6S5A

CRYSTAL STRUCTURE OF FC P329G LALA WITH ANTI FC P329G FAB

PDB DOI: https://doi.org/10.2210/pdb6S5A/pdb

Classification: IMMUNE SYSTEM
Organism(s): Homo sapiens, Mus musculus
Expression System: Cricetulus griseus
Mutation(s): No

Deposited: 2019-07-01 Released: 2019-09-25
Deposition Author(s): Ehler, A., Darowski, D., Jost, C., Stubenrauch, K., Wessels, U., Benz, J., Birk, M., Freimoser-Grundschober, A., Bruenker, P., Moessner, E., Umana, P., Kobold, S., Klein, C.

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 1.72 Å
R-Value Free: 0.226
R-Value Work: 0.196
R-Value Observed: 0.197

wwPDB Validation 3D Report Full Report

This is version 2.1 of the entry. See complete history.

Literature

P329G-CAR-J: a novel Jurkat-NFAT-based CAR-T reporter system recognizing the P329G Fc mutation.

Darowski, D., Jost, C., Stubenrauch, K., Wessels, U., Benz, J., Ehler, A., Freimoser-Grundschober, A., Brunker, P., Mossner, E., Umana, P., Kobold, S., Klein, C.

(2019) Protein Eng Des Sel 32: 207-218

PubMed: 31504896 Search on PubMed
DOI: https://doi.org/10.1093/protein/gzz027
Primary Citation of Related Structures:
6S5A

PubMed Abstract:
Monoclonal antibody-based therapeutics are an integral part of treatment of different human diseases, and the selection of suitable antibody candidates during the discovery phase is essential. Here, we describe a novel, cellular screening approach for the identification and characterization of therapeutic antibodies suitable for conversion into T cell bispecific antibodies using chimeric antigen receptor (CAR) transduced Jurkat-NFAT-luciferase reporter cells (CAR-J). For that purpose, we equipped a Jurkat-NFAT reporter cell line with a universal CAR, based on a monoclonal antibody recognizing the P329G mutation in the Fc-part of effector-silenced human IgG1-antibodies. In addition to scFv-based second generation CARs, Fab-based CARs employing the P329G-binder were generated. Using these anti-P329G-CAR-J cells together with the respective P329G-mutated IgG1-antibodies, we established a system, which facilitates the rapid testing of therapeutic antibody candidates in a flexible, high throughput setting during early stage discovery. We show that both, scFv- and Fab-based anti-P329G-CAR-J cells elicit a robust and dose-dependent luciferase signal if the respective antibody acts as an adaptor between tumor target and P329G-CAR-J cells. Importantly, we could demonstrate that functional characteristics of the antibody candidates, derived from the anti-P329G-CAR-J screening assay, are predictive for the functionality of these antibodies in the T cell bispecific antibody format.

Organizational Affiliation

Roche Innovation Center Zurich, Roche Pharma Research & Early Development, Wagistrasse 10 CH-8952 Schlieren, Switzerland.

Macromolecule Content

Total Structure Weight: 103.63 kDa
Atom Count: 7,720
Modelled Residue Count: 855
Deposited Residue Count: 916
Unique protein chains: 3

Macromolecules

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(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
Fc P329G LALA	A, B [auth D]	225	Homo sapiens	Mutation(s): 0
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Glycosylation
Glycosylation Sites: 1	Glycosylation Focus chain A Focus chain B [auth D]
Sequence Annotations Expand
Reference Sequence

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(by identity cutoff) | 3D Structure

Entity ID: 2
Molecule	Chains	Sequence Length	Organism	Details	Image
anti P329G LALA Fab heavy chain	C [auth H]	251	Mus musculus	Mutation(s): 0
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations Expand
Reference Sequence

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(by identity cutoff) | 3D Structure

Entity ID: 3
Molecule	Chains	Sequence Length	Organism	Details	Image
anti P329G LALA Fab light chain	D [auth L]	215	Mus musculus	Mutation(s): 0
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations Expand
Reference Sequence

Oligosaccharides

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Entity ID: 4
Molecule	Chains	Length	2D Diagram	Glycosylation	3D Interactions
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose	E [auth B]	6		N-Glycosylation	Interactions Focus chain E [auth B] Interactions & Density Focus chain E [auth B]
Glycosylation Resources
GlyTouCan: G39203UC GlyCosmos: G39203UC GlyGen: G39203UC

Entity ID: 5
Molecule	Chains	Length	2D Diagram	Glycosylation	3D Interactions
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose	F [auth C]	7		N-Glycosylation	Interactions Focus chain F [auth C] Interactions & Density Focus chain F [auth C]
Glycosylation Resources
GlyTouCan: G45889JQ GlyCosmos: G45889JQ GlyGen: G45889JQ

Small Molecules

Ligands 2 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
PO4 Query on PO4 Download Ideal Coordinates CCD File SDF format, chain G [auth D] SDF format, chain J [auth L] SDF format, chain K [auth L] MOL2 format, chain G [auth D] MOL2 format, chain J [auth L] MOL2 format, chain K [auth L] mmCIF format, chain G [auth D] mmCIF format, chain J [auth L] mmCIF format, chain K [auth L]	G [auth D], J [auth L], K [auth L]	PHOSPHATE ION O₄ P NBIIXXVUZAFLBC-UHFFFAOYSA-K		Interactions Focus chain G [auth D] Focus chain J [auth L] Focus chain K [auth L] Interactions & Density Focus chain G [auth D] Focus chain J [auth L] Focus chain K [auth L]
GOL Query on GOL Download Ideal Coordinates CCD File SDF format, chain I [auth H] SDF format, chain L SDF format, chain H [auth D] MOL2 format, chain I [auth H] MOL2 format, chain L MOL2 format, chain H [auth D] mmCIF format, chain I [auth H] mmCIF format, chain L mmCIF format, chain H [auth D]	H [auth D], I [auth H], L	GLYCEROL C₃ H₈ O₃ PEDCQBHIVMGVHV-UHFFFAOYSA-N		Interactions Focus chain H [auth D] Focus chain I [auth H] Focus chain L Interactions & Density Focus chain H [auth D] Focus chain I [auth H] Focus chain L

Experimental Data & Validation

Experimental Data

Method: X-RAY DIFFRACTION
Resolution: 1.72 Å
R-Value Free: 0.226
R-Value Work: 0.196
R-Value Observed: 0.197
Space Group: P 2₁ 2₁ 2₁

Unit Cell:

Length ( Å )	Angle ( ˚ )
a = 71.022	α = 90
b = 135.545	β = 90
c = 137.259	γ = 90

Software Package:

Software Name	Purpose
BUSTER	refinement
PDB_EXTRACT	data extraction
XDS	data reduction
SADABS	data scaling
PHASER	phasing

Structure Validation

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Entry History

Deposition Data

Released Date: 2019-09-25

Deposition Author(s):

Freimoser-Grundschober, A.

Revision History (Full details and data files)

Version 1.0: 2019-09-25
Type: Initial release
Version 1.1: 2020-02-26
Changes: Data collection, Database references
Version 2.0: 2020-07-29
Type: Remediation
Reason: Carbohydrate remediation
Changes: Atomic model, Data collection, Derived calculations, Structure summary
Version 2.1: 2024-11-13
Changes: Data collection, Database references, Derived calculations, Structure summary