Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering

Schoot, Johan M S van der; Fennemann, Felix L.; Valente, Michael; Dölen, Yusuf; Hagemans, Iris M; Becker, Anouk M. D.; Gall, Camille Le; Dalen, Duco van; Cevirgel, Alper; Bruggen, Jaco A. C. van; Engelfriet, Melanie; Čaval, Tomislav; Bentlage, Arthur E. H.; Fransen, Marieke F.; Nederend, Maaike; Leusen, Jeanette H.W.; Heck, Albert J. R.; Vidarsson, Gestur; Figdor, Carl G.; Verdoes, Martijn; Scheeren, Ferenc A.

doi:10.1101/551382

Cited by 6 publications

(12 citation statements)

References 44 publications

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“…In this study, we present for the first time a strategy to generate dual-tagged Fab' fragments bearing two orthogonal site-specific modification tags (DTFab'), which can readily be utilized for the chemoenzymatic conjugation with two distinct cargos. Extending on our previous work 23 , we demonstrate the successful engineering of a hybridoma secreting mIgG1 antibodies (anti-CD20 WT) to a stable daughter cell line producing Fab' fragments carrying an eSrt2A-9 (LAETGG) motif on its HC, and an eSrt4S-9 (LPESGG) motif on its κ LC (anti-CD20 DTFab'). This method enables robust biorthogonal engineering of virtually any antibody-secreting hybridoma to reproducibly produce high yields of modified DTFab' molecules.…”

Section: Discussionmentioning

confidence: 68%

“…Transfection. Nucleofection of the HDR template and CRISPR-Cas9 vectors was performed as described previously 23 . Briefly, hybridoma cells were assessed for viability, centrifuged (90 g, 5 min), resuspended in phosphate buffered saline (PBS)/1% FBS, and centrifuged again (90 g, 5 min).…”

Section: Methodsmentioning

confidence: 99%

“…To produce Fab' fragments suitable for dual site-specific labelling we sought to alter the immunoglobulin domain within the genome of an anti-hCD20-producing hybridoma cell line, which expresses a mAb of the mIgG1 isotype with a κ LC (Cκ). We hypothesized this could be achieved by adapting our recently developed CRISPR/HDR approach 23 , in which we genetically engineered the rat IgG2a IgH locus, for modification of the murine IgH and IgK loci. We started with the genetic modification of the HC ( figure 1A and B).…”

Section: Generation Of a Genetically Engineered Cell Line Secreting Fmentioning

confidence: 99%

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Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

Gall

Schoot

Ramos-Tomillero

et al. 2020

Preprint

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I.AbstractFunctionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theragnostics, and antibody-drug conjugates (ADC). Antibody functionalization is classically achieved by coupling payloads onto lysine or cysteine residues. However, such stochastic strategies typically lead to heterogenous products, bearing a varying number of payloads. This affects bioconjugate efficacy and stability, as well as its in vivo biodistribution, and therapeutic index, while potentially obstructing the binding sites and leading to off-target toxicity. In addition, therapeutic and theragnostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labelling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogenous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab’ fragments bearing two distinct site-specific labelling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab’ molecule (DTFab’), which can be easily isolated in high yields. To demonstrate feasibility, we functionalized the DTFab’ with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theragnostics, and next-generation ADCs.

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Section: Discussionmentioning

confidence: 68%

Section: Methodsmentioning

confidence: 99%

Section: Generation Of a Genetically Engineered Cell Line Secreting Fmentioning

confidence: 99%

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Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

Gall

Schoot

Ramos-Tomillero

et al. 2020

Preprint

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“…Here hybridomas were designed to remove endogenous copies of heavy and light chains, and full-length light and heavy chains, under the native IgH promoter, were expressed as a single transcript. Similar hybridoma CRISPR/HDR reprogramming has expanded upon this idea to generate on-demand class switching of recombinant monoclonal antibodies 271 .…”

Section: Efficient Modification Of Immune Receptor Activity In Vitro ...mentioning

confidence: 99%

Augmenting adaptive immunity: progress and challenges in the quantitative engineering and analysis of adaptive immune receptor repertoires

Brown

Snapkov

Akbar

et al. 2019

Mol. Syst. Des. Eng.

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The adaptive immune system is a natural diagnostic and therapeutic. It recognizes threats earlier than clinical symptoms manifest and neutralizes antigen with exquisite specificity. Recognition specificity and broad reactivity is enabled via adaptive B-and T-cell receptors: the immune receptor repertoire. The human immune system, however, is not omnipotent. Our natural defense system sometimes loses the battle to parasites and microbes and even turns against us in the case of cancer and (autoimmune) inflammatory disease. A long-standing dream of immunoengineers has been, therefore, to mechanistically understand how the immune system "sees", "reacts" and "remembers" (auto)antigens. Only very recently, experimental and computational methods have achieved sufficient quantitative resolution to start querying and engineering adaptive immunity with great precision. In specific, these innovations have been applied with the greatest fervency and success in immunotherapy, autoimmunity and vaccine design. The work here highlights advances, challenges and future directions of quantitative approaches which seek to advance the fundamental understanding of immunological phenomena, and reverse engineer the immune system to produce auspicious biopharmaceutical drugs and immunodiagnostics. Our review indicates that the merger of fundamental immunology, computational immunology and (digital) biotechnology minimizes black box engineering, thereby advancing both immunological knowledge and as well immunoengineering methodologies. Introduction 3Advancing immunology through engineering innovations 3Adaptive immune receptors are natural diagnostics and therapeutics 3Engineering the vast immune receptor sequence space requires quantitative approaches 4Current approaches for immune repertoire analysis and immunoengineering 4Computational immunology and immunoinformatics of adaptive immunity 4 B-and T-cell pattern mining using machine and deep learning 6Mathematical modeling of immune receptor recognition 8Computational modeling of immune receptor 3D structure 9Computational modeling of antibody-epitope interaction 10Genomic sequencing of immune repertoires 12Identifying candidate TCRs or antibodies via high-throughput library screens 13Proteomic sequencing and serological profiling of antibody repertoires 14 Future directions for quantitative immunoengineering and immune receptor analysis 15Setting targets on public and private immune receptors 15Efficient modification of immune receptor activity in vitro and in vivo 16De novo design of immune receptor sequences 18Closing the data gap between immune receptor sequence and cognate epitope for immune receptor and epitope engineering 21Challenges in machine learning analysis on immune receptor repertoires 21Relating immune receptor antigen specificity to cellular transcriptomic profile 24 Conclusion 25 Conflicts of Interest 25 Focus Boxes 26Focus Box 1: Brief summary of deep learning and its architectures. 26Focus Box 2: Recognition holes in the immune repertoire 26 Notes and references 30mo...

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“…Here, we report a widely applicable strategy to introduce two orthogonal siteselective labeling tags on a Fab′ fragment by capitalizing on our recently reported Clustered Regularly Interspaced Short Palindromic Repeats/Homology Directed Repair (CRISPR/ HDR) hybridoma genomic engineering approach. 24 In this work, we expand the genomic engineering toolbox to enable modification of the HC and LC loci of the mouse IgG1 (mIgG1) hybridoma, available for a plethora of targets. With this, dual-tagged Fab′ (DTFab′) are generated equipped with two distinct sortase A recognition motifs (sortags) on the HC and LC, each orthogonally recognized by a specific variant of the "evolved sortase A" (eSrtA) enzyme (eSrt2A-9 or eSrt4S-9).…”

Section: ■ Introductionmentioning

confidence: 99%

Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering

et al. 2021

Self Cite

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Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theranostics, and antibody–drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab′ fragments bearing two distinct site-specific labeling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab′ molecule (DTFab′), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab′ with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theranostics, and next-generation ADCs.

show abstract

Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering

Cited by 6 publications

References 44 publications

Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

Augmenting adaptive immunity: progress and challenges in the quantitative engineering and analysis of adaptive immune receptor repertoires

Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering

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