirect immunofluorescence assay (IFA) and Western blotting analysis confirmed that these VHHs specifically bind to both PEDV and its S protein. Neutralization assays demonstrated that seven PEDV S-VHHs exhibited potent neutralizing activity against PEDV. Additionally, a combination of these seven antibodies showed enhanced antiviral effects. Preliminary predictions were also made regarding the binding sites between these VHHs and PEDV. The PEDV S-VHHs described in this study hold potential as candidates for the prevention and treatment of PEDV infection.

Quantitative SARS-CoV-2 Spike Receptor-Binding Domain and Neutralizing Antibody Titers in Previously Infected Persons, United States, January 2021-February 2022.

We studied SARS-CoV-2 binding and neutralizing antibody titers among previously infected persons in the United States over time. We assayed SARS-CoV-2 spike protein receptor-binding domain and neutralizing antibody titers for a convenience sample of residual clinical serum specimens that had evidence of prior SARS-CoV-2 infection gathered during January 2021-February 2022. We correlated titers and examined them by age group (<18, 18-49, 50-64, and >65 years) across 4 different SARS-CoV-2 variant epochs. Among selected specimens, 30,967 had binding antibody titers and 744 had neutralizing titers available. Titers in specimens from children and adults correlated. In addition, mean binding antibody titers increased over time for all age groups, and mean neutralization titers increased over time for persons 16-49 and >65 years of age. Incorporating binding and neutralization antibody titers into infectious disease surveillance could provide a clearer picture of overall immunity and help target vaccination campaigns.

Safety, efficacy and immunogenicity of aerosolized Ad5-nCoV COVID-19 vaccine in a non-inferiority randomized controlled trial

This phase 3, observer-blinded, non-inferiority randomized trial (ClinicalTrials.gov: NCT05517642), conducted from September 2022 to May 2023 at three Malaysian sites, involved 540 adults previously vaccinated with three COVID-19 doses. Participants were randomized 1:1 to receive either one dose of inhaled Recombinant COVID-19 Vaccine (Ad5-nCoV-IH) or intramuscular tozinameran (BNT-IM). The study assessed safety, vaccine efficacy (VE) and immunogenicity against SARS-CoV-2 variants. The primary outcome was the non-inferiority of anti-spike protein receptor-binding domain (S-RBD IgG) antibodies, with a 97.5% confidence interval lower limit for the geometric mean concentration (GMC) ratio >0.67. Ad5-nCoV-IH showed lower immunogenicity than BNT-IM, with a GMC ratio of 0.22 and a seroconversion rate difference of -71.91%. Adverse drug reactions (ADRs) were less frequent with Ad5-nCoV-IH (39.26%) compared to BNT-IM (64.68%). No serious vaccine-related adverse events were reported. Both vaccines had comparable efficacy against COVID-19 variants. This study was funded by Tianjin Biomedical Science and Technology Major Project.

Humoral and cellular response to the third COVID-19 vaccination in patients with inborn errors of immunity or mannose-binding lectin deficiency : Aprospective controlled open-label trial.

Impaired immune response to COVID-19 (coronavirus disease 2019) vaccination has been reported in patients with inborn errors of immunity (IEI). Repetitive vaccinations are recommended for this vulnerable group. Due to the high diversity within IEI patients, additional safety and immunogenicity data are needed to better understand these aspects especially in less common immunodeficiency syndromes. In this prospective open-label clinical trial, we assessed the humoral immune response and the T‑cell response in patients with IEI or severe MBL (mannose-binding lectin) deficiency (IEI/MBLdef) after three vaccinations. A total of 16 patients and 16 matched healthy controls (HC) with suboptimal humoral response defined by anti-SARS-CoV‑2 RBD (severe acute respiratory syndrome coronavirus type 2 receptor binding domain) antibodies below 1500 BAU/ml (binding antibody units per ml) after the second COVID-19 vaccination were enrolled in this study and qualified for athird mRNA vaccine dose. After 4 weeks following vaccination, 100% of HC and 75% of IEI/MBLdef patients exhibited anti-SARS-CoV‑2 RBD antibodies > 1500 BAU/ml, although the difference was not statistically significant (75% vs. 100%; p = 0.109). Although post-vaccination IEI/MBLdef patients demonstrated significantly increased anti-SARS-CoV‑2 RBD antibodies and neutralizing antibodies compared to baseline, these responses were significantly lower in IEI/MBLdef patients compared to HCs. Notably, the third vaccination augmented the cellular immune response to both wild-type and omicron peptide stimulation. No serious adverse events were reported within the 4‑week follow-up period and, importantly, vaccination had little to no effect on the long-term disease activity and fatigue. This trial strongly supports the recommendation of repeated COVID-19vaccinations for patients suffering from immunodeficiencies, especially when they exhibit an initially limited response to the vaccine.

Cryo-electron microscopy reveals a single domain antibody with a unique binding epitope on fibroblast activation protein alpha.

Fibroblast activation protein alpha (FAP) is a serine protease that is expressed at basal levels in benign tissues but is overexpressed in a variety of pathologies, including cancer. Despite this unique expression profile, designing effective diagnostic and therapeutic agents that effectively target this biomarker remain elusive. Here we report the structural characterization of the interaction between a novel single domain antibody (sdAbs), I3, and FAP using cryo-electron microscopy. The reconstructions were determined to a resolution of 2.7 Å and contained two distinct populations; one I3 bound and two I3 molecules bound to the FAP dimer. In both cases, the sdAbs bound a unique epitope that was distinct from the active site of the enzyme. Furthermore, this report describes the rational mutation of specific residues within the complementarity determining region 3 (CDR3) loop to enhance affinity and selectivity of the I3 molecule for FAP. This report represents the first sdAb-FAP structure to be described in the literature.

Vδ2 T-cell engagers bivalent for Vδ2-TCR binding provide anti-tumor immunity and support robust Vγ9Vδ2 T-cell expansion.

Vγ9Vδ2 T-cells are antitumor immune effector cells that can detect metabolic dysregulation in cancer cells through phosphoantigen-induced conformational changes in the butyrophilin (BTN) 2A1/3A1 complex. In order to clinically exploit the anticancer properties of Vγ9Vδ2 T-cells, various approaches have been studied including phosphoantigen stimulation, agonistic BTN3A-specific antibodies, adoptive transfer of expanded Vγ9Vδ2 T-cells, and more recently bispecific antibodies. While Vγ9Vδ2 T-cells constitute a sizeable population, typically making up ~1-10% of the total T cell population, lower numbers have been observed with increasing age and in the context of disease. We evaluated whether bivalent single domain antibodies (VHHs) that link Vδ2-TCR specific VHHs with different affinities could support Vγ9Vδ2 T-cell expansion and could be incorporated in a bispecific engager format when additionally linked to a tumor antigen specific VHH. Bivalent VHHs that link a high and low affinity Vδ2-TCR specific VHH can support Vγ9Vδ2 T-cell expansion. The majority of Vγ9Vδ2 T-cells that expanded following exposure to these bivalent VHHs had an effector or central memory phenotype and expressed relatively low levels of PD-1. Bispecific engagers that incorporated the bivalent Vδ2-TCR specific VHH as well as a tumor antigen specific VHH triggered antitumor effector functions and supported expansion of Vγ9Vδ2 T-cells in vitro and in an in vivo model in NOG-hIL-15 mice. By enhancing the number of Vγ9Vδ2 T-cells available to exert antitumor effector functions, these novel Vδ2-bivalent bispecific T cell engagers may promote the overall efficacy of bispecific Vγ9Vδ2 T-cell engagement, particularly in patients with relatively low levels of Vγ9Vδ2 T-cells.

Targeting esophageal carcinoma: molecular mechanisms and clinical studies.

Esophageal cancer (EC)is identified as a predominant health threat worldwide, with its highest incidence and mortality rates reported in China. The complex molecular mechanisms underlying EC, coupled with the differential incidence of esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) across various regions, highlight the necessity for in-depth research targeting molecular pathogenesis and innovative treatment strategies. Despite recent progress in targeted therapy and immunotherapy, challenges such as drug resistance and the lack of effective biomarkers for patient selection persist, impeding the optimization of therapeutic outcomes. Our review delves into the molecular pathology of EC, emphasizing genetic and epigenetic alterations, aberrant signaling pathways, tumor microenvironment factors, and the mechanisms of metastasis and immune evasion. We further scrutinize the current landscape of targeted therapies, including the roles of EGFR, HER2, and VEGFR, alongside the transformative impact of ICIs. The discussion extends to evaluating combination therapies, spotlighting the synergy between targeted and immune-mediated treatments, and introduces the burgeoning domain of antibody-drug conjugates, bispecific antibodies, and multitarget-directed ligands. This review lies in its holistic synthesis of EC's molecular underpinnings and therapeutic interventions, fused with an outlook on future directions including overcoming resistance mechanisms, biomarker discovery, and the potential of novel drug formulations.

Making the effect visible - OX40 targeting nanobodies for in vivo imaging of activated T cells.

Human OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging. Nbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro, including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model. Our selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. Considering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40+ T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.

A Simple Analysis of the Second (Extra) Disulfide Bridge of VHHs.

Camelids produce a special type of antibody, known as VHHs, that has lost the VL domain, providing a more optimised VH domain. This particular highly stable antibody domain has interesting properties for biotechnological development. Ordinarily, those molecules possess only one disulphide bridge, but surprisingly, at least a quarter of these VHHs have a second one. Curiously, this does not seem to be essential for the stability and the function of this domain. In an attempt to characterise precisely the role and impact of this disulphide bridge at the molecular level, several in silico mutants of a VHH were created to disrupt this second disulphide bridge and those systems were submitted to molecular dynamics simulation. The loss of the second disulphide bridge leads to an increase in the flexibility of CDR1 and CDR3 and an unexpected rigidification of CDR2. Local conformational analysis shows local differences in the observed protein conformations. However, in fact, there is no exploration of new conformations but a change in the equilibrium between the different observed conformations. This explains why the interaction of VHHs is not really affected by the presence or absence of this second bridge, but their rigidity is slightly reduced. Therefore, the additional disulphide bridge does not seem to be an essential part of VHHs function.

The development of single-domain VHH nanobodies that target the Candida albicans cell surface.

Candida albicans causes life-threatening invasive infections that are hard to diagnose and treat, with drug resistance leading to treatment failure. The goal of this study was to develop VHH (single variable domain on a heavy chain) nanobodies to detect drug-resistant infections. Llamas were immunized with a mixture of heat killed and fixed C. albicans cells of different morphologies. Llama lymphocyte RNA was used to generate phage display libraries that were tested for binding to C. albicans cells or cell wall fractions, and single antibody domains were isolated. The libraries were panned against echinocandin-resistant C. albicans isolates and counter-selected against echinocandin-susceptible isolates with the aim of isolating binding domains specific for antigens on drug-resistant cells. Thirty diverse VHH nanobodies were selected, and binding characteristics were assessed via dose-response ELISA. Binding was tested against a variety of C. albicans isolates and other Candida species, indicating that the VHHs were specific for C. albicans. The VHH nanobodies were sorted into four distinct groups based on their binding patterns. Two of the groups bound preferentially to the yeast cell poles and hyphae, respectively. Nanobody binding to C. albicans deletion mutants was tested by fluorescence microscopy and ELISA to identify the antigen targets. VHH19 nanobody, belonging to the largest group, recognized the Als4 adhesin. VHH14 antibody in the hyphae-specific group recognized Als3. None of the isolated VHH nanobodies was selective for drug-resistant clinical isolates. Our data indicate that this approach can generate valuable single-domain antibodies specific to C. albicans proteins.IMPORTANCEThe human fungal pathogen Candida albicans causes a range of diseases from superficial mucosal infections such as oral and vaginal thrush to life-threatening, systemic infections. Accurate and rapid diagnosis of these infections remains challenging, and currently, there are no rapid ways to diagnose drug-resistant infections without performing drug susceptibility testing from blood culture, which can take several days. In this proof-of-concept study, we have generated a diverse set of single domain VHH antibodies (nanobodies) from llamas that recognize and bind specifically to C. albicans cell surface. The nanobodies were classified into four groups based on their binding patterns, for example, cell poles or hyphae. Specific nanobodies were verified as recognizing the important adhesin Als4 or the hyphae associated invasin Als3, respectively. The data validate the approach that small VHH antibody domains hold future promise for diagnostic applications and as probes to study the fungal cell surface.