Generation of Lamprey Monoclonal Antibodies (Lampribodies) Using the Phage Display System.

The variable lymphocyte receptors (VLRs) consist of leucine rich repeats (LRRs) and comprise the humoral antibodies produced by lampreys and hagfishes. The diversity of the molecules is generated by stepwise genomic rearrangements of LRR cassettes dispersed throughout the VLRB locus. Previously, target-specific monovalent VLRB antibodies were isolated from sea lamprey larvae after immunization with model antigens. Further, the cloned VLR cDNAs from activated lamprey leukocytes were transfected into human cell lines or yeast to select best binders. Here, we expand on the overall utility of the VLRB technology by introducing it into a filamentous phage display system. We first tested the efficacy of isolating phage into which known VLRB molecules were cloned after a series of dilutions. These experiments showed that targeted VLRB clones could easily be recovered even after extensive dilutions (1 to 109). We further utilized the system to isolate target-specific "lampribodies" from phage display libraries from immunized animals and observed an amplification of binders with relative high affinities by competitive binding. The lampribodies can be individually purified and ostensibly utilized for applications for which conventional monoclonal antibodies are employed

Recombinant antibodies for the study of livestock infection: from basic genetics to single-chain Fvs

Molecular biology has provided new opportunities to understand better the functioning of the immune system and to exploit this information for the construction of specific antibodies against a wide variety of antigens including the pathogens of humans and animals. In spite of the economic importance of cattle, many aspects of the immunology of this animal remain uncharacterised and tools to understand better bovine infections are lacking. This project has addressed aspects of both issues. The bovine immunoglobulin (Ig) system resembles that of other domesticated mammals in some respects, but other properties (eg the length of the third antigen-binding region of the heavy chain) appear unique. The first area for investigation in this project was to characterise the bovine JH locus and to understand why Ig rearrangement apparently favours a single JH segment. PCR was used to recover JH sequence from genomic DNA, either from non-lymphoid tissues or lambda vectors isolated and studied by other investigators. A region of 3200 bp was characterised which included the DQ52 segment, 6 JH segments and the heavy chain enhancer. The bovine DQ52 sequence is longer than those of other species and differs in sequence from a common consensus. For the most part, the JH locus is homologous to that of the sheep. The sixth JH segment identified appears to undergo rearrangement and is expressed in a minority of cattle antibodies. However, none of the segments carried the sequence which is most commonly expressed in bovine Ig. To identify which segment participates in this process, sequence was recovered from the rearranged genomic DNA of isolated bovine B cells using PCR with primers against VH and JH regions. This implicated the rearrangement of the fourth JH segment in the formation of bovine Igs but as the sequence differed between germline and rearranged copies, it appears that a non-conventional process operates in cattle. It is proposed that a gene conversion event or modified rearrangement process introduces sequence to form the fourth framework region of bovine Ig which does not exist at the JH locus in the germline. The mechanism of this modification requires more investigation. The second part of this project aimed to construct a library of recombinant bovine Fab antibodies from the Ig repertoire of a calf vaccinated against Mannheimia haemolytica (previously named Pasteurella haemolyticd)

Application of M13 Phage Display for Identifying Immunogenic Proteins from Tick (\u3cem\u3eIxodes scapularis\u3c/em\u3e) Saliva

Background: Ticks act as vectors for a large number of different pathogens, perhaps most notably Borrelia burgdorferi, the causative agent of Lyme disease. The most prominent tick vector in the United States is the blacklegged tick, Ixodes scapularis. Tick bites are of special public health concern since there are no vaccines available against most tick-transmitted pathogens. Based on the observation that certain non-natural host animals such as guinea pigs or humans can develop adaptive immune responses to tick bites, anti-tick vaccination is a potential approach to tackle health risks associated with tick bites. Results: The aim of this study was to use an oligopeptide phage display strategy to identify immunogenic salivary gland proteins from I. scapularis that are recognized by human immune sera. Oligopeptide libraries were generated from salivary gland mRNA of 18 h fed nymphal I. scapularis. Eight immunogenic oligopeptides were selected using human immune sera. Three selected immunogenic oligopeptides were cloned and produced as recombinant proteins. The immunogenic character of an identified metalloprotease (MP1) was validated with human sera. This enzyme has been described previously and was hypothesized as immunogenic which was confirmed in this study. Interestingly, it also has close homologs in other Ixodes species. Conclusion: An immunogenic protein of I. scapularis was identified by oligopeptide phage display. MP1 is a potential candidate for vaccine development

NEXT-GENERATION SEQUENCING AND MOTIF GRAFTING APPLICATIONS IN SYNTHETIC ANTIBODY DISCOVERY

The overall objective of this PhD project was to develop and validate methods for advancing the applications of two techniques, next-generation sequencing (NGS) and motif grafting, in synthetic antibody discovery. In the first part of this project, we developed an NGS-assisted antibody discovery platform by integrating phage-displayed single-framework synthetic antigen- binding fragment (Fab) libraries with Ion Torrent sequencing. We constructed a new single- framework synthetic Fab library containing 8.5 billion unique Fab clones, and validated its functionality by generating high affinity Fabs against Notch and Jagged receptors. We developed a rapid and simple method to link and sequence all diversified complementarity-determining regions (CDRs) in phage Fab pools without losing the CDR pairing information. We identified and reconstructed low-frequency rare Fab clones from NGS information in a reliable and high- throughput manner. In some cases, reconstructed rare clones (frequency ~0.1%) showed higher affinity and better specificity than high-frequency top clones isolated by Sanger sequencing, highlighting the importance of NGS in synthetic antibody discovery. In the second part of this project, we employed motif grafting to semi-rationally design phage-displayed synthetic Fab libraries that are biased towards interacting with a specific site on a receptor. We used structural information on the epidermal growth factor receptor (EGFR) homo-dimerization interaction to design a structure-guided Fab library that was biased towards interacting with domain II of EGFR. We used this structure-guided Fab library to obtain Fabs against the EGFR extracellular domain. For comparison, we used a naïve synthetic Fab library to generate an anti-EGFR Fab whose binding overlapped with the Fab isolated from the structure-guided Fab library. Both Fabs possessed low-nM binding values for recombinant and cell-surface EGFR and inhibited EGF- mediated EGFR activation. Epitope mapping showed that domain II is partially responsible for the interaction of Fabs with EGFR. Further, both Fabs target unique epitopes that are different from previously validated epitopes on EGFR. In total, this PhD project resulted in novel methods for discovering synthetic antibodies using NGS and motif grafting techniques, three functional Fab libraries and numerous high-affinity Fabs against Notch, Jagged and EGF receptors

Identifikation neuer tumorassoziierter Proteine in Kopf-Halskrebs mittels aus Patienten abgeleiteten cDNA-Bibliotheken aus frischem Gewebe und Antikörperrepertoiren

There is an increasing demand for the discovery of novel therapeutic targets in immunotherapy. Novel applications of immunotherapies in turn require specific biomarkers to guide treatment, monitor disease and to predict patient outcome, further increasing the need for discovery. Typical discovery ventures make use of proteomics and transcriptomics approaches that try to provide solutions via big data or rely on screening of common peptide or protein libraries against large numbers of patient sera. Though effective in some cases, such large scope techniques generally do not take into account patient-specific phenotypes and might thereby miss individual indications. This study presents a novel approach for the selection of potential biomarkers and therapeutic targets using fresh tumor tissue and patient antibody repertoires. Patient specific cDNA antigen libraries were created from head and neck cancer tissues. Autologous antibody repertoires obtained from tumor infiltrating B cells (TIBC) or patient sera were used for selection of protein fragments via M13 phage display. The technology was tested successfully by selecting mesothelin (MSLN) peptides from HCT116 cell line antigen libraries using anti-MSLN antibody SUW57-D11. Panning in solution in combination with phage ELISA screening was found to be the most promising workflow for biomarker and target discovery. High throughput applicability of this method was confirmed through successful selections in 96-well format and by employing next generation sequencing (NGS) as a single massively parallel screening method to replace ELISA. Using antibodies from TIBC, protein fragments of known cancer therapy target MMP9 were isolated from the antigen libraries, providing proof of principle for the identification of potential therapeutic targets. Furthermore, SHTN1 and WWC2 protein fragments were enriched in a similar approach. Using patient sera, EEA1 emerged as a protein of interest. The presented technology allows target and biomarker discovery based on patient specific local and systemic immune responses. This approach yields candidates with an implied relevance by basing the selection process on a biological mechanism within the disease context. The workflow has the capacity to be advanced towards a target and biomarker discovery platform that is applicable not only to cancer, but to diseases that require immunotherapeutic interventions in general.In der Immuntherapie existiert eine steigende Nachfrage an neuen therapeutisch angreifbaren Zielstrukturen. In gleichem Maße sind Biomarker gefordert, um Behandlungsentscheidungen zu leiten, den Krankheitsfortschritt zu überwachen und Folgen für die Patienten vorherzusagen. Übliche Unternehmungen zur Entdeckung neuer Zielstrukturen basieren auf Proteomik und Transkriptomik, welche aus großen Datenmengen Lösungen ableiten, oder auf der Selektion von einzelnen Peptid- oder Proteinbibliotheken gegen eine Vielzahl von Patientenseren. Diese umfangreichen Techniken erfassen normalerweise keine patientenspezifischen Phänotypen und können deshalb individuelle Indikationen übersehen. Diese Studie präsentiert einen neuartigen Ansatz zur Selektion von potentiellen Biomarkern und therapeutischen Zielstrukturen mit Hilfe von Tumorgeweben und Patientenantikörpern. Aus frischen Kopf-, Halstumorgeweben wurden cDNA-Antigenbibliotheken generiert. Autologe Antikörperrepertoire aus tumorinfiltrierenden B-Zellen (TIBZ) oder Patientenseren wurden zu deren Selektion im M13 Phagendisplay verwendet. Durch die Selektion von Mesothelinpeptiden aus HCT116 Zellinien-Antigenbibliotheken mittels des anti-Mesothelin Antikörpers SUW57-D11 konnte die Technologie erfolgreich getestet werden. Phagendisplay in Lösung und Selektion mittels Phagen-ELISA wurde als vielversprechender Arbeitsablauf für die Entdeckung von Biomarkern und Zielstrukturen erarbeitet. Die Anwendbarkeit der Methode im Hochdurchsatzverfahren wurde durch erfolgreiche Selektionen im 96-well-Format und mittels der Anwendung hochparalleler Sequenzierung zur Selektion bestätigt. Mit Antikörpern aus TIBZ konnte MMP9 als Zielstruktur isoliert werden. Dies ist ein prinzipieller Beweis für die Funktionalität der Methode. Des Weiteren wurden Proteinfragmente von SHTN1 und WWC2 in einem ähnlichen Ansatz angereichert. Unter Verwendung von Patientenseren, hat sich EEA1 als interessantes Protein herausgestellt. Die präsentierte Technologie erlaubt die Entdeckung von therapeutischen Zielstrukturen und Biomarkern basierend auf patientenspezifischen Immunreaktionen. Dieser Ansatz bringt Kandidaten mit implizierter Relevanz hervor, da der Selektionsprozess auf einem biologischen Mechanismus im Krankheitskontext beruht. Der Arbeitsablauf besitzt die Kapazität zu einer Plattform für die Entdeckung von therapeutischen Zielstrukturen und Biomarkern ausgebaut zu werden, die auf alle Krankheiten, die immuntherapeutische Interventionen benötigen, anwendbar ist

지도 학습 기반 바이오패닝 클론 증폭 패턴 분석을 통한 항원 결합 반응성 예측

학위논문(박사) -- 서울대학교대학원 : 의과대학 의과학과, 2021.8. 정준호.Background: Monoclonal antibodies (mAbs) are produced by B cells and specifically binds to target antigens. Technical advances in molecular and cellular cloning made it possible to purify recombinant mAbs in a large scale, enhancing the multiple research area and potential for their clinical application. Since the importance of therapeutic mAbs is increasing, mAbs have become the predominant drug classes for various diseases over the past decades. During that time, immense technological advances have made the discovery and development of mAb therapeutics more efficient. Owing to advances in high-throughput methodology in genomic sequencing, phenotype screening, and computational data analysis, it is conceivable to generate the panel of antibodies with annotated characteristics without experiments. Thesis objective: This thesis aims to develop the next-generation antibody discovery methods utilizing high-throughput antibody repertoire sequencing and bioinformatics analysis. I developed novel methods for construction of in vitro display antibody library, and machine learning based antibody discovery. In chapter 3, I described a new method for generating immunoglobulin (Ig) gene repertoire, which minimizes the amplification bias originated from a large number of primers targeting diverse Ig germline genes. Universal primer-based amplification method was employed in generating Ig gene repertoire then validated by high-throughput antibody repertoire sequencing, in the aspect of clonal diversity and immune repertoire reproducibility. A result of this research work is published in ‘Journal of Immunological Methods (2021). doi: 10.1016/j.jim.2021. 113089’. In chapter 4, I described a novel machine learning based antibody discovery method. In conventional colony screening approach, it is impossible to identify antigen specific binders having low clonal abundance, or hindered by non-specific phage particles having antigen reactivity on p8 coat protein. To overcome the limitations, I applied the supervised learning algorithm on high-throughput sequencing data annotated with binding property and clonal frequency through bio-panning. NGS analysis was performed to generate large number of antibody sequences annotated with its’ clonal frequency at each selection round of the bio-panning. By using random forest (RF) algorithm, antigen reactive binders were predicted and validated with in vitro screening experiment. A result of this research work is published in ‘Experimental & Molecular Medicine (2017). doi:0.1038/emm.2017.22’ and ‘Biomolecule (2020). doi:10.3390/biom10030421’. Conclusion: By combining conventional antibody discovery techniques and high-throughput antibody repertoire sequencing, it was able to make advances in multiple attributes of the previous methodology. Multi-cycle amplification with Ig germline gene specific primers showed the high level of repertoire distortion, but could be improved by employing universal primer-based amplification method. RF model generates the large number of antigen reactive antibody sequences having various clonal enrichment pattern. This result offers the new insight in interpreting clonal enrichment process, frequency of antigen specific binder does not increase gradually but depends on the multiple selection rounds. Supervised learning-based method also provides the more diverse antigen specific clonotypes than conventional antibody discovery methods.연구의 배경: 단일 클론 항체 (monoclonal antibody, mAb) 는 B 세포에서 생산되어 표적 항원에 특이적으로 결합하는 폴리펩타이드 복합체 이다. 분자 및 세포 클로닝 기술의 발전으로 재조합 단일 클론 항체를 대용량으로 생산하는것이 가능해졌으며, 이를 바탕으로 다양한 연구 및 임상 분야에서의 활용이 확대되고 있다. 또한 치료용 항체를 효율적으로 발굴하고 개발하는 기술에 대한 비약적인 발전이 이루어졌다. 유전자 서열 분석, 표현형 스크리닝, 컴퓨팅 기반 분석법 분야에서 이루어진 고집적 방법론 (high-throughput methodology) 의 발전과 이의 응용을 통해, 비실험적 방법을 통해 항원 반응성 항체 패널을 생산하는것이 가능해졌다. 연구의 목표: 본 박사 학위 논문은 고집적 항체 레퍼토어 시퀀싱 (high-throughput antibody repertoire sequencing) 과 생물정보학 (bioinformatics) 기법을 활용하여 신규한 (novel) 차세대 항체 발굴법 (next-generation antibody discovery method) 을 개발하는것을 목표로 하고 있다. 본 연구를 통해 in vitro display 항체 라이브러리를 제작하기 위한 신규 프로토콜 및 기계 학습을 기반으로한 항체 발굴법을 개발 하였다. Chapter 3: 항체 레퍼토어를 증폭하는 과정에서, 다수의 생식세포 면역 글로불린 유전자 (germline immunoglobulin gene) 특이적 프라이머 사용에 의해 발생하는 증폭 편차 (amplification bias) 를 최소화 하는 방법론에 대해 기술하였다. 유니버셜 (universal) 프라이머를 사용한 다중 사이클 증폭 (multi-cycle amplification) 법이 사용되었으며, 고집적 항체 레퍼토어 시퀀싱을 통해, 클론 다양성 (clonal diversity) 및 면역 레퍼토어 재구성도 (immune repertoire reproducibility) 를 생물정보학적 기법으로 측정하여 신규 방법론에 대한 검증을 수행하였다. 본 연구의 연구결과는 다음의 학술지에 출판 되었다: Journal of Immunological Methods (2021). doi: 10.1016/j.jim.2021. 113089. Chapter 4: 기계 학습 기반의 항체 발굴법 개발에 대해 기술하였다. 전통적 콜로니 스크리닝 (colony screening) 방법에서는, 클론 빈도 (clonal abundance) 가 낮은 클론을 발굴 하거나 선택압 (selective pressure) 이 부여되는 과정에서, p8 표면 단백질의 비 특이적 항원 특이성을 제거할 수 없다. 이러한 제한점을 극복하기 위해서 항원 결합능 및 바이오패닝 에서의 클론 빈도가 측정 되어있는 고집적 항체 서열 데이터를 대상으로 지도 학습 알고리즘을 적용하였다. 랜덤 포레스트 (random forest, RF) 알고리즘을 적용하여 항원 특이적 항체 클론을 예측하였으며, 시험관 내 스크리닝을 통해 항원 특이성을 검증하였다. 본 연구의 연구 결과는 다음의 학술지에 출판되었다: 1) Experimental & Molecular Medicine (2017). doi:0.1038/emm.2017.22., 2) Biomolecule (2020). doi:10.3390/biom10030421. 결론: 전통적 항체 발굴 기술과 고집적 항체 레퍼토어 시퀀싱 기술을 융합함으로써, 기존 방법론의 다양한 한계점을 개선할 수 있었다. 면역 글로불린 생식세포 유전자 특이적 프라이머를 사용한 다중 사이클 증폭은 클론 빈도 및 다양성에 왜곡을 유도 하였으나, 유니버셜 프라이머를 사용한 증폭법을 통해 높은 효율로 레퍼토어 왜곡을 개선시킬 수 있음을 관찰할 수 있었다. RF 모델은 다양한 클론 증폭 패턴 (enrichment pattern) 을 가지는 항원 반응성 항체 서열을 생성하였다. 이를 통해 항원에 특이적으로 결합하는 클론이 단계적으로 증폭되는 것이 아니라 초기 및 후기의 다수의 선별 단계 (selection round) 에 의존함을 확인할 수 있었으며, 바이오패닝 에서의 클론 증폭에 대한 새로운 해석을 제시하였다. 또한 지도 학습을 기반으로 발굴 된 클론들에서, 전통적 콜로니 스크리닝 방법과 대비하여 더 높은 서열 다양성을 관찰할 수 있었다.1. Introduction 8 1.1. Antibody and immunoglobulin repertoire 8 1.2. Antibody therapeutics 16 1.3. Methodology: antibody discovery and engineering 21 2. Thesis objective 28 3. Establishment of minimally biased phage display library construction method for antibody discovery 29 3.1. Abstract 29 3.2. Introduction 30 3.3. Results 32 3.4. Discussion 44 3.5. Methods 47 4. In silico identification of target specific antibodies by high-throughput antibody repertoire sequencing and machine learning 58 4.1. Abstract 58 4.2. Introduction 60 4.3. Results 64 4.4. Discussion 111 4.5. Methods 116 5. Future perspectives 129 6. References 135 7. Abstract in Korean 150박

Development of a Dusky kob scFv gene phage display library for the discovery of antibodies to Brome mosaic virus - a proxy for a novel, emerging fish pathogen

Fish farming is rapidly becoming the world's fastest growing production sector, achieving an annual growth rate of approximately 8.9% since the early 1970s. However, high stocking densities result in elevated stress levels in farmed fish, leading to increased susceptibility to infection by opportunistic pathogens and parasites. Antibody phage display is a method that allows foreign peptides or proteins to be expressed on the phage surface through translational fusion with phage coat proteins. Consequently, antibodies expressed by a diverse repertoire of genes coding for the single chain variable fragment (scFv) of immunoglobulin M can be isolated and screened for affinity to a specific infectious agent or parasite. In this study, a phage display library displaying scFvs derived from combination pairings of Dusky kob (Argyrosomus japonicas) variable heavy and light chain fragments, sourced from the splenic B cells of healthy Dusky kob, was constructed. The library was subjected to two rounds of biopanning against brome mosaic virus (BMV), a grass virus to which Dusky kob would have no prior exposure that served as a proxy for an emerging fish pathogen. Five clones were identified as having high affinity and specificity to BMV, as determined by phage enzymelinked immunosorbent assay (ELISA) and phage western blot analysis, respectively. To validate the diagnostic and therapeutic potential of antibody fragments isolated from this phage display library, the gene encoding the antibody fragment of the clone displaying the highest affinity to BMV was selected and expressed using a yeast surface display system. ELISA analysis of serum sampled from Dusky kob exposed to BMV by injection demonstrated that the yeast displayed anti-BMV antibody could successfully detect BMV in the blood serum of BMV-infected Dusky kob with similar sensitivity to a commercially available counterpart. Similarly, this study demonstrated the neutralising effect of yeast displayed anti-BMV antibodies which were found to successfully reduce BMV infection in barley. Overall, these findings demonstrate the feasibility of a Dusky kob phage display library as a source of diagnostically and therapeutically important antibodies against emerging fish pathogens or parasites that threaten the fish farming industry of South Africa