Dual targeting strategies with bispecific antibodies

Kontermann, Roland E.

doi:10.4161/mabs.4.2.19000

Cited by 397 publications

(309 citation statements)

References 149 publications

(162 reference statements)

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“…14 To overcome the light chain exchange problem, which often happens when the two arms of a bispecific antibody are co-expressed in one cell line, techniques such as common light chain, 13 Crossmab 13 and additional knob-into-hole between heavy and light chains of both arms 15 have been introduced. We used a different approach to manufacture our bispecific anti-HER2 antibody: expressing the two arms (half antibodies), namely MBS301-hole and MBS301-knob, in two separate fucose-knockout host CHO cell lines, followed by assembling them together to produce the integrated MBS301.…”

Section: Resultsmentioning

confidence: 99%

“…13 So far, two bispecific antibody therapeutics, blinatumomab by Amgen and emicizumab by Roche and Genentech, have been approved by US FDA for marketing. Unlike blinatumomab, which is a bispecific tandem single-chain variable fragment with a 2.11 hours average half life in humans (www.drugbank.ca/drugs/DB09052 ) , native IgG1-like emicizumab presents a long half life of 27.8 to 34.4 days (www.drugbank.ca/drugs/DB13923).…”

Section: Discussionmentioning

confidence: 99%

“…As shown in Figure 1, the knobs-into-holes technique 13 was used for heterodimerizing the heavy chains of trastuzumab and pertuzumab in this molecular design. A knob variant called MBS301-knob was obtained by replacing a small amino acid with a larger one, T366W, in pertuzumab as the first half arm of MBS301.…”

Section: Introductionmentioning

confidence: 99%

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Structural and functional characterization of MBS301, an afucosylated bispecific anti-HER2 antibody

Huang

Liu

et al. 2018

mAbs

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MBS301, a glyco-engineered bispecific anti-human epidermal growth factor receptor 2 (HER2) antibody with a typical IgG1 monoclonal antibody structure, was developed through dual-cell expression and in vitro assembling process. MBS301 consists of two half antibodies engineered from trastuzumab and pertuzumab, respectively. Integrity and purity profiles of MB301 indicated that the heterodimerization of the two half antibodies was successful. The high and similar melting temperatures (Tm1,72.0°C and Tm2, 84.8°C) of MBS301 compared with those of its parental monoclonal antibodies trastuzumab and pertuzumab (in-house made T-mab and P-mab, respectively) revealed its structural compactness. With computer-modeling experiments and Biacore binding and competition kinetics studies, the binding stoichiometry between MBS301 and HER2-ECD was determined to be 1:1 and the two arms of MBS301 were shown to bind to domains II and IV of HER2-ECD antigen simultaneously. MBS301 displayed synergistic bioactivities as the combination of T-mab and P-mab in vitro in multiple cancer cell lines and in vivo in xenograft mouse model studies, and showed more effective activity than T-mab or P-mab used individually. Moreover, fucose-knockout dramatically increased MBS301’s binding affinity to low affinity FcγRIIIa allotype 158F (KD = 2.35 × 10−7M) to near the high affinity level of allotype V158 (KD = 1.17 × 10−7M). This resulted in far more effective ADCC activity of MBS301 than the combination of T-mab and P-mab in killing HER2-positive cancer cells. Hence, a novel fully afucosylated anti-HER2 bispecific antibody with improved antitumor activities was generated and shown to have the potential to be used for treating HER2-positive but trastuzumab-resistant solid tumors.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Structural and functional characterization of MBS301, an afucosylated bispecific anti-HER2 antibody

Huang

Liu

et al. 2018

mAbs

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“…Not only are these antibodies able to bind to 2 different antigens such as 2 cell surface proteins or 2 ligands, they are also able to, for example, simultaneously bind to tumor proteins and cytotoxic T-cells, thus directing therapeutic agents to the intended target (for recent reviews, see Riethm€ uller, 1 Kontermann, 2 and Spiess et al 3 ). Among the first attempts to produce such compounds, the so-called "Quadroma" approach has been used, where 2 antibody-secreting cell lines were combined by somatic fusion.…”

Section: Introductionmentioning

confidence: 99%

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

Schaefer

Völger

Lorenz

et al. 2015

mAbs

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(2016) Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry, mAbs, 8:1, 49-55, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: bispecific antibody, CrossMab, chain pairing, ESI-QTOF mass spectrometry, knobs-into-holes, quadromaAbbreviations: Ang2, angiopoietin-2; bsAbs, bispecific antibodies; ESI, electrospray ionization; GuA HCl, guanidine hydrochloride; HEK, human embryonic kidney; HC, heavy chain; IgG1, immunoglobulin G1; ISCID, ion source collision induced dissociation; KiH, knobs-into-holes; LC, light chain; MS, mass spectrometry; TCEP, tris(2-carboxyethyl)phosphine; UHR, ultrahigh-resolution; VEGF-A, vascular endothelial growth factor A; QTOF, quadrupole time-of-flightThe quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.

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“…While monoclonal antibodies with novel targets and antibody based derivatives such as antibody-drug conjugates, antibody fragments, antibody-based fusion proteins, bispecific antibodies, nanobodies and similar constructs have received much attention in recent years [2,3], refining and optimizing the protein structure and subsequently pharmacologic properties of established, clinically used monoclonal antibodies is gaining increasing interest. While this effort is likely partly driven by patent expirations and the growing momentum for establishing a viable and effective biosimilar market for monoclonal antibodies in many nations [4], it also opens a pathway to apply recent advances in protein engineering and manufacturing to create derivatives that form a new, improved generation of established therapeutic proteins.…”

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confidence: 99%

Protein Engineering for Improved Pharmacologic Characteristics of Established Monoclonal Antibody-Based Therapeutics

Meibohm

2014

Clin Pharmacokinet

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Dual targeting strategies with bispecific antibodies

Cited by 397 publications

References 149 publications

Structural and functional characterization of MBS301, an afucosylated bispecific anti-HER2 antibody

Structural and functional characterization of MBS301, an afucosylated bispecific anti-HER2 antibody

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

Protein Engineering for Improved Pharmacologic Characteristics of Established Monoclonal Antibody-Based Therapeutics

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