Phage Display Research Articles

Characterization and humanization of VNARs targeting human serum albumin from the whitespotted bamboo shark (Chiloscyllium plagiosum)

The Shark-derived immunoglobulin new antigen receptors (IgNARs) have gained increasing attention for their high solubility, exceptional thermal stability, and intricate sequence variation. In this study, we immunized whitespotted bamboo shark (Chiloscyllium plagiosum) to create phage display library of variable domains of IgNAR (VNARs) for screening against Human Serum Albumin (HSA), a versatile vehicle in circulation due to its long in vivo half-life. We identified two HSA-binding VNAR clones, 2G5 and 2G6, and enhanced their expression in E. coli with the FKPA chaperone. 2G6 exhibited a strong binding affinity of 13 nM with HSA and an EC50 of 1 nM. In vivo study with a murine model further provided initial validation of 2G6's ability to prolong circulation time by binding to HSA. Additionally, we employed computational molecular docking to predict the binding affinities of both 2G6 and its humanized derivative, H2G6, to HSA. Our analysis unveiled that the complementarity-determining regions (CDR1 and CDR3) are pivotal in the antigen recognition process. Therefore, our study has advanced the understanding of the potential applications of VNARs in biomedical research aimed at extending drug half-life, holding promise for future therapeutic and diagnostic progressions.

Discovery of a new class of cell-penetrating peptides by novel phage display platform

The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol.

Selection and evaluation of single domain antibody against p19 subunit of IL-23 by phage display for potential use as an autoinflammatory therapeutic

IL-23 is a double-subunit cytokine that plays an important role in shaping the immune response. IL-23 was found to be associated with several autoinflammatory diseases by generating sustained inflammatory loops that lead to tissue damage. Antibody neutralization of IL-23 was proven to be effective in ameliorating associated diseases. However, antibodies as large proteins have limited tissue penetration and tend to elicit anti-drug antibodies. Additionally, anti-IL-23 antibodies target only one subunit of IL-23 leaving the other one unneutralized. Here, we attempted to isolate a recycling single domain antibody by phage display. One of IL-23 subunits, p19, was expressed in E. coli fused to Gamillus protein to stabilize the α-helix-only p19. To remove Gamillus binders, two biopanning methods were investigated, first, preselection with Gamillus and second, challenge with IL-23 then on the subsequent round challenge with p19-Gam. The isolation of calcium-dependent and pH-dependent recycling binders was performed with EDTA and citrate buffers respectively. Both methods of panning failed to isolate high-affinity and specific p19 recycling binders, while from the second panning method, a high affinity and specific p19 standard binder, namely H11, was successfully isolated. H11 significantly inhibited the gene expression of IL-17 and IL-22 in IL-23-challenged PBMCs indicating H11 specificity and neutralizing ability for IL-23. The new binder due to its small size can overcome antibodies limitations, also, it can be further engineered in the future for antigen clearance such as fusing it to cell penetrating peptides, granting H11 the ability to clear excess IL-23 and enhancing its potential therapeutic effect.

Rapid Evolutionary Characteristics of P1 and T5 Infection in E. coli Strains

The emergence of superbug has once again thrust century-old phage into the spotlight as a hot research topic. While the "phage display" technology has enabled targeted evolution of viruses, the successful application of phage remains uncertain due to their replicative and mutative nature, as well as the complexity of phage-bacteria interactions and evolutionary mechanisms. In this study, we used two rapidly evolving phages, P1 and T5, along with 18 types of E. coli strains, as representatives to explore the evolutionary characteristics of phage-infected bacteria. Our findings confirm that the evolutionary of phages and their hosts is a co-evolutionary process, resembling an "arms race", after the early rapid evolution of P1 and T5, the host range expands to infect different E. coli strains, but continued rapid evolution reaches a certain stage where P1, T5, and E. coli strains all experience a fitness cost, and the host range no longer expands. Three new findings in this study differ from previous studies. Firstly, although E. coli strains were not infected in the same way, there was no statistically significant difference between P1 and T5 infection in different E. coli strains at different evolutionary stages. Secondly, 11 types of E. coli strains that had been infected by T5 during co-evolution all showed E. coli tolerant plaques on the medium at the fifth stage of evolution, whether they had been infected by T5 at that stage of evolution, and at the sixth stage of evolution, all of these tolerant plaques disappeared again. Thirdly, after the fitness cost of the co-evolutionary process had already occurred, although the host range of P1 and T5 no longer continued to expand, the types of E. coli strains infected by P1 and T5 at the same evolutionary stage did not decrease in focus with the continued prolongation of the co-evolutionary time, but instead showed a gradual incremental trend. This study provides experimental evidence for the resolution of phage-bacteria co-evolutionary mechanism and new ideas for phagotherapy strategies for the superbug.

Discovery of a potent, selective, and tumor-suppressing antibody antagonist of adenosine A2A receptor.

New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A2A receptor (A2AR) has been proposed as a target for immunotherapeutic development due to its participation in immunosuppression of the tumor microenvironment. Blockade of A2AR could restore tumor immunity and, consequently, improve patient outcomes. Here, we describe the discovery of a potent, selective, and tumor-suppressing antibody antagonist of human A2AR (hA2AR) by phage display. We constructed and screened four single-chain variable fragment (scFv) libraries-two synthetic and two immunized-against hA2AR and antagonist-stabilized hA2AR. After biopanning and ELISA screening, scFv hits were reformatted to human IgG and triaged in a series of cellular binding and functional assays to identify a lead candidate. Lead candidate TB206-001 displayed nanomolar binding of hA2AR-overexpressing HEK293 cells; cross-reactivity with mouse and cynomolgus A2AR but not human A1, A2B, or A3 receptors; functional antagonism of hA2AR in hA2AR-overexpressing HEK293 cells and peripheral blood mononuclear cells (PBMCs); and tumor-suppressing activity in colon tumor-bearing HuCD34-NCG mice. Given its therapeutic properties, TB206-001 is a good candidate for incorporation into next-generation bispecific immunotherapeutics.

Phage Display as a Medium for Target Therapy Based Drug Discovery, Review and Update.

Phage libraries are now amongst the most prominent approaches for the identification of high-affinity antibodies/peptides from billions of displayed phages in a specific library through the biopanning process. Due to its ability to discover potential therapeutic candidates that bind specifically to targets, phage display has gained considerable attention in targeted therapy. Using this approach, peptides with high-affinity and specificity can be identified for potential therapeutic or diagnostic use. Furthermore, phage libraries can be used to rapidly screen and identify novel antibodies to develop immunotherapeutics. The Food and Drug Administration (FDA) has approved several phage display-derived peptides and antibodies for the treatment of different diseases. In the current review, we provided a comprehensive insight into the role of phage display-derived peptides and antibodies in the treatment of different diseases including cancers, infectious diseases and neurological disorders. We also explored the applications of phage display in targeted drug delivery, gene therapy, and CAR T-cell.

A novel co-receptor with mutated TIGIT to enhance PSCA CAR-T therapy for bladder cancer.

e14574 Background: TIGIT plays a crucial role in immune regulation, particularly in tumor environments, by acting as an inhibitory receptor. Leveraging its interaction with ligands like CD155 on tumor cells, we developed an innovative CAR-T cell therapy using a mutated TIGIT co-receptor to overcome inhibitory effect induced by CD155, together with targeting Prostate Stem Cell Antigen (PSCA) CAR, chosen for its high expression in bladder and other cancers versus its minimal presence in normal tissues. Methods: We initiated our approach by inducing strategic mutations in TIGIT, selecting a variant with heightened affinity for CD155. Bio-Layer Interferometry (BLI) analysis confirmed the variant's high binding affinity (KD = 1.635 nM). This mutated TIGIT, combined with CD28's signaling components, was integrated as a co-receptor in our CAR construct. For PSCA targeting, we isolated a high-affinity ScFv against PSCA from a fully human phage display library. The specificity of this ScFv for PSCA was rigorously validated using Cell Membrane Protein Targeting Catcher (CMPTC) technology, covering over 6000 human cell membrane proteins. Results: In our comparative analyses, CAR-T cells featuring a mutated TIGIT co-receptor (T-PSCA-CAR-T) matched the cytotoxicity of traditional second-generation BBz-structured CAR-T cells (PSCA-CAR-T) against PSCA-expressing HT-1376 bladder cancer cells. However, T-PSCA-CAR-T cells distinguished themselves by producing significantly higher IFN-γ levels (3805.683±65.506 vs 1271.712±39.768 pg/mL). Our in vivo tumor model, utilizing luciferase-expressing HT-1376 cells, demonstrated T-PSCA-CAR-T's enhanced anti-tumor efficacy, as evidenced by a more significant reduction in bioluminescence signal intensity (average decreasing ratio: 0.02 for T-PSCA-CAR-T vs 0.35 for PSCA-CAR-T; p < 0.05). These results underscore the efficacy of the mutated TIGIT co-receptor in improving CAR-T cell therapy. Conclusions: Our study signifies a transformative advancement in CAR-T cell therapy by incorporating a mutated TIGIT co-receptor, markedly improving therapeutic outcomes in bladder cancer treatment. Utilizing PSCA as a model target not only validates the efficacy of this novel approach but also paves the way for its application in a broader range of cancers, representing a significant leap in the field of immunotherapy.

Reduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvs.

We aimed to assess the effects of a cocktail comprising three specific anti- HER2 scFvs on breast tumor formation in a xenograft mouse model and to evaluate quantitative changes in the tumor using stereological analysis. Three specific anti-HER2 phage antibodies were produced from a scFv-library using phage display technology. The cell binding capacities of the antibodies were assessed via FACS analysis. Soluble forms of the antibodies were prepared by infecting HB2151-E. coli cells and purified using a centrifugal ultrafiltration method. The purification process was evaluated by SDSPAGE analysis. Two forms of scFv cocktails were prepared, soluble scFv and phage-scFv cocktail, which contained an equal amount/phage of each of the three antibodies. Inbred female BALB/c mice were pretreated with 5 and 20 mg/kg of the soluble scFv cocktail and 1011 phage-scFv cocktail/ kg. The mice were then injected with 2×106 SKBR-3 human breast cancer cells. Total tumor, inflammatory and non-inflammatory volumes were estimated using the Cavalieri principle after preparing photomicrograph slides. The anti-HER2 scFvs showed significantly higher binding to SKBR-3 cells compared to the isotype control. SDS-PAGE analysis confirmed the high purification of the scFvs. Stereological analysis revealed that the group pretreated with 20 mg/kg of the soluble scFv cocktail exhibited the highest reductions in total tumor volume, non-inflammatory volume, and inflammatory volume, with reductions of 73%, 78%, and 72%, respectively, compared to PBS-pretreated mice (P-value < 0.0001). The volumetric ratio of necrotic tissue to total tumor volume increased by 2.2-fold and 2- fold in the 20 mg/kg of soluble scFv cocktail and phage-scFv cocktail groups, respectively, compared to the PBS-treated mice (P-value < 0.05). Pre-treatment with a 20 mg/kg anti-HER2 scFv cocktail resulted in a significant reduction in tumor volume and increased necrotic area in a human breast cancer xenograft model, indicating the remarkable anti-tumor effect of the cocktail in vivo.

Design, Construction, and Validation of a Yeast-Displayed Chemically Expanded Antibody Library.

In vitro display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photo-reactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing O-(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multi-modal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads.

Construction and characterization of murine single-chain variable fragment (MuscFv) antibody against acrylamide in coffee

Ingredients of food, especially sugar and starch at high-temperature cooking processes could lead to the formation of acrylamide (AA). This chemical is a harmful carcinogen, a neurotoxicant, a reproductive toxicant, and a carcinogen in animal species. However, the detection of acrylamide contamination in food goes unnoticed. In this work, the mouse monoclonal antibody in the form of a single chain variable fragment (scFv) specific to acrylamide was selected from the murine scfv (muscfv) phage-displayed library. Acrylamide (AA) was used as an antigen for bio-panning. The murine single-chain variable fragment (MuscFv) antibody specific to acrylamide in coffee was successfully constructed, which was determined by ELISA and HPLC. Currently, this is the first study, which describes the selection of antibodies against acrylamide from the muscfv phage-displayed library and could be used as a tool for the detection of acrylamide in coffee.

Advancements in ovarian cancer immunodiagnostics and therapeutics via phage display technology.

Ovarian cancer, ranking as the seventh most prevalent malignancy among women globally, faces significant challenges in diagnosis and therapeutic intervention. The difficulties in early detection are amplified by the limitations and inefficacies inherent in current screening methodologies, highlighting a pressing need for more efficacious diagnostic and treatment strategies. Phage display technology emerges as a pivotal innovation in this context, utilizing extensive phage-peptide libraries to identify ligands with specificity for cancer cell markers, thus enabling precision-targeted therapeutic strategies. This technology promises a paradigm shift in ovarian cancer management, concentrating on targeted drug delivery systems to improve treatment accuracy and efficacy while minimizing adverse effects. Through a meticulous review, this paper evaluates the revolutionary potential of phage display in enhancing ovarian cancer therapy, representing a significant advancement in combating this challenging disease. Phage display technology is heralded as an essential instrument for developing effective immunodiagnostic and therapeutic approaches in ovarian cancer, facilitating early detection, precision-targeted medication, and the implementation of customized treatment plans.

In vivo phage display identifies novel peptides for cardiac targeting

Heart failure remains a leading cause of mortality. Therapeutic intervention for heart failure would benefit from targeted delivery to the damaged heart tissue. Here, we applied in vivo peptide phage display coupled with high-throughput Next-Generation Sequencing (NGS) and identified peptides specifically targeting damaged cardiac tissue. We established a bioinformatics pipeline for the identification of cardiac targeting peptides. Hit peptides demonstrated preferential uptake by human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and immortalized mouse HL1 cardiomyocytes, without substantial uptake in human liver HepG2 cells. These novel peptides hold promise for use in targeted drug delivery and regenerative strategies and open new avenues in cardiovascular research and clinical practice.

Development of a highly effective combination monoclonal antibody therapy against Herpes simplex virus

BackgroundInfections with Herpes simplex virus (HSV)-1 or -2 usually present as mild chronic recurrent disease, however in rare cases can result in life-threatening conditions with a large spectrum of pathology. Monoclonal antibody therapy has great potential especially to treat infections with virus resistant to standard therapies. HDIT101, a humanized IgG targeting HSV-1/2 gB was previously investigated in phase 2 clinical trials. The aim of this study was to develop a next-generation therapy by combining different antiviral monoclonal antibodies.MethodsA lymph-node derived phage display library (LYNDAL) was screened against recombinant gB from Herpes simplex virus (HSV) -1 and HDIT102 scFv was selected for its binding characteristics using bio-layer interferometry. HDIT102 was further developed as fully human IgG and tested alone or in combination with HDIT101, a clinically tested humanized anti-HSV IgG, in vitro and in vivo. T-cell stimulating activities by antigen-presenting cells treated with IgG-HSV immune complexes were analyzed using primary human cells. To determine the epitopes, the cryo-EM structures of HDIT101 or HDIT102 Fab bound to HSV-1F as well as HSV-2G gB protein were solved at resolutions < 3.5 Å.ResultsHDIT102 Fab showed strong binding to HSV-1F gB with Kd of 8.95 × 10–11 M and to HSV-2G gB with Kd of 3.29 × 10–11 M. Neutralization of cell-free virus and inhibition of cell-to-cell spread were comparable between HDIT101 and HDIT102. Both antibodies induced internalization of gB from the cell surface into acidic endosomes by binding distinct epitopes in domain I of gB and compete for binding. CryoEM analyses revealed the ability to form heterogenic immune complexes consisting of two HDIT102 and one HDIT101 Fab bound to one gB trimeric molecule. Both antibodies mediated antibody-dependent phagocytosis by antigen presenting cells which stimulated autologous T-cell activation. In vivo, the combination of HDIT101 and HDIT102 demonstrated synergistic effects on survival and clinical outcome in immunocompetent BALB/cOlaHsd mice.ConclusionThis biochemical and immunological study showcases the potential of an effective combination therapy with two monoclonal anti-gB IgGs for the treatment of HSV-1/2 induced disease conditions.

AttABseq: an attention-based deep learning prediction method for antigen-antibody binding affinity changes based on protein sequences.

The optimization of therapeutic antibodies through traditional techniques, such as candidate screening via hybridoma or phage display, is resource-intensive and time-consuming. In recent years, computational and artificial intelligence-based methods have been actively developed to accelerate and improve the development of therapeutic antibodies. In this study, we developed an end-to-end sequence-based deep learning model, termed AttABseq, for the predictions of the antigen-antibody binding affinity changes connected with antibody mutations. AttABseq is a highly efficient and generic attention-based model by utilizing diverse antigen-antibody complex sequences as the input to predict the binding affinity changes of residue mutations. The assessment on the three benchmark datasets illustrates that AttABseq is 120% more accurate than other sequence-based models in terms of the Pearson correlation coefficient between the predicted and experimental binding affinity changes. Moreover, AttABseq also either outperforms or competes favorably with the structure-based approaches. Furthermore, AttABseq consistently demonstrates robust predictive capabilities across a diverse array of conditions, underscoring its remarkable capacity for generalization across a wide spectrum of antigen-antibody complexes. It imposes no constraints on the quantity of altered residues, rendering it particularly applicable in scenarios where crystallographic structures remain unavailable. The attention-based interpretability analysis indicates that the causal effects of point mutations on antibody-antigen binding affinity changes can be visualized at the residue level, which might assist automated antibody sequence optimization. We believe that AttABseq provides a fiercely competitive answer to therapeutic antibody optimization.

BCMA/CD47-directed universal CAR-T cells exhibit excellent antitumor activity in multiple myeloma

BackgroundBCMA-directed autologous chimeric antigen receptor T (CAR-T) cells have shown excellent clinical efficacy in relapsed or refractory multiple myeloma (RRMM), however, the current preparation process for autologous CAR-T cells is complicated and costly. Moreover, the upregulation of CD47 expression has been observed in multiple myeloma, and anti-CD47 antibodies have shown remarkable results in clinical trials. Therefore, we focus on the development of BCMA/CD47-directed universal CAR-T (UCAR-T) cells to improve these limitations.MethodsIn this study, we employed phage display technology to screen nanobodies against BCMA and CD47 protein, and determined the characterization of nanobodies. Furthermore, we simultaneously disrupted the endogenous TRAC and B2M genes of T cells using CRISPR/Cas9 system to generate TCR and HLA double knock-out T cells, and developed BCMA/CD47-directed UCAR-T cells and detected the antitumor activity in vitro and in vivo.ResultsWe obtained fourteen and one specific nanobodies against BCMA and CD47 protein from the immunized VHH library, respectively. BCMA/CD47-directed UCAR-T cells exhibited superior CAR expression (89.13-98.03%), and effectively killing primary human MM cells and MM cell lines. BCMA/CD47-directed UCAR-T cells demonstrated excellent antitumor activity against MM and prolonged the survival of tumor-engrafted NCG mice in vivo.ConclusionsThis work demonstrated that BCMA/CD47-directed UCAR-T cells exhibited potent antitumor activity against MM in vitro and in vivo, which provides a potential strategy for the development of a novel “off-the-shelf” cellular immunotherapies for the treatment of multiple myeloma.Graphic

Оптимизация технологии фагового дисплея для получения антител, специфичных к тетрациклину

Large-scale production and use of antimicrobials in human, veterinary and agricultural applications has led to antibiotic contamination of water resources. Therefore, it is relevant to develop methods for monitoring the content of antibiotics, especially in water resources. Biosensor methods are successful for the analysis of antibiotics. One of the main elements of a biosensor system is the selection of a recognition element. An alternative tool for selecting a sensitive element (recognition element) is phage display of antibodies, which makes it possible to obtain antibodies to low molecular weight antigens. The purpose of the work was to conduct research to develop and optimize the methodology for obtaining antibodies specific to tetracycline by using phage display technology, and to evaluate the possibility of their use for the indication of tetracycline. The work included studies of optimization of conditions for the production of phage antibodies specific to tetracycline, as well as the preservation of their activity during storage. The promise of using phage display technology for the production of anti-tetracycline antibodies has been shown. Using the dot immunoassay method, the possibility of using the obtained phage antibodies for tetracycline detection was shown.

Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region.

Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1' site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.

A novel LAG3 neutralizing antibody improves cancer immunotherapy by dual inhibition of MHC-II and FGL1 ligand binding

LAG3 is an inhibitory immune checkpoint expressed on activated T and NK cells. Blocking the interaction of LAG3 with its ligands MHC-II and FGL1 renders T cells improved cytotoxicity to cancer cells. Current study generated a panel of LAG3 monoclonal antibodies (mAbs) through immunization of mice followed by phage display. Some of them bound to the D1-D2 domain of LAG3, which is known for the engagement of its ligands FGL1 and MHC-II. Three outperformers, M208, M226, and M234, showed stronger blocking activity than Relatlimab in the FGL1 binding. Furthermore, M234 showed dual inhibition of FGL1 (IC50 of 20.6 nM) and MHC-II binding (IC50 of 6.2 nM) to LAG3. In vitro functional tests showed that M234 significantly stimulated IFN-γ secretion from activated PBMC cells. In vivo studies in a mouse model of hepatocellular carcinoma xenografts demonstrated that combining M234 IgG with GPC3-targeted bispecific antibodies significantly improved efficacy. In addition, GPC3-targeted CAR-T cells secreting IL-21-M234 scFv fusion protein exhibited enhanced activity in inhibiting tumor growth and greatly increased the survival rate of mice. Taken together, M234 has potential in cancer immunotherapy and warrants further clinical trial.

In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model

Oligoclonal mixtures of broadly-neutralizing antibodies can neutralize complex compositions of similar and dissimilar antigens, making them versatile tools for the treatment of e.g., infectious diseases and animal envenomations. However, these biotherapeutics are complicated to develop due to their complex nature. In this work, we describe the application of various strategies for the discovery of cross-neutralizing nanobodies against key toxins in coral snake venoms using phage display technology. We prepare two oligoclonal mixtures of nanobodies and demonstrate their ability to neutralize the lethality induced by two North American coral snake venoms in mice, while individual nanobodies fail to do so. We thus show that an oligoclonal mixture of nanobodies can neutralize the lethality of venoms where the clinical syndrome is caused by more than one toxin family in a murine challenge model. The approaches described may find utility for the development of advanced biotherapeutics against snakebite envenomation and other pathologies where multi-epitope targeting is beneficial.

A humanized trivalent Nectin-4-targeting nanobody drug conjugate displays potent antitumor activity in gastric cancer

BackgroundGastric cancer represents a highly lethal malignancy with an elevated mortality rate among cancer patients, coupled with a suboptimal postoperative survival prognosis. Nectin-4, an overexpressed oncological target for various cancers, has been exploited to create antibody-drug conjugates (ADCs) to treat solid tumors. However, there is limited research on Nectin-4 ADCs specifically for gastric cancer, and conventional immunoglobulin G (IgG)-based ADCs frequently encounter binding site barriers. Based on the excellent tumor penetration capabilities inherent in nanobodies (Nbs), we developed Nectin-4-targeting Nb drug conjugates (NDCs) for the treatment of gastric cancer.ResultsAn immunized phage display library was established and employed for the selection of Nectin-4-specific Nbs using phage display technology. Subsequently, these Nbs were engineered into homodimers to enhance Nb affinity. To prolong in vivo half-life and reduce immunogenicity, we fused an Nb targeting human serum albumin (HSA), resulting in the development of trivalent humanized Nbs. Further, we site-specifically conjugated a monomethyl auristatin E (MMAE) at the C-terminus of the trivalent Nbs, creating Nectin-4 NDC (huNb26/Nb26-Nbh-MMAE) with a drug-to-antibody ratio (DAR) of 1. Nectin-4 NDC demonstrated excellent in vitro cell-binding activities and cytotoxic efficacy against cells with high Nectin-4 expression. Subsequent administration of Nectin-4 NDC to mice bearing NCI-N87 human gastric cancer xenografts demonstrated rapid tissue penetration and high tumor uptake through in vivo imaging. Moreover, Nectin-4 NDC exhibited noteworthy dose-dependent anti-tumor efficacy in in vivo studies.ConclusionWe have engineered a Nectin-4 NDC with elevated affinity and effective tumor uptake, further establishing its potential as a therapeutic agent for gastric cancer.Graphical abstract