Preparation of VCSM13 Helper Phage for Display Library Reamplification and Bio-Panning.

Phage-displayed antibody libraries can be constructed using any species that is easily immunized. The pComb3XSS phagemid vector is commonly used for library cloning and phage display. This phagemid encodes the origin of replication of the filamentous bacteriophage f1 but lacks all the genes required for replication and assembly of phage particles. The replication and the assembly of phage from these phagemids thus requires a "helper" phage that provides the genes essential for those steps during library production and bio-panning. One of those helper phages is VCSM13. In this protocol, we describe the preparation of VCSM13 helper phage. Users should prepare VCSM13 helper phage for library reamplification and for bio-panning.

Generation of Chicken Antibody Libraries and Selection of Antigen Binders.

Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.

Chicken Immunization followed by RNA Extraction and cDNA Synthesis for Antibody Library Preparation.

Effective isolation of specific antibodies from immunological repertoires requires the generation of a diverse library against a specific antigen of interest, as well as efficient selection procedures, such as bio-panning and phage ELISA. Key to this is the generation of a good immune response in the host, followed by preparation of high-quality RNA and cDNA from which a library can be constructed by the amplification and cloning of immunoglobulin heavy and light chain genes. The first step in the construction of such an "immune library" is a successful course of immunization. Detection of a strong serum antibody titer will theoretically then result in a pool of extracted RNA that is enriched for transcripts of genes encoding the antibody of interest. Chicken antibodies have been widely used for research and diagnostic purposes, largely because of both their cross-reactivity to epitopes shared by humans, mice, primates, and other mammals, and their simple characteristics, with chickens featuring single functional copies of V H /J H and V λ /J λ gene pairs. In chickens, antibodies against an antigen of interest can be detected in the serum as soon as 5-7 d after immunization. Once the antibody titer reaches an appropriate level in the serum, the spleen, bursa of Fabricius, and bone marrow are then harvested, and antibody libraries can be prepared from extracted RNA. Here, we describe a protocol for chicken immunization with an antigen of interest, followed by RNA extraction from the relevant tissues and cDNA synthesis, which users can use for antibody library construction.

Generation of a Phage Display Chicken Single-Chain Variable Fragment Library.

Phage-displayed antibody fragment libraries can be constructed using essentially any species that is easily immunized, as long as the immunoglobulin variable region gene sequences are known. This protocol describes the procedures for the generation of a phage-displayed chicken single-chain variable fragment (scFv) library after immunization with a target antigen. Briefly, the rearranged heavy chain variable region (V H ) genes and the λ light chain variable region (V λ ) genes are amplified separately and are linked through two separate PCR steps to give the final scFv genes. The genes are then cloned into pComb3XSS to generate the phage display chicken scFv library, which can then be used for test and final library ligations.

Selection of Antigen Binders from a Chicken Single-Chain Variable Fragment Library.

Antibody production against an antigen of interest is highly efficient in chickens, and the use of chicken antibody libraries in phage display can result in high-affinity single-chain variable fragments (scFvs) for multiple applications. After library preparation from an animal immunized with the antigen of interest, the next step involves the identification of antigen binders. Here, we describe a process for the screening of a phage display chicken library using a technique called bio-panning. It consists of several rounds of binding scFv-displaying phage to antigens, followed by washing, elution, and reamplification. We also describe the steps for assessing clone pools obtained after bio-panning via an ELISA-based procedure known as "phage ELISA" to identify single clones. Last, we provide the steps for using high-throughput sequencing to analyze the pool of selected clones.

Polymeric immunoglobulin receptor (pIgR) in cancer.

BACKGROUND: The polymeric immunoglobulin receptor (pIgR) is a transmembrane transporter of polymeric IgA through the intestinal epithelium. Its overexpression has been reported in several cancers, but its role as a diagnostic and prognostic biomarker of oncogenesis is currently unclear. METHOD: A literature search was conducted to summarize the functions of pIgR, its expression levels, and its clinical implications. RESULTS: pIgR expression has previously been investigated by proteomic analysis, RNA sequencing, and tissue microarray at the level of both RNA and protein in various cancers including pancreatic, esophageal, gastric, lung, and liver. However, studies have reported inconsistent results on how pIgR levels affect clinical outcomes such as survival rate and chemotherapy resistance. Possible explanations include pIgR mRNA levels being minimally correlated with the rate of downstream pIgR protein synthesis, and the diversity of antibodies used in immunohistochemistry studies further magnifying this ambiguity. In ovarian cancer cells, the transcytosis of IgA accompanied a series of transcriptional changes in intracellular inflammatory pathways that inhibit the progression of cancer, including the upregulation of IFN-gamma and downregulation of tumor-promoting ephrins. These findings suggest that both the levels of pIgR and secreted IgA from tumor-infiltrating B cells affect clinical outcomes. CONCLUSION: Overall, no direct correlation was observed between the levels of pIgR inside tumor tissue and the clinical features in cancer patients. Measuring pIgR protein levels with a more specific and possibly chemically defined antibody, along with tumoral IgA, is a potential solution to better understand the pathways and consequences of pIgR overexpression in cancer cells.

Specific ablation of PDGFRß-overexpressing pericytes with antibody-drug conjugate potently inhibits pathologic ocular neovascularization in mouse models.

Background: Crosstalk between pericytes and endothelial cells is critical for ocular neovascularization. Endothelial cells secrete platelet-derived growth factor (PDGF)-BB and recruit PDGF receptor ß (PDGFRß)-overexpressing pericytes, which in turn cover and stabilize neovessels, independent of vascular endothelial growth factor (VEGF). Therapeutic agents inhibiting PDGF-BB/PDGFRß signaling were tested in clinical trials but failed to provide additional benefits over anti-VEGF agents. We tested whether an antibody-drug conjugate (ADC) - an engineered monoclonal antibody linked to a cytotoxic agent - could selectively ablate pericytes and suppress retinal and choroidal neovascularization. Methods: Immunoblotting, flow cytometry, cell viability test, and confocal microscopy were conducted to assess the internalization and cytotoxic effect of ADC targeting mPDGFRß in an in vitro setting. Immunofluorescence staining of whole-mount retinas and retinal pigment epithelium-choroid-scleral complexes, electroretinography, and OptoMotry test were used to evaluate the effect and safety of ADC targeting mPDGFRß in the mouse models of pathologic ocular neovascularization. Results: ADC targeting mPDGFRß is effectively internalized into mouse brain vascular pericytes and showed significant cytotoxicity compared with the control ADC. We also show that specific ablation of PDGFRß-overexpressing pericytes using an ADC potently inhibits pathologic ocular neovascularization in mouse models of oxygen-induced retinopathy and laser-induced choroidal neovascularization, while not provoking generalized retinal toxicity. Conclusion: Our results suggest that removing PDGFRß-expressing pericytes by an ADC targeting PDGFRß could be a potential therapeutic strategy for pathologic ocular neovascularization.

Machine Learning-Guided Prediction of Antigen-Reactive In Silico Clonotypes Based on Changes in Clonal Abundance through Bio-Panning.

c-Met is a promising target in cancer therapy for its intrinsic oncogenic properties. However, there are currently no c-Met-specific inhibitors available in the clinic. Antibodies blocking the interaction with its only known ligand, hepatocyte growth factor, and/or inducing receptor internalization have been clinically tested. To explore other therapeutic antibody mechanisms like Fc-mediated effector function, bispecific T cell engagement, and chimeric antigen T cell receptors, a diverse panel of antibodies is essential. We prepared a chicken immune scFv library, performed four rounds of bio-panning, obtained 641 clones using a high-throughput clonal retrieval system (TrueRepertoireTM, TR), and found 149 antigen-reactive scFv clones. We also prepared phagemid DNA before the start of bio-panning (round 0) and, after each round of bio-panning (round 1-4), performed next-generation sequencing of these five sets of phagemid DNA, and identified 860,207 HCDR3 clonotypes and 443,292 LCDR3 clonotypes along with their clonal abundance data. We then established a TR data set consisting of antigen reactivity for scFv clones found in TR analysis and the clonal abundance of their HCDR3 and LCDR3 clonotypes in five sets of phagemid DNA. Using the TR data set, a random forest machine learning algorithm was trained to predict the binding properties of in silico HCDR3 and LCDR3 clonotypes. Subsequently, we synthesized 40 HCDR3 and 40 LCDR3 clonotypes predicted to be antigen reactive (AR) and constructed a phage-displayed scFv library called the AR library. In parallel, we also prepared an antigen non-reactive (NR) library using 10 HCDR3 and 10 LCDR3 clonotypes predicted to be NR. After a single round of bio-panning, we screened 96 randomly-selected phage clones from the AR library and found out 14 AR scFv clones consisting of 5 HCDR3 and 11 LCDR3 AR clonotypes. We also screened 96 randomly-selected phage clones from the NR library, but did not identify any AR clones. In summary, machine learning algorithms can provide a method for identifying AR antibodies, which allows for the characterization of diverse antibody libraries inaccessible by traditional methods.