Journal: Proc Natl Acad Sci U S A / Year: 2023 Title: Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins. Authors: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan ...Authors: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan Verma / Jingjia Liu / Robyn L Stanfield / Xueyong Zhu / Hannah L Turner / Devin Sok / Po-Ssu Huang / Dennis R Burton / Andrew B Ward / Ian A Wilson / Joseph G Jardine / Abstract: Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple ...Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human V3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the V and V domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.
Entire : LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex wi...
Entire
Name: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex with SARS-CoV-2-6P-Mut7 S protein
Components
Complex: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex with SARS-CoV-2-6P-Mut7 S protein
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Supramolecule #1: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex wi...
Supramolecule
Name: LM18/Nb136 bispecific tetra-nanobody immunoglobulin in complex with SARS-CoV-2-6P-Mut7 S protein type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)
Organism: Severe acute respiratory syndrome coronavirus 2
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
0.85 mg/mL
Buffer
pH: 7.4 Component:
Concentration
Name
Formula
50.0 mM
Tris
150.0 mM
sodium chloride
NaCl
0.06 mM
n-Dodecyl-B-D-Maltopyranoside
Details: Detergent added shortly before grid application
Grid
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details
incubated complex for 100 minutes at room temperature prior to freezing
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 4296 / Average exposure time: 9.0 sec. / Average electron dose: 49.6 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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