Bispecific antibodies: a mechanistic review of the pipeline

Labrijn, Aran F.; Janmaat, Maarten L.; Reichert, Janice M.; Parren, Paul W.H.I.

doi:10.1038/s41573-019-0028-1

Cited by 931 publications

(873 citation statements)

References 188 publications

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“…Alternatively, a recently developed SampleStream device (17) can also afford rapid ion sampling through a process similar to asymmetric flow field flow fractionation. However, highthroughput ESI screening of antibody mixtures (>140 kDa) from complex matrices at the intact level, an unmet need in biopharmaceutical drug discovery, has not been reported.The major impact of antibody therapeutics in treating diseases, such as cancer, has led to the rise of many new format large molecules (18), such as bispecific antibodies (19,20), which enables the binding of multiple antigens that are useful for many therapeutic applications. Here, we report a robust method for high-throughput screening to characterize bispecific immunoglobulin G (IgG) (BsIgG) generated by coexpressing two different light and heavy chains in a single host cell (21,22).…”

mentioning

confidence: 99%

“…The major impact of antibody therapeutics in treating diseases, such as cancer, has led to the rise of many new format large molecules (18), such as bispecific antibodies (19,20), which enables the binding of multiple antigens that are useful for many therapeutic applications. Here, we report a robust method for high-throughput screening to characterize bispecific immunoglobulin G (IgG) (BsIgG) generated by coexpressing two different light and heavy chains in a single host cell (21,22).…”

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confidence: 99%

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High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

Sawyer¹,

Srikumar²,

Carver³

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFiretime-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.antibody screening | high throughput | intact protein mass spectrometry | electrospray U nambiguous characterization of analytes from complex matrices with high content information in a label-free format continues to expand the application of mass spectrometry (MS) in drug discovery (1-3). With the need for high accuracy, sensitivity, and selectivity, the rapidly improving MS instrumentations are emerging at the forefront of analyzing analytes with limited alternative assays for biologics developability (4-6), biotransformation, and high-throughput screening (HTS). For ultra-high-throughput screening, matrix-assisted laser desorption/ionization (MALDI) is amenable to speeds >100,000 samples per day (7-11). Through incorporating self-assembled monolayers for MALDI-time-of-flight (TOF) (SAMDI) technology (12-14), it is also possible to infer small molecule noncovalent hit identification from MALDI-MS detection under native conditions. While such MALDI-MS approaches have the capability for screening small molecule and peptide analytes, larger molecular weight proteins suffer from limited mass resolution and quantitative challenges. Alternatively, electrospray ionization MS (ESI-MS) can provide isotopic resolution for molecules as large as antibodies (15). Although modern mass spectrometers can scan as rapidly as tens of microseconds, highthroughput antibody analyses using ESI-MS is challenged by the relatively low ion sampling rate from chromatographic elution coupled to MS. For rapid sampling, Agilent RapidFire MS (RF-MS) utilizes a rapid trap and elute strategy to enable desalting and ion sampling coupled to a MS ion source. RF-MS has been shown to afford HTS triage for small molecules and proteins <30 kDa from biochemical buffers as fast as 15 s as opposed to minutes observed with conventional chromatography (16). Altern...

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confidence: 99%

mentioning

confidence: 99%

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

Sawyer¹,

Srikumar²,

Carver³

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…The approach described in this article should be generally applicable to members of the TNF superfamily. Alternative approaches may involve bispecific antibodies 93,94 , the modular use of small protein domains 85,95 or of chemically-modified bicyclic peptides 96 . Members of the TNF receptor superfamily are particularly suited for cooperative activation strategies in view of their homotrimeric structure and clustering-driven activation properties [97][98][99] .…”

Section: Discussionmentioning

confidence: 99%

An engineered 4-1BBL fusion protein with “activity-on-demand”

Mock

Stringhini

Villa

et al. 2020

Preprint

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ABSTRACTEngineered cytokines are gaining importance for cancer therapy but those products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein (termed F8-4-1BBL), which does not exhibit cytokine activity in solution but regains biological activity upon antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively-spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent anti-tumor activity in various mouse models of cancer, without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity-on-demand” properties at the site of disease upon antigen binding and reduce toxicity to normal tissues.

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“…creative methods have been developed to achieve this property. [1,2] In many of the early proof-of-concept studies, BsAbs were generated by chemically crosslinking two different IgGs or Fabs using bifunctional crosslinking reagents that react specifically with the thiol and primary amine groups of the antibody. [3,4] Although several of BsAbs prepared in this way have advanced to clinical trials, [5][6][7] large majority of BsAbs currently being developed are generated with recombinant antibody engineering.…”

Section: Doi: 101002/advs201900973mentioning

confidence: 99%

DNA‐Mediated Assembly of Multispecific Antibodies for T Cell Engaging and Tumor Killing

Pan

Cao²,

Run³

et al. 2019

Advanced Science

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Targeting T‐cells against cancer cells is a direct means of treating cancer, and has already shown great responses in clinical treatment of B‐cell malignancies. A simple way to redirect T‐cells to cancer cells is by using multispecific antibody (MsAb) that contains different arms for specifically “grabbing” the T‐cells and cancer cells; as such, the T‐cells are activated upon target engagement and the killing begins. Here, a nucleic acid mediated protein–protein assembly (NAPPA) approach is implemented to construct a MsAb for T‐cell engaging and tumor killing. Anti ‐CD19 and ‐CD3 single‐chain variable fragments (scFvs) are conjugated to different l‐DNAs with sequences that form the Holliday junction, thus allowing spontaneous assembly of homogeneous protein–DNA oligomers containing two anti‐CD19 and one anti‐CD3 scFvs. The new MsAb shows strong efficacy in inducing Raji tumor cell cytotoxicity in the presence of T‐cells with EC50 ≈ 0.2 × 10−9 m; it also suppresses tumor growth in a Raji xenograft mouse model. The data indicates that MsAbs assembled from protein–DNA conjugates are effective macromolecules for directing T‐cells for tumor killing. The modular nature of the NAPPA platform allows rapid generation of complex MsAbs from simple antibody fragments, while offering a general solution for preparing antibodies with high‐order specificity.

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Bispecific antibodies: a mechanistic review of the pipeline

Cited by 931 publications

References 188 publications

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry

An engineered 4-1BBL fusion protein with “activity-on-demand”

DNA‐Mediated Assembly of Multispecific Antibodies for T Cell Engaging and Tumor Killing

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