Coupling brain perfusion screens and next generation sequencing to identify blood–brain barrier binding antibodies

Stutz, Charles C.; Georgieva, Julia V.; Shusta, Eric V.

doi:10.1002/aic.16360

Cited by 11 publications

(9 citation statements)

References 32 publications

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“…10 However, unlike genomic and proteomic approaches in which the BBB target receptor is known, phenotypic screening requires downstream target receptor identification. 12 To date, phenotypic screening of large libraries in vivo 13,14 and in vitro 15,16 for new antibody-RMT pairs has shown limited success, with only a handful of new BBB targeting antibodies isolated. In vivo screening challenges include the finding that phage antibody libraries are plagued by high background recoveries masking relevant clones, 16 while antibodies identified from in vitro biopanning often do no not cross-react with in vivo antigens, due to potential alteration of protein expression profiles in culture.…”

Section: Introductionmentioning

confidence: 99%

“…12 To date, phenotypic screening of large libraries in vivo 13,14 and in vitro 15,16 for new antibody-RMT pairs has shown limited success, with only a handful of new BBB targeting antibodies isolated. In vivo screening challenges include the finding that phage antibody libraries are plagued by high background recoveries masking relevant clones, 16 while antibodies identified from in vitro biopanning often do no not cross-react with in vivo antigens, due to potential alteration of protein expression profiles in culture. Further, human in vitro BBB models based on primary or immortalized BMECs are inherently leaky, limiting the effectiveness of functional transcytosis screens of antibody libraries.…”

Section: Introductionmentioning

confidence: 99%

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Antibody screening using a human iPSC‐based blood‐brain barrier model identifies antibodies that accumulate in the CNS

et al. 2020

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Drug delivery across the blood‐brain barrier (BBB) remains a significant obstacle for the development of neurological disease therapies. The low penetration of blood‐borne therapeutics into the brain can oftentimes be attributed to the restrictive nature of the brain microvascular endothelial cells (BMECs) that comprise the BBB. One strategy beginning to be successfully leveraged is the use of endogenous receptor‐mediated transcytosis (RMT) systems as a means to shuttle a targeted therapeutic into the brain. Limitations of known RMT targets and their cognate targeting reagents include brain specificity, brain uptake levels, and off‐target effects, driving the search for new and potentially improved brain targeting reagent‐RMT pairs. To this end, we deployed human‐induced pluripotent stem cell (iPSC)‐derived BMEC‐like cells as a model BBB substrate on which to mine for new RMT‐targeting antibody pairs. A nonimmune, human single‐chain variable fragment (scFv) phage display library was screened for binding, internalization, and transcytosis across iPSC‐derived BMECs. Lead candidates exhibited binding and internalization into BMECs as well as binding to both human and mouse BBB in brain tissue sections. Antibodies targeted the murine BBB after intravenous administration with one particular clone, 46.1‐scFv, exhibiting a 26‐fold increase in brain accumulation (8.1 nM). Moreover, clone 46.1‐scFv was found to associate with postvascular, parenchymal cells, indicating its successful receptor‐mediated transport across the BBB. Such a new BBB targeting ligand could enhance the transport of therapeutic molecules into the brain.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Antibody screening using a human iPSC‐based blood‐brain barrier model identifies antibodies that accumulate in the CNS

et al. 2020

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“…In recent years, a variety of methods using NGS for antibody discovery have been developed. Most of the methods exclusively explore CDR-H3 repertoires 55 , 57 – 59 , 61 , 62 . The identified potential binders in NGS data are discovered using naïve approaches, including enrichment ranking or interrogation of antibody databases 55 – 62 .…”

Section: Discussionmentioning

confidence: 99%

“…It also implies that the combination of CDR-L3 and CDR-H3 offers higher diversities than CDR-H3 alone (Fig. S2D ), with increased numbers of potential binders when compared to the methods that explore CDR-H3 repertoires exclusively 55 , 57 – 59 , 61 , 62 .…”

Section: Discussionmentioning

confidence: 99%

CellectSeq: In silico discovery of antibodies targeting integral membrane proteins combining in situ selections and next-generation sequencing

et al. 2021

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Synthetic antibody (Ab) technologies are efficient and cost-effective platforms for the generation of monoclonal Abs against human antigens. Yet, they typically depend on purified proteins, which exclude integral membrane proteins that require the lipid bilayers to support their native structure and function. Here, we present an Ab discovery strategy, termed CellectSeq, for targeting integral membrane proteins on native cells in complex environment. As proof of concept, we targeted three transmembrane proteins linked to cancer, tetraspanin CD151, carbonic anhydrase 9, and integrin-α11. First, we performed in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders. Then, we designed next-generation sequencing procedures to explore Ab diversities and abundances. Finally, we developed motif-based scoring and sequencing error-filtering algorithms for the comprehensive interrogation of next-generation sequencing pools to identify Abs with high diversities and specificities, even at extremely low abundances, which are very difficult to identify using manual sampling or sequence abundances.

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“…NGS has previously been used for discovery of antibodies from phage display libraries (Ravn et al, 2010; Zhang et al, 2011; Ravn et al, 2013; Lopez et al, 2017; Yang et al, 2017; Stutz et al, 2018; Barreto et al, 2019). These studies have shown that compared with traditional screening, NGS enables discovery of more clones (Ravn et al, 2010; Ravn et al, 2013; Yang et al, 2017; Stutz et al, 2018), clones with higher affinity (Barreto et al, 2019), or clones binding functionally more interesting epitopes (Lopez et al, 2017; Stutz et al, 2018; Barreto et al, 2019). In many of these studies, the most enriched antibody sequences, against one or several proteins, although sometimes in a complex context (Zhang et al, 2011), have been recovered and used for downstream analysis.…”

Section: Discussionmentioning

confidence: 99%

Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

et al. 2019

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Phage display technology is a common approach for discovery of therapeutic antibodies. Drug candidates are typically isolated in two steps: First, a pool of antibodies is enriched through consecutive rounds of selection on a target antigen, and then individual clones are characterized in a screening procedure. When whole cells are used as targets, as in phenotypic discovery, the output phage pool typically contains thousands of antibodies, binding, in theory, hundreds of different cell surface receptors. Clonal expansion throughout the phage display enrichment process is affected by multiple factors resulting in extremely complex output phage pools where a few antibodies are highly abundant and the majority is very rare. This is a huge challenge in the screening where only a fraction of the antibodies can be tested using a conventional binding analysis, identifying mainly the most abundant clones typically binding only one or a few targets. As the expected number of antibodies and specificities in the pool is much higher, complementing methods, to reach deeper into the pool, are required, called deep mining methods. In this study, four deep mining methods were evaluated: 1) isolation of rare sub-pools of specific antibodies through selection on recombinant proteins predicted to be expressed on the target cells, 2) isolation of a sub-pool enriched for antibodies of unknown specificities through depletion of the primary phage pool on recombinant proteins corresponding to receptors known to generate many binders, 3) isolation of a sub-pool enriched for antibodies through selection on cells blocked with antibodies dominating the primary phage pool, and 4) next-generation sequencing-based analysis of isolated antibody pools followed by antibody gene synthesis and production of rare but enriched clones. We demonstrate that antibodies binding new targets and epitopes, not discovered through screening alone, can be discovered using described deep mining methods. Overall, we demonstrate the complexity of phage pools generated through selection on cells and show that a combination of conventional screening and deep mining methods are needed to fully utilize such pools. Deep mining will be important in future phenotypic antibody drug discovery efforts to increase the diversity of identified antibodies and targets.

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Coupling brain perfusion screens and next generation sequencing to identify blood–brain barrier binding antibodies

Cited by 11 publications

References 32 publications

Antibody screening using a human iPSC‐based blood‐brain barrier model identifies antibodies that accumulate in the CNS

Antibody screening using a human iPSC‐based blood‐brain barrier model identifies antibodies that accumulate in the CNS

CellectSeq: In silico discovery of antibodies targeting integral membrane proteins combining in situ selections and next-generation sequencing

Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

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