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In memoriam: Jefferson Foote

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Written by:
E. Sally Ward (a), Peter Jones (b), Tim Buss (c), Cristina Rada (d), Gregory Winter (e) and Richard Willson (f)

a Centre for Cancer Immunology, University of Southampton, Southampton, UK
b Lode, Cambridge, UK
c Proteogenomics Research Institute for Systems Medicine, San Diego, USA
d MRC Laboratory of Molecular Biology, Cambridge, UK
e Trinity College, Cambridge, UK
f Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, USA

Photo courtesy of Kathleen Foote.

Jefferson (Jeff) Foote sadly passed away of pancreatic cancer on January 17, 2020 at the age of 64.  He was a leading figure in physical immunochemistry and antibody humanization, a polymath of broad interests, and a wonderful friend and colleague.  Jeff was born in Chicago and grew up in Tarrytown, NY. Following graduation from Harvard University where he worked in the laboratory of William Lipscomb, he earned his Ph.D. at Berkeley with Howard Schachman, studying the canonical aspartate transcarbamylase system.  In 1985 he moved to the Laboratory of Molecular Biology (LMB) in Cambridge, where he worked with (now Sir) Greg Winter and then with Cesar Milstein. During his time in Cambridge, Jeff applied his understanding of protein biophysics and interaction kinetics to address problems in immunochemistry, increasingly leveraging the availability of the first emerging crystal structures of antibody-antigen complexes. This was before the BIAcore/surface plasmon resonance era that started in the early 1990s, and the work required a comprehensive knowledge of the inner workings of fluorometers, including stop-flow, and the associated mathematical tools. Jeff imported a Macintosh (“Mac”) culture to the laboratory, which was well-received by other local Mac fans in days when benchtop computers were still something of a novelty and there was a threat of other personal computer models becoming the norm.

Whilst at the LMB, Jeff made significant contributions in areas ranging from state-of-the-art antibody engineering to fundamental aspects of B cell biology, including the first description of the CDR grafting, or humanization, of an antibody specific for a hapten.[1] Jeff applied his expertise to determine the affinities of the test grafts, enabling the design principles of the engineered antibodies to be verified in precise, quantitative terms. This seminal study formed the foundation for the subsequent avalanche of therapeutic antibody humanizations, the first of which was the CD52-specific antibody Campath-1 (Alemtuzumab) generated in the Winter/Waldmann laboratories and used to treat chronic lymphocytic leukemia and multiple sclerosis. In addition, Jeff used the first antibody to be structurally solved in complex with antigen, the anti-lysozyme antibody D1.3, to define how framework residue modifications could restore binding behavior close to that of the donor (rodent) antibody to a humanized antibody.[2] As well as the biophysical characterization of framework mutants, he was also the first to synthesize a “consensus” framework.[2,3]

In parallel to Jeff’s work on antibody humanization, he carried out an extensive analysis with Cesar Milstein on how the maturation of the immune response is accompanied by an increased on-rate of antibodies for binding to their antigen. This study led to the paradigm that the selection of the “fittest” B cells is driven by interaction kinetics.[4] Subsequently, in a second publication with Cesar, Jeff observed that antibodies could undergo switching between different conformations (“conformational isomerism”), resulting in bi- or triphasic interaction kinetics.[5] This not only provided a molecular mechanism for the further diversification of antibodies, but also challenged the longstanding axiom that each lymphocyte produces an antibody with a single combining site.

Jeff was one of those more civilized members of the LMB who drove into work, rather than arriving with the appearance of a half-drowned rat following a cycle ride in the wintry, wet days that were common in Cambridge. Whilst working with Greg Winter in the tiny 5-6 person laboratory known as T4, Jeff relished being in the thick of the day-to-day, frequently frenetic activities. The day usually started with copious quantities of “Java”, an almost toxic, viscous dark brown liquid that kept the group members charged and running. Given that antibody humanization and, subsequently, antibody repertoire work were ongoing in the laboratory at this time, there was rarely a dull moment.

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Antibody Engineering & Therapeutics Poster Competition Winners Announced

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Congratulations to our winners!

To recognize the research activities of promising student and postdoctoral attendees of Antibody Engineering & Therapeutics, The Antibody Society sponsors a competition for members who submit posters for display at the meeting. Our judges select the best work based on originality, relevance and perceived impact on the field of antibody research and development.

This year, our judges selected one student and one postdoc winners who receive: 1) complimentary registration to all conference sessions; 2) an opportunity to give a short oral presentation of their work in one of the conference sessions; and 3) a lovely crystal award.

The winners of the contest are:

Felix Goerdeler, Max Planck Institute of Colloids and Interfaces, Germany

Poster title: Fighting protozoan parasites using carbohydrate-binding nanobodies

Dr. Nicolas Bery, Cancer Research Centre of Toulouse, France

Poster title: Discovery of a potent KRAS macromolecule degrader specifically targeting tumours with mutant KRAS

Please join us for the virtual Antibody Engineering & Therapeutics  conference on December 14-16, 2020.

Society members receive a 15% discount on the registration fee. Contact us at membership@antibodysociety.org for the code.

James S. Huston – In Memoriam

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Post written by: Richard Begent, Ph.D., Emeritus Professor of Oncology, University College London

James S. Huston Jr, Ph.D., antibody engineer and founding President of The Antibody Society, died in Boston on March 25, 2020.

Jim Huston was a distinguished biophysicist and a pioneering antibody engineer; his creation of the single chain Fv (scFv) antibody was a seminal advance. These genetically encoded molecules could express the vast diversity of antibody repertoires, and could be used for specific target binding by themselves, incorporated into multifunctional molecules, attached to cell surfaces or applied in any number of formats relevant to biomedicine.

Antibodies with their multiple functions, including the capacity for specific binding to a range of targets, became practical pharmaceuticals with the advent of monoclonal antibodies as described by Köhler and Milstein in 1975. Genetic manipulation humanized the constant regions, making repeated administration feasible with widespread benefits for human health.

It was evident that the smallest target recognition moiety of antibodies, the variable region (Fv), if produced separately could be linked to many agents, conferring exquisite binding specificity. Since the VH and VL domains are separate in the native form of the Fv, they needed to be joined in a way that retained stability, the binding performance of the two components together, and appropriate flexibility.

Jim proposed doing this with one genetic construct that encoded a single-chain Fv (scFv) in which the VH and VL were joined by a flexible linker. The design issues were complex, but, consummate biophysicist that he was, he translated the requirements into a successful design for the linker. Working with colleagues at Creative Biomolecules, Massachusetts General Hospital and Harvard Medical School, an sFv reactive with digoxin was successfully produced and tested. The report (Huston et al 1988) of this work has been cited more than 2,300 times.

scFvs are readily expressed on the surface of filamentous bacteriophage and have often been the basis for naïve human antibody libraries with potential for rapid selection of desired binders from diverse libraries of many millions. This technology can be used for antibody discovery and humanization, and it has been the foundation of many successful commercial ventures. Jim’s own work included the demonstration of scFv fusion proteins and the first scFv intrabody therapy for the neurodegenerative condition, Huntington’s disease, an approach that is now being investigated in Parkinson’s disease.

The scFv format itself forms the targeting basis of T-cell recruiting agents, bispecific T-cell engagers (BiTEs), and chimeric antigen receptors (CARs), the antigen binding moiety of CAR-T cells. A number of products based on these formats are already licensed for clinical use, while several others are in development. This is an important beginning, but the potential for further applications is great because of the diversity of antibody repertoires and the robust nature of the sFv format.

Jim graduated in Chemistry from the University of Michigan and was awarded his Ph.D. for work on the Fd fragment of IgG and its domains, supervised by Professor Charles Tanford at Duke University. After postdoctoral research at Stanford and Harvard Medical Schools, he joined Creative Biomolecules in Boston in 1983 where he undertook the original work on scFvs. There followed numerous publications and patents relating to engineered antibodies and their applications.

Jim was one of the first people to see the long-term potential of antibody engineering and recognize how broad the applications could be. His lectures on this topic gave a unique experience in that one sometimes seemed to be discovering the meaning of his data simultaneously with him. Those who had the privilege of working with him benefited greatly not only from his generosity, enthusiasm, intellectual rigor and encouragement, but also from his ability to advise wisely or find the appropriate expert. He took the mission of advancing antibody engineering to an international level by serving as the scientific adviser to the Antibody Engineering & Therapeutics meetings for nearly 30 years. Over time, he brought the antibody engineering and therapeutics community together at meetings in San Diego and elsewhere. His insistence that scientific quality and education were the principal criteria for the program resulted in progressive growth and helped to cement a culture encompassing academia and industry. Building on this, he co-founded The Antibody Society in 2007 and was the Founding President and Chairman, remaining a Board Member until his death. He shared the gratification of many that, after a long gestation, antibody engineering is proving so beneficial to human health, with the promise of much more to come.

Jim’s many friends around the world will remember his love of life based on a deep Episcopalian faith, his pride and joy in his family, and the fortitude with which he bore illness over recent years.

The Antibody Society will honor Jim Huston and his many contributions to the field of antibody engineering at our next annual meeting.

Antibody Engineering & Therapeutics, December 2019

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Thank you for joining us at The Antibody Society’s annual Antibody Engineering & Therapeutics meeting held December 9-13, 2019 in San Diego. The meeting was a great opportunity for Society members to connect with industry and academic scientists and executives from around the world, and learn about advances in antibody discovery and development.

As always, The Antibody Society’s members designed the scientific program and acted as session Chairs. The meeting featured sessions on Antibody Libraries, Selection, Screening and Engineering; Bioinformatics and Computational Tools for Antibody Optimization and Engineering; Clinical Data and Lessons from Cancer Immunotherapy; Systems Immunology for Target Discovery; and Targeting Subcellular Trafficking Pathways to Generate Antibody Therapeutics.

AE&T Student/Postdoc Poster Competition

The Society sponsored a poster competition for students and postdocs, with winners receiving complimentary registration, support for travel expenses, and an opportunity to present at the conference. Congratulations to the winners:

Timothy Czajka, University of New York at Albany. Poster title: RIP-Off: An Intrabody-based Strategy to Neutralize Ricin and other Ribosome-Inactivating Protein (RIP) Toxins.

Kamal Joshi, PhD., Genentech. Poster title: Toward Deeper Understanding of Bispecific Antibodies

“Thank you again to the Antibody Society for this recognition and opportunity to speak here.  This is my third time attending the Antibody Engineering and Therapeutics conference and each time I’ve learned more than I could possibly remember and return to the lab with a huge surge of excitement and several new ideas that I’d like to apply to my own project.  I’m honored to be able to present my own research this year alongside so many fascinating talks and to be able to engage with many different experts in the field.  I would also like to thank my advisor, Nicholas Mantis, and Anne Messer for their help with my research and encouragement to attend and apply for this award.” Timothy Czajka

 

“It is an honor to receive this award. I would like to thank the judges for conferring this recognition on my work. I would also like to thank the Antibody Society, of course for funding my trip here but more importantly for organizing these fantastic meetings and providing a solid platform for the exchange of the latest information on antibody research and development benefiting all including early stage career scientists like me. It’s a real treat coming back to this meeting every time. Not only do I learn what is ongoing in the field, this meeting also provides me the opportunity to network with fellow scientists and facilitate connections. So again, thank you to the Antibody Society for this award. Thank you all.” Kamal Joshi, Ph.D.

 

Moments at Antibody Engineering & Therapeutics

 

We look forward to seeing you at AE&T in December 2020!

All Society members receive discounts on registration to Antibody Engineering & Therapeutics (US), as well as registration at many antibody-related meetings.

Most read from mAbs, Nov/Dec 2019

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The Antibody Society is pleased to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue.

All the articles are open access; PDFs can be freely downloaded by following the links below.

Issue 11.8 (Nov/Dec 2019)

Insights into the IgG heavy chain engineering patent landscape as applied to IgG4 antibody development

In this new Perspective, Dumet et al., present the results from their study of the patent landscape of IgG4 Fc engineering, i.e., patents claiming modifications in the heavy chain. Thirty-seven relevant patent families were identified, comprising hundreds of IgG4 Fc variants focusing on removal of residual effector functions (since IgG4s bind to FcγRI and weakly to other FcγRs), half-life enhancement and IgG4 stability. Given the number of expired or soon to expire major patents in those 3 areas, companies developing blocking antibodies now have, or will in the near future, access to free tools to design silenced, half-life extended and stable IgG4 antibodies.

Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

Parola et al. describe an antibody engineering and screening approach where complete variable light (VL) and heavy (VH) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion. By developing an improved HDR donor format that utilizes in situ linearization, they were able to achieve >15-fold improvement of genomic integration, resulting in a screening workflow that only requires a simple plasmid electroporation. This proved suitable for different applications in antibody discovery and engineering. By integrating and screening an immune library obtained from the variable gene repertoire of an immunized mouse, they isolated a diverse panel of >40 unique antigen-binding variants. They also successfully performed affinity maturation by directed evolution screening of an antibody library based on random mutagenesis, leading to the isolation of several clones with affinities in the picomolar range.

DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity

In this new Report,  Sustmann et al. present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one Fc, whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the ‘Fc-load’ on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their ADCC competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.

Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for generating highly pure heterodimers in a single step are needed. Ollier et al. describe two different purification methods that exploit differences in Protein A (PA) or Protein G (PG) avidity between homo- and heterodimers. Differential elution between species was enabled by removing PA or PG binding in one of the Hcs of the bsAb. The PA method allowed the avidity purification of heterodimers based on the VH3 subclass, which naturally binds PA and interferes with separation, by using a combination of IgG3 Fc and a single amino acid change in VH3, N82aS. The PG method relied on a combination of three mutations that completely disrupts PG binding, M428G/N434A in IgG1 Fc and K213V in IgG1 CH1. Both methods achieved a high level of heterodimer purity as single-step techniques without Hc HD (93–98%). Since PA and PG have overlapping binding sites with the neonatal Fc receptor (FcRn), they investigated the effects of the engineering both in vitro and in vivo. Mild to moderate differences in FcRn binding and Fc thermal stability were observed, but these did not significantly change the serum half-lives of engineered control antibodies and heterodimers. The methods are conceptually compatible with various Hc HD platforms such as BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor), in which the PA method has already been successfully implemented.