Selective engagement of FcγRIV by a M2e-specific single domain antibody construct protects against influenza A virus infection

Lower respiratory tract infections, such as infections caused by influenza A viruses, are a constant threat for public health. Antivirals are indispensable to control disease caused by epidemic as well as pandemic influenza A. We developed a novel anti-influenza A virus approach based on an engineered single-domain antibody (VHH) construct that can selectively recruit innate immune cells to the sites of virus replication. This protective construct comprises two VHHs. One VHH binds with nanomolar affinity to the conserved influenza A matrix protein 2 (M2) ectodomain (M2e). Co-crystal structure analysis revealed that the complementarity determining regions 2 and 3 of this VHH embrace M2e. The second selected VHH specifically binds to the mouse Fc gamma Receptor IV (Fc gamma RIV) and was genetically fused to the M2e-specific VHH, which resulted in a bi-specific VHH-based construct that could be efficiently expressed in Pichia pastoris. In the presence of M2 expressing or influenza A virus-infected target cells, this single domain antibody construct selectively activated the mouse Fc gamma RIV. Moreover, intranasal delivery of this bispecific Fc gamma RIV-engaging VHH construct protected wild type but not Fc gamma RIV-/- mice against challenge with an H3N2 influenza virus. These results provide proof of concept that VHHs directed against a surface exposed viral antigen can be readily armed with effector functions that trigger protective antiviral activity beyond direct virus neutralization

Immunogenicity Study of Biosimilar Candidates

Therapeutic proteins can induce undesirable immune reactions in the patient and constitute a major concern as they may compromise therapy safety and efficacy. During the comparability study between a biosimilar product and the innovator, several attributes are considered. Among them, the comparative immunogenicity analysis in preclinical and clinical stages has a major relevance. In this chapter, we will describe the most used experimental platforms for biotherapeutic immunogenicity characterization. Special emphasis will be placed on in vitro assays for the detection of contaminants that modulate innate immune responses, as well as tools for the identification of biologic-derived T-cell epitopes. Likewise, we will also review the current trials used for the detection of host cell proteins (HCPs) and their potential impact on protein immunogenicity. Finally, we will analyze the admissibility criteria established by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), when comparing the immunogenicity of reference products and biosimilar candidates

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

T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation

Protein therapeutics hold a prominent and rapidly expanding place among medicinal products. Purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, and chimeric fusion proteins are all examples of therapeutic proteins that have been developed in the past few decades and approved for use in the treatment of human disease. Despite early belief that the fully human nature of these proteins would represent a significant advantage, adverse effects associated with immune responses to some biologic therapies have become a topic of some concern. As a result, drug developers are devising strategies to assess immune responses to protein therapeutics during both the preclinical and the clinical phases of development. While there are many factors that contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) responses appear to play a critical role in the development of antibody responses to biologic therapeutics. A range of methodologies to predict and measure Td immune responses to protein drugs has been developed. This review will focus on the Td contribution to immunogenicity, summarizing current approaches for the prediction and measurement of T cell-dependent immune responses to protein biologics, discussing the advantages and limitations of these technologies, and suggesting a practical approach for assessing and mitigating Td immunogenicity

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring.

The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of "lab-on-a-chip" methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research

An empirical approach towards the efficient and optimal production of influenza-neutralizing ovine polyclonal antibodies demonstrates that the novel adjuvant CoVaccine HT(TM) is functionally superior to Freund's adjuvant

Passive immunotherapies utilising polyclonal antibodies could have a valuable role in preventing and treating infectious diseases such as influenza, particularly in pandemic situations but also in immunocompromised populations such as the elderly, the chronically immunosuppressed, pregnant women, infants and those with chronic diseases. The aim of this study was to optimise current methods used to generate ovine polyclonal antibodies. Polyclonal antibodies to baculovirus-expressed recombinant influenza haemagglutinin from A/Puerto Rico/8/1934 H1N1 (PR8) were elicited in sheep using various immunisation regimens designed to investigate the priming immunisation route, adjuvant formulation, sheep age, and antigen dose, and to empirically ascertain which combination maximised antibody output. The novel adjuvant CoVaccine HT™ was compared to Freund’s adjuvant which is currently the adjuvant of choice for commercial production of ovine polyclonal Fab therapies. CoVaccine HT™ induced significantly higher titres of functional ovine anti-haemagglutinin IgG than Freund’s adjuvant but with fewer side effects, including reduced site reactions. Polyclonal hyperimmune sheep sera effectively neutralised influenza virus in vitro and, when given before or after influenza virus challenge, prevented the death of infected mice. Neither the age of the sheep nor the route of antigen administration appeared to influence antibody titre. Moreover, reducing the administrated dose of haemagglutinin antigen minimally affected antibody titre. Together, these results suggest a cost effective way of producing high and sustained yields of functional ovine polyclonal antibodies specifically for the prevention and treatment of globally significant diseases.Natalie E. Stevens, Cara K. Fraser, Mohammed Alsharifi, Michael P. Brown, Kerrilyn R. Diener, John D. Haybal

Voices of biotech leaders

Nature Biotechnology asks a selection of leaders from across biotech to look at the future of the sector and make some predictions for the coming years.</p