Biophysical and biochemical characterization of a VHH-based IgG-like bi- and trispecific antibody platform

Pekar, Lukas; Busch, Michael; Valldorf, Bernhard; Hinz, Steffen C.; Toleikis, Lars; Krah, Simon; Zielonka, Stefan

doi:10.1080/19420862.2020.1812210

Cited by 27 publications

(30 citation statements)

References 41 publications

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“… 27 In this regard, our group recently developed a platform process to generate multispecific antibodies with flexible valencies by exchanging VH and VL of a conventional Fab with camelid derived VHH single domain antibodies. 28 By applying SEED for heavy chain heterodimerization, trispecific antibodies have readily been produced.…”

Section: Introductionmentioning

confidence: 99%

Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

Yanakieva

Pekar

Evers

et al. 2022

mAbs

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Controlled Fab arm exchange (cFAE) has proven to be a generic and versatile technology for the efficient generation of IgG-like bispecific antibodies (DuoBodies or DBs), with several in clinical development and one product, amivantamab, approved by the Food and Drug Administration. In this study, we expand the cFAE-toolbox by incorporating VHH-modules at the C-termini of DB-IgGs, termed DB-VHHs. This approach enables the combinatorial generation of tri- and tetraspecific molecules with flexible valencies in a straightforward fashion. Using cFAE, a variety of multispecific molecules was produced and assessed for manufacturability and physicochemical characteristics. In addition, we were able to generate DB-VHHs that efficiently triggered natural killer cell mediated lysis of tumor cells, demonstrating the utility of this format for potential therapeutic applications.

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Section: Introductionmentioning

confidence: 99%

Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

Yanakieva

Pekar

Evers

et al. 2022

mAbs

Self Cite

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“…The ADDC assays were performed according to Pekar et al. ( 31 ). EasySep Human NK isolation Kit was used to isolate NK cells.…”

Section: Methodsmentioning

confidence: 99%

“…For NK cell based ADCC assays, whole blood samples of healthy human donors were used to isolate PBMCs via gradient centrifugation. The ADDC assays were performed according to Pekar et al (31). EasySep Human NK isolation Kit was used to isolate NK cells.…”

Section: Antibody-dependent Cell-mediated Cytotoxicitymentioning

confidence: 99%

Protease-Activation of Fc-Masked Therapeutic Antibodies to Alleviate Off-Tumor Cytotoxicity

et al. 2021

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The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats.

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“…Several other trispecific platforms also exist, either bulkier, such as the older Dock-and-Lock (DNL, Figure 1 g) method based on the dimerization domain of cAMP-dependent protein kinase A and the anchoring domain of an A-kinase anchoring protein [ 183 , 184 ], or the newer, more compact VHH-based trispecific constructs, which are still at the preclinical stage [ 185 ]. In addition, IgG/scFV-based tetraspecifics (scFv6-IgG, Figure 1 d and Table 1 ), have recently crossed the border to clinical testing.…”

Section: Trispecific and Other Multispecific Antibodiesmentioning

confidence: 99%

Principles and Current Clinical Landscape of Multispecific Antibodies against Cancer

Elshiaty

Schindler

Christopoulos

2021

IJMS

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Building upon the resounding therapeutic success of monoclonal antibodies, and supported by accelerating progress in engineering methods, the field of multispecific therapeutic antibodies is growing rapidly. Over 140 different molecules are currently in clinical testing, with excellent results in recent phase 1–3 clinical trials for several of them. Multivalent bispecific IgG-modified formats predominate today, with a clear tendency for more target antigens and further increased valency in newer constructs. The strategies to augment anticancer efficacy are currently equally divided between disruption of multiple surface antigens, and additional redirection of cytotoxic T or NK lymphocytes against the tumor. Both effects complement other modern modalities, such as tyrosine kinase inhibitors and adoptive cell therapies, with which multispecifics are increasingly applied in combination or merged, for example, in the form of antibody producing CAR-T cells and oncolytics. While mainly focused on B-cell malignancies early on, the contemporary multispecific antibody sector accommodates twice as many trials against solid compared to hematologic cancers. An exciting emerging prospect is the targeting of intracellular neoantigens using T-cell receptor (TCR) fusion proteins or TCR-mimic antibody fragments. Considering the fact that introduction of PD-(L)1 inhibitors only a few years ago has already facilitated 5-year survival rates of 30–50% for per se highly lethal neoplasms, such as metastatic melanoma and non-small-cell lung carcinoma, the upcoming enforcement of current treatments with “next-generation” immunotherapeutics, offers a justified hope for the cure of some advanced cancers in the near future.

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Biophysical and biochemical characterization of a VHH-based IgG-like bi- and trispecific antibody platform

Cited by 27 publications

References 41 publications

Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

Protease-Activation of Fc-Masked Therapeutic Antibodies to Alleviate Off-Tumor Cytotoxicity

Principles and Current Clinical Landscape of Multispecific Antibodies against Cancer

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