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- EMDB-26719: FMC63 scFv in complex with soluble CD19 -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-26719
TitleFMC63 scFv in complex with soluble CD19
Map dataFMC63-scFv and soluble CD19
Sample
  • Complex: FMC63 scFv in complex with soluble CD19
    • Complex: B-lymphocyte antigen CD19
      • Protein or peptide: B-lymphocyte antigen CD19
    • Complex: FMC63 single-chain variable fragment
      • Protein or peptide: FMC63 single-chain variable fragment
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsMeyerson J / He C
Funding support1 items
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, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateMar 15, 2023-
Current statusMar 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26719.png

Downloads & links

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Map

FileDownload / File: emd_26719.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFMC63-scFv and soluble CD19
Voxel sizeX=Y=Z: 1.096 Å
Density
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-3.435229 - 5.971818
Average (Standard dev.)0.0016382375 (±0.06886415)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 350.71997 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: FMC63 scFv in complex with soluble CD19

Fileemd_26719_half_map_1.map
AnnotationFMC63 scFv in complex with soluble CD19
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: FMC63 scFv in complex with soluble CD19

Fileemd_26719_half_map_2.map
AnnotationFMC63 scFv in complex with soluble CD19
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : FMC63 scFv in complex with soluble CD19

EntireName: FMC63 scFv in complex with soluble CD19
Components
  • Complex: FMC63 scFv in complex with soluble CD19
    • Complex: B-lymphocyte antigen CD19
      • Protein or peptide: B-lymphocyte antigen CD19
    • Complex: FMC63 single-chain variable fragment
      • Protein or peptide: FMC63 single-chain variable fragment

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Supramolecule #1: FMC63 scFv in complex with soluble CD19

SupramoleculeName: FMC63 scFv in complex with soluble CD19 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all

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Supramolecule #2: B-lymphocyte antigen CD19

SupramoleculeName: B-lymphocyte antigen CD19 / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: FMC63 single-chain variable fragment

SupramoleculeName: FMC63 single-chain variable fragment / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: B-lymphocyte antigen CD19

MacromoleculeName: B-lymphocyte antigen CD19 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.153135 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EEPLVVKVEE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP FLKLSLGLPG LGIHVSPLAI WLFISNVSQQ MGGFYLCQPG PPSEKAWQP GWTVNVEGSG ELFRWNVSDL GGLGCGLKNR SSEGPSSPSG KLMSPKLYVW AKDRPEIWEG EPPCLPPRDS L NQSLSQDL ...String:
EEPLVVKVEE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP FLKLSLGLPG LGIHVSPLAI WLFISNVSQQ MGGFYLCQPG PPSEKAWQP GWTVNVEGSG ELFRWNVSDL GGLGCGLKNR SSEGPSSPSG KLMSPKLYVW AKDRPEIWEG EPPCLPPRDS L NQSLSQDL TMAPGSTLWL SCGVPPDSVS RGPLSWTHVH PKGPKSLLSL ELKDDRPARD MWVMETGLLL PRATAQDAGK YY CHRGNLT MSFHLEITAR

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Macromolecule #2: FMC63 single-chain variable fragment

MacromoleculeName: FMC63 single-chain variable fragment / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.948705 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DIQMTQTTSS LSASLGDRVT ISCRASQDIS KYLNWYQQKP DGTVKLLIYH TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFCQ QGNTLPYTFG GGTKLEITGG GGSGGGGSGG GGSEVKLQES GPGLVAPSQS LSVTCTVSGV SLPDYGVSWI R QPPRKGLE ...String:
DIQMTQTTSS LSASLGDRVT ISCRASQDIS KYLNWYQQKP DGTVKLLIYH TSRLHSGVPS RFSGSGSGTD YSLTISNLEQ EDIATYFCQ QGNTLPYTFG GGTKLEITGG GGSGGGGSGG GGSEVKLQES GPGLVAPSQS LSVTCTVSGV SLPDYGVSWI R QPPRKGLE WLGVIWGSET TYYNSALKSR LTIIKDNSKS QVFLKMNSLQ TDDTAIYYCA KHYYYGGSYA MDYWGQGTSV TV SS

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.82 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.9 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 442863
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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