Biochemical and Functional Characterization of the Three Zebrafish Transglutaminases 2.

Transglutaminase 2 (TG2) is a multifunctional protein widely distributed in various tissues and involved in many physiological and pathological processes. However, its actual role in biological processes is often controversial as TG2 shows different effects in these processes depending on its localization, cell type, or experimental conditions. We characterized the enzymatic and functional properties of TG2 proteins expressed in Danio rerio (zebrafish) to provide the basis for using this established animal model as a reliable tool to characterize TG2 functions in vivo. We confirmed the existence of three genes orthologous to human TG2 (zTGs2) in the zebrafish genome and their expression and function during embryonic development. We produced and purified the zTGs2s as recombinant proteins and showed that, like the human enzyme, zTGs2 catalyzes a Ca2+ dependent transamidation reaction that can be inhibited with TG2-specific inhibitors. In a cell model of human fibroblasts, we also demonstrated that zTGs2 can mediate RGD-independent cell adhesion in the extracellular environment. Finally, we transfected and selected zTGs2-overexpressing HEK293 cells and demonstrated that intracellular zTGs2 plays a very comparable protective/damaging role in the apoptotic process, as hTG2. Overall, our results suggest that zTGs2 proteins behave very similarly to the human ortholog and pave the way for future in vivo studies of TG2 functions in zebrafish.

A Functional Idiotype/Anti-Idiotype Network Is Active in Genetically Gluten-Intolerant Individuals Negative for Both Celiac Disease-Related Intestinal Damage and Serum Autoantibodies.

An unbalance between Abs that recognize an autoantigen (idiotypes; IDs) and Igs that bind such Abs (anti-IDs) is considered a functional event in autoimmune disorders. We investigated the presence of an ID/anti-ID network in celiac disease (CD), a condition in which antitissue transglutaminase 2 (TG2) Abs are suspected to contribute to CD pathogenesis. To characterize the ID side, we reproduced by in vitro yeast display the intestine-resident Abs from CD and control patients. These TG2-specific IDs were used to identify potential anti-IDs in the serum. We observed elevated titers of anti-IDs in asymptomatic patients with predisposition to CD and demonstrated that anti-ID depletion from the serum restores a detectable humoral response against TG2. Our study provides an alternative approach to quantify CD-related autoantibodies in cases that would be defined "negative serology" with current diagnostic applications. Therefore, we suggest that developments of this technology could be designed for perspective routine tests.

Integrated nanotechnology platform for tumor-targeted multimodal imaging and therapeutic cargo release.

A major challenge of targeted molecular imaging and drug delivery in cancer is establishing a functional combination of ligand-directed cargo with a triggered release system. Here we develop a hydrogel-based nanotechnology platform that integrates tumor targeting, photon-to-heat conversion, and triggered drug delivery within a single nanostructure to enable multimodal imaging and controlled release of therapeutic cargo. In proof-of-concept experiments, we show a broad range of ligand peptide-based applications with phage particles, heat-sensitive liposomes, or mesoporous silica nanoparticles that self-assemble into a hydrogel for tumor-targeted drug delivery. Because nanoparticles pack densely within the nanocarrier, their surface plasmon resonance shifts to near-infrared, thereby enabling a laser-mediated photothermal mechanism of cargo release. We demonstrate both noninvasive imaging and targeted drug delivery in preclinical mouse models of breast and prostate cancer. Finally, we applied mathematical modeling to predict and confirm tumor targeting and drug delivery. These results are meaningful steps toward the design and initial translation of an enabling nanotechnology platform with potential for broad clinical applications.

Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes.

Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.

Targeted molecular-genetic imaging and ligand-directed therapy in aggressive variant prostate cancer.

Aggressive variant prostate cancers (AVPC) are a clinically defined group of tumors of heterogeneous morphologies, characterized by poor patient survival and for which limited diagnostic and treatment options are currently available. We show that the cell surface 78-kDa glucose-regulated protein (GRP78), a receptor that binds to phage-display-selected ligands, such as the SNTRVAP motif, is a candidate target in AVPC. We report the presence and accessibility of this receptor in clinical specimens from index patients. We also demonstrate that human AVPC cells displaying GRP78 on their surface could be effectively targeted both in vitro and in vivo by SNTRVAP, which also enabled specific delivery of siRNA species to tumor xenografts in mice. Finally, we evaluated ligand-directed strategies based on SNTRVAP-displaying adeno-associated virus/phage (AAVP) particles in mice bearing MDA-PCa-118b, a patient-derived xenograft (PDX) of castration-resistant prostate cancer bone metastasis that we exploited as a model of AVPC. For theranostic (a merging of the terms therapeutic and diagnostic) studies, GRP78-targeting AAVP particles served to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which has a dual function as a molecular-genetic sensor/reporter and a cell suicide-inducing transgene. We observed specific and simultaneous PET imaging and treatment of tumors in this preclinical model of AVPC. Our findings demonstrate the feasibility of GPR78-targeting, ligand-directed theranostics for translational applications in AVPC.

Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer.

Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. Finally, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.

Profiling celiac disease antibody repertoire.

The aim of this study was to dissect the autoantibody response in celiac disease (CD) that remains largely unknown, with the goal of identifying the disease-specific autoantigenic protein pattern or the so called epitome. Sera from CD patients were used to select immunoreactive antigens from a cDNA phage-display library. Candidate genes were identified, the corresponding proteins produced and their immunoreactivity validated with sera from CD patients and controls. Thirteen CD-specific antigens were identified and further validated by protein microarray. The specificity for 6 of these antigens was confirmed by ELISA. Furthermore we showed that this antibody response was not abolished on a gluten free diet and was not shared with other autoimmune diseases. These antigens appear to be CD specific and independent of gluten induction. The utility of this panel extends beyond its diagnostic value and it may drive the attention to new targets for unbiased screens in autoimmunity research.

Using phage display selected antibodies to dissect microbiomes for complete de novo genome sequencing of low abundance microbes.

BACKGROUND: Single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes. METHODS: We describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS). Single chain (scFv) antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in FACS for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing. RESULTS: We selected antibodies against Lactobacillus acidophilus and demonstrate a FACS-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. The ability to selectively enrich for L. acidophilus when it is present at a very low abundance (<0.2%) leads to complete (>99.8%) de novo genome coverage whereas the standard single-cell sequencing approach is incomplete (<68%). We show that specific antibodies can be selected against L. acidophilus when the monoculture is used as antigen as well as when a community of 10 closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities. CONCLUSIONS: The approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. Combining antibody specificity with FACS provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.

High throughput purification of monoclonal recombinant antibodies using a Protein-A coated membrane plate system.

The therapeutic use of monoclonal antibodies (mAbs) ranges from cancer treatment to immune-mediated conditions, covering infectious and cardiovascular disorders, among others. The development of improved methods for therapeutic antibody discovery has accelerated the identification of numerous mAbs: a discovery campaign can be deeply mined, resulting in hundreds, even thousands, of potential antibody leads for a given target of interest. High throughput mAb expression and purification methods are required for the rapid validation of those leads. In this work, we describe the implementation of a Protein-A coated membrane plate system, the Purexa™ AHT membrane plate, for robust preparative purification of hundreds of recombinant mAbs, without the need for automation. The high efficiency (>80%) recovery generated sufficient mAb for downstream screening analyses such as ELISA and surface plasmon resonance (SPR). This new system allows the functional validation of hundreds of lead antibodies from discovery campaigns in a timely manner regardless of operational size.

Determining the affinities of high-affinity antibodies using KinExA and surface plasmon resonance.

Accurate and efficient affinity measurement techniques are essential for the biophysical characterization of therapeutic monoclonal antibodies, one of the fastest growing drug classes. Surface plasmon resonance (SPR) is widely used for determining antibody affinity, but does not perform well with extremely high affinity (low picomolar to femtomolar range) molecules. In this study, we compare the SPR-based Carterra LSA and the kinetic exclusion assay (KinExA) for measuring the affinities of 48 antibodies generated against the SARS-CoV-2 receptor-binding domain. These data reveal that high-affinity antibodies can be generated straight from selections using high-quality in vitro library platforms with 54% correspondence between affinities measured using LSA and KinExA. Generally, where there was a 2-fold or greater difference between LSA and KinExA, KinExA reported that affinities were tighter. We highlight the differences between LSA and KinExA, identifying the benefits and pitfalls of each in terms of dynamic range and throughput. Furthermore, we demonstrate for the first time that single-point screening with KinExA can significantly improve throughput while maintaining a strong correlation with full binding curve equilibrium measurements, enabling the accurate rank-ordering of clones with exceptionally tight binding properties.

Direct selection of functional fluorescent-protein antibody fusions by yeast display.

Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras. Here, we describe a high throughput method based on yeast display to directly select antibodies most suitable for conversion to fluorescent chimera. A library of scFv binders was converted to a fluorescent chimeric form, by cloning thermal green protein into the linker between VH and VL, and directly selecting for both binding and fluorescent functionality. This allowed us to directly identify antibodies functional in the single chain TGP format, that manifest higher protein expression, easier protein purification, and one-step binding assays.

Insights into next generation sequencing guided antibody selection strategies.

Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random colony picking and Sanger sequencing, which has limitations. Next-generation sequencing (NGS) offers a cost-effective solution with increased read depth, allowing a comprehensive understanding of diversity. Our study establishes NGS guidelines for antibody drug discovery, demonstrating its advantages in expanding the number of unique HCDR3 clusters, broadening the number of high affinity antibodies, expanding the total number of antibodies recognizing different epitopes, and improving lead prioritization. Surprisingly, our investigation into the correlation between NGS-derived frequencies of CDRs and affinity revealed a lack of association, although this limitation could be moderately mitigated by leveraging NGS clustering, enrichment and/or relative abundance across different regions to enhance lead prioritization. This study highlights NGS benefits, offering insights, recommendations, and the most effective approach to leverage NGS in therapeutic antibody discovery.

Cryptic genetic gluten intolerance revealed by intestinal antitransglutaminase antibodies and response to gluten-free diet.

BACKGROUND AND OBJECTIVE: Antitransglutaminase (anti-TG2) antibodies are synthesised in the intestine and their presence seems predictive of future coeliac disease (CD). This study investigates whether mucosal antibodies represent an early stage of gluten intolerance even in the absence of intestinal damage and serum anti-TG2 antibodies. METHODS: This study investigated 22 relatives of patients with CD genetically predisposed to gluten intolerance but negative for both serum anti-TG2 antibodies and intestinal abnormalities. Fifteen subjects were symptomatic and seven were asymptomatic. The presence of immunoglobulin A anti-TG2 antibodies in the intestine was studied by creating phage-antibody libraries against TG-2. The presence of intestinal anti-TG2 antibodies was compared with the serum concentration of the intestinal fatty acid-binding protein (I-FABP), a marker for early intestinal mucosal damage. The effects of a 12-month gluten-free diet on anti-TG2 antibody production and the subjects' clinical condition was monitored. Twelve subjects entered the study as controls. RESULTS: The intestinal mucosa appeared normal in 18/22; 4 had a slight increase in intraepithelial lymphocytes. Mucosal anti-TG2 antibodies were isolated in 15/22 subjects (68%); in particular symptomatic subjects were positive in 13/15 cases and asymptomatic subjects in 2/7 cases (p=0.01). No mucosal antibodies were selected from the controls' biopsies. There was significant correlation between the presence of intestinal anti-TG2 antibodies and positive concentrations of I-FABP (p=0.0008). After a gluten-free diet, 19/22 subjects underwent a second intestinal biopsy, which showed that anti-TG2 antibodies had disappeared in 12/15 (p=0.002), while I-FABP decreased significantly (p<0.0001). The diet resolved both extraintestinal and intestinal symptoms. CONCLUSIONS: A new form of genetic-dependent gluten intolerance has been described in which none of the usual diagnostic markers is present. Symptoms and intestinal anti-TG2 antibodies respond to a gluten free-diet. The detection of intestinal anti-TG2 antibodies by the phage-antibody libraries has an important diagnostic and therapeutic impact for the subjects with gluten-dependent intestinal or extraintestinal symptoms. Clinical trial number NCT00677495.

Pandemic's silver lining.

The intense international focus on the COVID-19 pandemic has provided a unique opportunity to use a wide array of novel tools to carry out scientific studies on the SARS-CoV-2 virus. The value of these comparative studies extends far beyond their consequences for SARS-CoV-2, providing broad implications for health-related science. Here we specifically discuss the impacts of these comparisons on advances in vaccines, the analysis of host humoral immunity, and antibody discovery. As an extension, we also discuss potential synergies between these areas.Abbreviations: CoVIC: The Coronavirus Immunotherapeutic Consortium; EUA: Emergency Use Authorization.

A pandemic-enabled comparison of discovery platforms demonstrates a naïve antibody library can match the best immune-sourced antibodies.

As a result of the SARS-CoV-2 pandemic numerous scientific groups have generated antibodies against a single target: the CoV-2 spike antigen. This has provided an unprecedented opportunity to compare the efficacy of different methods and the specificities and qualities of the antibodies generated by those methods. Generally, the most potent neutralizing antibodies have been generated from convalescent patients and immunized animals, with non-immune phage libraries usually yielding significantly less potent antibodies. Here, we show that it is possible to generate ultra-potent (IC50 < 2 ng/ml) human neutralizing antibodies directly from a unique semisynthetic naïve antibody library format with affinities, developability properties and neutralization activities comparable to the best from hyperimmune sources. This demonstrates that appropriately designed and constructed naïve antibody libraries can effectively compete with immunization to directly provide therapeutic antibodies against a viral pathogen, without the need for immune sources or downstream optimization.

Simultaneous affinity maturation and developability enhancement using natural liability-free CDRs.

ABBREVIATIONS: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; ka: association rate; kd: dissociation rate; KD: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.

A single donor is sufficient to produce a highly functional in vitro antibody library.

Antibody complementarity determining region diversity has been considered to be the most important metric for the production of a functional antibody library. Generally, the greater the antibody library diversity, the greater the probability of selecting a diverse array of high affinity leads. According to this paradigm, the primary means of elevating library diversity has been by increasing the number of donors. In the present study we explored the possibility of creating an in vitro antibody library from a single healthy individual, showing that the number of lymphocytes, rather than the number of donors, is the key criterion in the production of a diverse and functional antibody library. We describe the construction of a high-quality phage display library comprising 5 × 109 human antibodies by applying an efficient B cell extraction protocol from a single donor and a targeted V-gene amplification strategy favoring specific antibody families for their improved developability profiles. Each step of the library generation process was followed and validated by next generation sequencing to monitor the library quality and diversity. The functionality of the library was tested using several therapeutically relevant targets for which a vast number of different antibodies with desired biophysical properties were obtained.

Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.

Drug-like antibodies with high affinity, diversity and developability directly from next-generation antibody libraries.

Therapeutic antibodies must have "drug-like" properties. These include high affinity and specificity for the intended target, biological activity, and additional characteristics now known as "developability properties": long-term stability and resistance to aggregation when in solution, thermodynamic stability to prevent unfolding, high expression yields to facilitate manufacturing, low self-interaction, among others. Sequence-based liabilities may affect one or more of these characteristics. Improving the stability and developability of a lead antibody is typically achieved by modifying its sequence, a time-consuming process that often results in reduced affinity. Here we present a new antibody library format that yields high-affinity binders with drug-like developability properties directly from initial selections, reducing the need for further engineering or affinity maturation. The innovative semi-synthetic design involves grafting natural complementarity-determining regions (CDRs) from human antibodies into scaffolds based on well-behaved clinical antibodies. HCDR3s were amplified directly from B cells, while the remaining CDRs, from which all sequence liabilities had been purged, were replicated from a large next-generation sequencing dataset. By combining two in vitro display techniques, phage and yeast display, we were able to routinely recover a large number of unique, highly developable antibodies against clinically relevant targets with affinities in the subnanomolar to low nanomolar range. We anticipate that the designs and approaches presented here will accelerate the drug development process by reducing the failure rate of leads due to poor antibody affinities and developability.Abbreviations: AC-SINS: affinity-capture self-interaction nanoparticle spectroscopy; CDR: complementarity-determining region; CQA: critical quality attribute; ELISA: enzyme-linked immunoassay; FACS: fluorescence-activated cell sorting; Fv: fragment variable; GM-CSF: granulocyte-macrophage colony-stimulating factor; HCDR3: heavy chain CDR3; IFN2a: interferon α-2; IL6: interleukin-6; MACS: magnetic-activated cell sorting; NGS: next generation sequencing; PCR: polymerase chain reaction; SEC: size-exclusion chromatography; SPR: surface plasmon resonance; TGFß-R2: transforming growth factor ß-R2; VH: variable heavy; VK: variable kappa; VL: variable light; Vl: variable lambda.

Exploiting next-generation sequencing in antibody selections - a simple PCR method to recover binders.

Antibody discovery using invitro display technologies such as phage and/or yeast display has become acornerstone in many research and development projects, including the creation of new drugs for clinical use. Traditionally, after the selection phase, random clones are isolated for binding validation and Sanger sequencing. More recently, next-generation sequencing (NGS) technology has allowed deeper insight into the antibody population after aselection campaign, enabling the identification of many more specific binders. However, this approach only provides the DNA sequences of potential binders, the properties of which need to be fully elucidated by obtaining corresponding clones and expressing them for further validation. Here we present arapid novel method to harvest potential clones identified by NGS that uses asimple PCR and yeast recombination approach. The protocol was tested in selections against three different targets and was able to recover clones at an abundance level that would be impractical to identify using traditional methods.