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- PDB-7urv: FMC63 scFv in complex with soluble CD19 -

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Basic information

Entry
Database: PDB / ID: 7urv
TitleFMC63 scFv in complex with soluble CD19
Components
  • B-lymphocyte antigen CD19
  • FMC63 single-chain variable fragment
KeywordsPROTEIN BINDING / Complex / CAR-T
Function / homology
Function and homology information


regulation of B cell activation / antigen receptor-mediated signaling pathway / B-1 B cell differentiation / regulation of B cell receptor signaling pathway / B cell proliferation involved in immune response / immunoglobulin mediated immune response / positive regulation of release of sequestered calcium ion into cytosol / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Regulation of Complement cascade / B cell receptor signaling pathway ...regulation of B cell activation / antigen receptor-mediated signaling pathway / B-1 B cell differentiation / regulation of B cell receptor signaling pathway / B cell proliferation involved in immune response / immunoglobulin mediated immune response / positive regulation of release of sequestered calcium ion into cytosol / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Regulation of Complement cascade / B cell receptor signaling pathway / Constitutive Signaling by Aberrant PI3K in Cancer / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / PIP3 activates AKT signaling / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / membrane raft / external side of plasma membrane / protein-containing complex / extracellular exosome / plasma membrane
Similarity search - Function
B-lymphocyte antigen CD19 / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
B-lymphocyte antigen CD19
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsMeyerson, J. / He, C.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Sci Immunol / Year: 2023
Title: CD19 CAR antigen engagement mechanisms and affinity tuning.
Authors: Changhao He / Jorge Mansilla-Soto / Nandish Khanra / Mohamad Hamieh / Victor Bustos / Alice J Paquette / Andreina Garcia Angus / Derek M Shore / William J Rice / George Khelashvili / Michel ...Authors: Changhao He / Jorge Mansilla-Soto / Nandish Khanra / Mohamad Hamieh / Victor Bustos / Alice J Paquette / Andreina Garcia Angus / Derek M Shore / William J Rice / George Khelashvili / Michel Sadelain / Joel R Meyerson /
Abstract: Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface antigens through an scFv (binder), ...Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface antigens through an scFv (binder), the affinity of which is central to determining CAR T cell function and therapeutic success. CAR T cells targeting CD19 were the first to achieve marked clinical responses in patients with relapsed/refractory B cell malignancies and to be approved by the U.S. Food and Drug Administration (FDA). We report cryo-EM structures of CD19 antigen with the binder FMC63, which is used in four FDA-approved CAR T cell therapies (Kymriah, Yescarta, Tecartus, and Breyanzi), and the binder SJ25C1, which has also been used extensively in multiple clinical trials. We used these structures for molecular dynamics simulations, which guided creation of lower- or higher-affinity binders, and ultimately produced CAR T cells endowed with distinct tumor recognition sensitivities. The CAR T cells exhibited different antigen density requirements to trigger cytolysis and differed in their propensity to prompt trogocytosis upon contacting tumor cells. Our work shows how structural information can be applied to tune CAR T cell performance to specific target antigen densities.
History
DepositionApr 22, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 15, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: B-lymphocyte antigen CD19
D: FMC63 single-chain variable fragment


Theoretical massNumber of molelcules
Total (without water)54,1022
Polymers54,1022
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein B-lymphocyte antigen CD19 / B-lymphocyte surface antigen B4 / Differentiation antigen CD19 / T-cell surface antigen Leu-12


Mass: 28153.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CD19 / Production host: Homo sapiens (human) / References: UniProt: P15391
#2: Antibody FMC63 single-chain variable fragment


Mass: 25948.705 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1FMC63 scFv in complex with soluble CD19COMPLEXall0RECOMBINANT
2B-lymphocyte antigen CD19COMPLEX#11RECOMBINANT
3FMC63 single-chain variable fragmentCOMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2900 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50.82 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 442863 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033528
ELECTRON MICROSCOPYf_angle_d0.5784800
ELECTRON MICROSCOPYf_dihedral_angle_d4.547474
ELECTRON MICROSCOPYf_chiral_restr0.042527
ELECTRON MICROSCOPYf_plane_restr0.005606

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