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You are here: Home / Archives for next-generation sequencing

Antibody Discovery in the Cloud: Using NGS to expand the universe of selectable antibodies

January 13, 2021 by The Antibody Society

Registration for this free event is now open!

January 21 2021, 9am PST/12 ET/6pm CET

Speakers: Drs. Andrew Bradbury and M. Frank Erasmus (Specifica)

Antibody Discovery in the Cloud: Using NGS to expand the universe of selectable antibodies

The Specifica Generation 3 platform is able to generate 500-5000 different antibody clonotypes against targets of interest, with over 80% of selected antibodies having no measurable biophysical liabilities and 20% having subnanomolar affinities. The most common approach to selecting antibodies from display technologies involves low-throughput random colony screening. However, this missed many potential therapeutic leads, particularly when diversity is high. Specifica uses next generation sequencing (NGS) to build its libraries as well as characterize selection outputs. In order to fully exploit the universe of selectable antibodies, Specifica has developed a cloud-based software platform, designed exclusively for antibody engineers and bioinformaticians, to enable a streamlined identification of leads with broad epitope coverage. Application of this to selection outputs has increased the number of clonotype leads by five to ten fold over random colony screening, significantly expanding the explorable paratope space.

Click here to register!

Filed Under: Antibody discovery Tagged With: antibody discovery, next-generation sequencing

New publication on OGRDB, the Open Germline Receptor Database, is available!

October 4, 2019 by Janice Reichert

Congratulations to The Antibody Society’s Adaptive Immune Repertoire Community on the publication of their newest paper, OGRDB: a reference database of inferred immune receptor genes, in Nucleic Acid Research. The paper is open access and can be freely downloaded here.

High-throughput sequencing of the adaptive immune receptor repertoire (AIRR-seq) is providing unprecedented insights into the immune response to disease and into the development of immune disorders. The accurate interpretation of AIRR-seq data depends on the existence of comprehensive germline gene reference sets. Current sets are known to be incomplete and unrepresentative of the degree of polymorphism and diversity in human and animal populations. A key issue is the complexity of the genomic regions in which they lie, which, because of the presence of multiple repeats, insertions and deletions, have not proved tractable with short-read whole genome sequencing. Recently, tools and methods for inferring such gene sequences from AIRR-seq datasets have become available, and a community approach has been developed for the expert review and publication of such inferences. Here, we present OGRDB, the Open Germline Receptor Database (https://ogrdb.airr-community.org), a public resource for the submission, review and publication of previously unknown receptor germline sequences together with supporting evidence.

Filed Under: AIRR Community, New articles Tagged With: adaptive immune receptor repertoire, Adaptive Immune Receptor Repertoire Community, next-generation sequencing

Most read from mAbs, October 2019

September 17, 2019 by Janice Reichert

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 11.7 (Oct 2019)

Glycoform-resolved FcɣRIIIa affinity chromatography–mass spectrometry

Lippold et al. describe a method to determination the impact of individual antibody glycoforms on FcɣRIIIa affinity, and consequently antibody-dependent cell-mediated cytotoxicity (ADCC) without performing high purity glycoengineering. They hyphenated FcɣRIIIa affinity chromatography to mass spectrometry, which allowed direct affinity comparison of glycoforms of intact monoclonal antibodies. The approach enabled reproduction and refinement of known glycosylation effects, and insights on afucosylation pairing as well as on low-abundant, unstudied glycoforms. Their method greatly improves the understanding of individual glycoform structure–function relationships, and it is highly relevant for assessing Fc-glycosylation critical quality attributes related to ADCC.

Looking for therapeutic antibodies in next-generation sequencing repositories

It is now possible to query the great diversity of natural antibody repertoires using next-generation sequencing (NGS) using methods capable of producing millions of sequences in a single experiment. In this new article, Krawczyk et al. compare clinical-stage therapeutic antibodies to the ~1b sequences from 60 independent sequencing studies in the Observed Antibody Space database, which includes antibody sequences from NGS analysis of immunoglobulin gene repertoires. Of 242 post-Phase 1 antibodies, they found 16 with sequence identity matches of 95% or better for both heavy and light chains. There were also 54 perfect matches to therapeutic CDR-H3 regions in the NGS outputs, suggesting a nontrivial amount of convergence between naturally observed sequences and those developed artificially. The authors discuss the potential implications for both the legal protection of commercial antibodies and the discovery of antibody therapeutics.

Elucidating heavy/light chain pairing preferences to facilitate the assembly of bispecific IgG in single cells

Joshi et al. report that a high yield (>65%) of bispecific IgG1 (BsIgG1) without Fab engineering can be a surprisingly common occurrence, i.e., observed for 33 of the 99 different antibody pairs evaluated. Installing charge mutations at both CH1/CL interfaces was sufficient for near quantitative yield (>90%) of BsIgG1 for most (9 of 11) antibody pairs tested with this inherent cognate chain pairing preference. Mechanistically, they demonstrate that a strong cognate pairing preference in one Fab arm can be sufficient for high BsIgG1 yield. These observed chain pairing preferences are apparently driven by variable domain sequences and can result from a few specific residues in the complementarity-determining region (CDR) L3 and H3. Transfer of these CDR residues into other antibodies increased BsIgG1 yield in most cases. Mutational analysis revealed that the disulfide bond between heavy and light chains did not affect the yield of BsIgG1. This study provides some mechanistic understanding of factors contributing to antibody heavy/light chain pairing preference and subsequently contributes to the efficient production of BsIgG in single host cells.

Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life

The neonatal Fc receptor (FcRn) promotes antibody recycling through rescue from normal lysosomal degradation. The binding interaction is pH-dependent with high affinity at low pH, but not under physiological pH conditions. In this new article, Mackness et al. describe how they combined rational design and saturation mutagenesis to generate novel antibody variants with prolonged half-life and acceptable development profiles. First, a panel of saturation point mutations was created at 11 key FcRn-interacting sites on the Fc region of an antibody. Multiple variants with slower FcRn dissociation kinetics than the wildtype (WT) antibody at pH 6.0 were successfully identified. The mutations were further combined and characterized for pH-dependent FcRn binding properties, thermal stability and the FcγRIIIa and rheumatoid factor binding. The most promising variants, YD (M252Y/T256D), DQ (T256D/T307Q) and DW (T256D/T307W), exhibited significantly improved binding to FcRn at pH 6.0 and retained similar binding properties as WT at pH 7.4. The pharmacokinetics in human FcRn transgenic mice and cynomolgus monkeys demonstrated that these properties translated to significantly prolonged plasma elimination half-life compared to the WT control. The novel variants exhibited thermal stability and binding to FcγRIIIa in the range comparable to clinically validated YTE and LS variants, and showed no enhanced binding to rheumatoid factor compared to the WT control. These engineered Fc mutants are promising new variants that are widely applicable to therapeutic antibodies, to extend their circulation half-life with obvious benefits of increased efficacy, and reduced dose and administration frequency.

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Filed Under: Antibody discovery, Antibody therapeutic, New articles Tagged With: antibody engineering, antibody therapeutics, bispecific, glycosylation, next-generation sequencing

The Adaptive Immune Receptor Repertoire Community is now part of The Antibody Society!

June 14, 2018 by The Antibody Society

We are pleased and proud to announce the incorporation of the Adaptive Immune Receptor Repertoire (AIRR) Community into the Society.

The AIRR Community is a research-driven group that is organizing and coordinating stakeholders in the use of next-generation sequencing (NGS) technologies to study antibody (Ab)/B-cell and T-cell receptor (TcR) repertoires. Recent advances in sequencing technology have made it possible to sample the immune repertoire in exquisite detail. AIRR sequencing has enormous promise for understanding the dynamics of the immune repertoire in vaccinology, infectious disease, autoimmunity, and cancer biology, but also poses substantial challenges. The AIRR Community was established to meet these challenges. The AIRR Community and its associated meetings and workshops are designed to develop standards and recommendations for: 1) obtaining, analyzing, curating and comparing/sharing NGS AIRR datasets; 2) using and validating tools for analyzing AIRR data; 3) relating AIRR NGS datasets to other “big data” sets, such as microarray, flow cytometric, and MiSeq gene-expression data; and 4) legal and ethical issues involving the use and sharing of AIRR data sets derived from human sources. The proceedings of the workshops, including the recommendations and action plans, will be published to benefit the larger scientific community.

To learn more about the AIRR Community and its work, please explore the items listed under the ‘AIRR Community’ tab above.

Filed Under: AIRR Community, Bioinformatics Tagged With: Adaptive Immune Receptor Repertoire Community, next-generation sequencing

Novel antibody display, selection and screening technologies

October 23, 2017 by The Antibody Society

The Antibody Society invites you to attend its annual meeting, Antibody Engineering & Therapeutics, on December 11-15, 2017 at the Manchester Grand Hyatt, San Diego, CA!

The session on “Novel antibody display, selection and screening technologies”, chaired by Andrew Bradbury, M.D., Ph.D. Research Scientist and Group Leader, Los Alamos National Laboratories; Chief Scientific Officer, Specifica, focusses on the new technologies expected to advance antibody library generation and selection in the future. Many of these reflect the rapidly growing role of next generation sequencing (NGS) in all aspects of in vitro antibody generation. Dr. Bradbury will discuss how NGS has enabled more informed discussions on antibody library sizes, and how traditional selection from antibody libraries does not address the full depth of possible positive antibodies. Tim Whitehead (Michigan State University) will discuss the power of NGS in protein engineering to analyze the outcomes of different selective pressures on antibody stability, affinity and function, and to use this information in antibody discovery programs, while Brandon DeKosky (The University of Kansas) will describe how the combination of cloning natural paired antibody responses to viruses with yeast display vectors provides insights into neutralizing HIV and Ebola responses. In addition to NGS, Benjamin Hackel (University of Minnesota) will describe the engineering of novel alternative novel yeast display vectors as applied to the development of novel small non-antibody scaffolds. James Wells (UCSF) will describe an innovative use of novel proteomic technologies involving phage display to both understand how cancer cells remodel their membrane proteomes, as well as to generate recombinant antibodies against them. Once potential therapeutic antibody leads have been identified, they need to be further developed before they can be used in the clinic. This involves understanding and overcoming fundamental challenges related to the design and selection of antibodies with high affinity, specificity, stability and solubility, a topic that will addressed by Peter Tessier (University of Michigan).

Interested in attending the meeting? Learn more from this PDF, which includes all session summaries written by the chairpersons.

Society members can save 15% on the registration fee! Not a member? Please join!

Membership is free for students and employees of the Society’s corporate sponsors.

 

Filed Under: Antibody discovery, Meetings Tagged With: antibody selection, antibody therapeutics, next-generation sequencing

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The Adaptive Immune Receptor Repertoire Community is a research-driven group organizing around the use of high-throughput sequencing technologies to study antibody/B-cell and T-cell receptor repertoires.

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