Grabbing the Bull by Both Horns: Bovine Ultralong CDR-H3 Paratopes Enable Engineering of ‘Almost Natural’ Common Light Chain Bispecific Antibodies Suitable For Effector Cell Redirection

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly, this type of immunoglobulin usually pairs with the single germline VL gene, V30 that is typically very conserved in sequence. In this work, we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection, EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly, engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-γ. Taken together, this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications.

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Expeditious Generation of Biparatopic Common Light Chain Antibodies via Chicken Immunization and Yeast Display Screening

Frontiers in Immunology ◽

10.3389/fimmu.2020.606878 ◽

2020 ◽

Vol 11 ◽

Author(s):

Jan P. Bogen ◽

Stefania C. Carrara ◽

David Fiebig ◽

Julius Grzeschik ◽

Björn Hock ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Light Chain ◽

Surface Display ◽

Growth Factor Receptor ◽

Yeast Surface Display ◽

Single Digit ◽

Dependent Cell ◽

Epidermal Growth ◽

Domain Iii

Bispecific (BsAb) and biparatopic (BpAb) antibodies emerged as promising formats for therapeutic biologics exhibiting tailor-made functional properties. Over recent years, chicken-derived antibodies have gained traction for diagnostic and therapeutic applications due to their broad epitope coverage and convenience of library generation. Here we report the first generation of a biparatopic common light chain (cLC) chicken-derived antibody by an epitope binning-based screening approach using yeast surface display. The resulting monospecific antibodies target conformational epitopes on domain II or III of the epidermal growth factor receptor (EGFR) with lower double- or single-digit nanomolar affinities, respectively. Furthermore, the domain III targeting variant was shown to interfere with epidermal growth factor (EGF) binding. Utilizing the Knob-into-Hole technology (KiH), a biparatopic antibody with subnanomolar affinity was generated that facilitates clustering of soluble and cell-bound EGFR and displayed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parental antibodies. This strategy for generating cLC-based biparatopic antibodies from immunized chickens may pave the way for their further development in therapeutic settings.

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Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning

Biological Chemistry ◽

10.1515/hsz-2018-0347 ◽

2019 ◽

Vol 400 (3) ◽

pp. 383-393 ◽

Cited By ~ 7

Author(s):

Lukas Roth ◽

Julius Grzeschik ◽

Steffen C. Hinz ◽

Stefan Becker ◽

Lars Toleikis ◽

...

Keyword(s):

Light Chain ◽

Surface Display ◽

Combinatorial Libraries ◽

Yeast Surface Display ◽

Golden Gate ◽

Yeast Display ◽

Step Method ◽

One Step ◽

Yeast Surface ◽

Golden Gate Cloning

Abstract Antibodies can be successfully engineered and isolated by yeast or phage display of combinatorial libraries. Still, generation of libraries comprising heavy chain as well as light chain diversities is a cumbersome process involving multiple steps. Within this study, we set out to compare the output of yeast display screening of antibody Fab libraries from immunized rodents that were generated by Golden Gate Cloning (GGC) with the conventional three-step method of individual heavy- and light-chain sub-library construction followed by chain combination via yeast mating (YM). We demonstrate that the GGC-based one-step process delivers libraries and antibodies from heavy- and light-chain diversities with similar quality to the traditional method while being significantly less complex and faster. Additionally, we show that this method can also be used to successfully screen and isolate chimeric chicken/human antibodies following avian immunization.

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Functional expression, production, and biochemical characterization of a laccase using yeast surface display technology

Fungal Biology ◽

10.1016/j.funbio.2016.08.009 ◽

2016 ◽

Vol 120 (12) ◽

pp. 1609-1622 ◽

Cited By ~ 6

Author(s):

Brandt Bertrand ◽

María R. Trejo-Hernández ◽

Daniel Morales-Guzmán ◽

Luis Caspeta ◽

Ramón Suárez Rodríguez ◽

...

Keyword(s):

Biochemical Characterization ◽

Surface Display ◽

Functional Expression ◽

Yeast Surface Display ◽

Display Technology ◽

Yeast Surface

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A functional mammalian display screen identifies rare antibodies that stimulate NK cell–mediated cytotoxicity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2104099118 ◽

2021 ◽

Vol 118 (31) ◽

pp. e2104099118

Author(s):

Emily Kang ◽

Cigall Kadoch ◽

James L. Rubenstein ◽

Lewis L. Lanier ◽

James A. Wells

Keyword(s):

Nk Cells ◽

Immune Cell ◽

Nk Cell ◽

B Cell Lymphoma ◽

Interferon Γ ◽

Bispecific Antibodies ◽

Effector Cells ◽

Cell Functions ◽

Her2 Breast Cancer ◽

Target Cell Line

Therapies that boost the antitumor immune response have shown a great deal of success. Although most of these therapies have focused on enhancing T cell functions, there is a growing interest in developing therapies that can target other immune cell subsets. Like T cells, natural killer (NK) cells are cytotoxic effector cells that play a key role in the antitumor response. To advance the development of NK-based therapies, we developed a functional screen to rapidly identify antibodies that can activate NK cells. We displayed antibodies on a mammalian target cell line and probed their ability to stimulate NK cell–mediated cytotoxicity. From this screen, we identified five antibodies that bound with high affinity to NK cells and stimulated NK cell–mediated cytotoxicity and interferon-γ (IFN-γ) secretion. We demonstrate that these antibodies can be further developed into bispecific antibodies to redirect NK cell–mediated cytotoxicity toward CD20+ B cell lymphoma cells and HER2+ breast cancer cells. While antibodies to two of the receptors, CD16 and NCR1, have previously been targeted as bispecific antibodies to redirect NK cell–mediated cytotoxicity, we demonstrate that bispecific antibodies targeting NCR3 can also potently activate NK cells. These results show that this screen can be used to directly identify antibodies that can enhance antitumor immune responses.

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