The making of bispecific antibodies

Brinkmann, Ulrich; Kontermann, Roland E.

doi:10.1080/19420862.2016.1268307

Cited by 697 publications

(766 citation statements)

References 293 publications

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“…PD-L1xEGFR is constructed in a so-called bispecific taFv-Fc format, 26 a class of symmetric tetravalent bispecific molecules that can be equipped with an Fc domain of choice. Our data demonstrate that the capacity of PD-L1xEGFR to simultaneously bind to PD-L1 and EGFR results in an significantly enhanced overall binding strength (avidity) for PD-L1 + /EGFR + cancer cells.…”

Section: Discussionmentioning

confidence: 99%

A novel bispecific antibody for EGFR-directed blockade of the PD-1/PD-L1 immune checkpoint

et al. 2018

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PD-L1-blocking antibodies produce significant clinical benefit in selected cancer patients by reactivating functionally-impaired antigen-experienced anticancer T cells. However, the efficacy of current PD-L1-blocking antibodies is potentially reduced by ‘on-target/off-tumor’ binding to PD-L1 widely expressed on normal cells. This lack of tumor selectivity may induce a generalized activation of all antigen-experienced T cells which may explain the frequent occurrence of autoimmune-related adverse events during and after treatment.To address these issues, we constructed a bispecific antibody (bsAb), designated PD-L1xEGFR, to direct PD-L1-blockade to EGFR-expressing cancer cells and to more selectively reactivate anticancer T cells. Indeed, the IC50 of PD-L1xEGFR for blocking PD-L1 on EGFR+ cancer cells was ∼140 fold lower compared to that of the analogous PD-L1-blocking bsAb PD-L1xMock with irrelevant target antigen specificity. Importantly, activation status, IFN-γ production, and oncolytic activity of anti-CD3xanti-EpCAM-redirected T cells was enhanced when cocultured with EGFR-expressing carcinoma cells. Similarly, the capacity of PD-L1xEGFR to promote proliferation and IFN-γ production by CMVpp65-directed CD8+ effector T cells was enhanced when cocultured with EGFR-expressing CMVpp65-transfected cancer cells. In contrast, the clinically-used PD-L1-blocking antibody MEDI4736 (durvalumab) promoted T cell activation indiscriminate of EGFR expression on cancer cells. Additionally, in mice xenografted with EGFR-expressing cancer cells 111In-PD-L1xEGFR showed a significantly higher tumor uptake compared to 111In-PD-L1xMock. In conclusion, PD-L1xEGFR blocks the PD-1/PD-L1 immune checkpoint in an EGFR-directed manner, thereby promoting the selective reactivation of anticancer T cells. This novel targeted approach may be useful to enhance efficacy and safety of PD-1/PD-L1 checkpoint blockade in EGFR-overexpressing malignancies.

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Section: Discussionmentioning

confidence: 99%

A novel bispecific antibody for EGFR-directed blockade of the PD-1/PD-L1 immune checkpoint

et al. 2018

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“…1-3 As of 2017, ~ 60 bispecific antibodies were in clinical studies, 4 and two have received approval from the US Food and Drug Administration: blinatumomab (Blincyto®; Amgen/Micromet) and emicizumab-kxwh (Hemlibra® Chugai/Genentech). The more than 100 bispecific antibody formats that have been reported in the literature 5,6 can be divided into three categories. The first group comprises bispecific antibodies possess two antigen binding sites connected by a linker, but no Fc region, as exemplified by the bispecific T-cell engager 7-10 format employed for Blincyto.…”

Section: Introductionmentioning

confidence: 99%

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

Wang¹,

Vemulapalli²,

Cao³

et al. 2018

mAbs

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Immunoglobulin G–like bispecific antibodies with asymmetric architecture are among the most widely used bispecific antibody formats for diagnostic and therapeutic applications. The primary technical challenge for this format is how to achieve correctly paired assembly of four unique polypeptide chains. Advances in protein engineering and process development are being used to overcome these challenges and are driving a corresponding demand for sensitive analytical tools to monitor and control mispaired species. Here, we report a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)–based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)–based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.

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“…9,11 This diversity is the result of a large number of bispecific “building blocks” that include antigen-binding fragments (Fabs), single-chain variable fragments (scFvs), and receptor ligands. Bispecific antibody formats can be broadly classified into three groups.…”

Section: Introductionmentioning

confidence: 99%

Characterization and analysis of scFv-IgG bispecific antibody size variants

Cao¹,

Wang²,

Chung³

et al. 2018

mAbs

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Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.

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The making of bispecific antibodies

Cited by 697 publications

References 293 publications

A novel bispecific antibody for EGFR-directed blockade of the PD-1/PD-L1 immune checkpoint

A novel bispecific antibody for EGFR-directed blockade of the PD-1/PD-L1 immune checkpoint

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

Characterization and analysis of scFv-IgG bispecific antibody size variants

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