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- EMDB-60767: HL-type bispecific diabody Ex3 composed of 528 and OKT3 Fvs in te... -

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Entry
Database: EMDB / ID: EMD-60767
TitleHL-type bispecific diabody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilon (open conformation)
Map datasharpened map
Sample
  • Complex: HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilon
    • Complex: Soluble Epidermal Growth Factor Receptor (sEGFR)
      • Protein or peptide: Epidermal growth factor receptor
    • Complex: HL-type bispecific diabody Ex3
      • Protein or peptide: HL-type bispecific diabody Ex3
    • Complex: CD3 gamma-epsilon
      • Protein or peptide: T-cell surface glycoprotein CD3 gamma chain,T-cell surface glycoprotein CD3 epsilon chain
Keywordsbispecific antibody / diabody / EGFR / CD3 / ternary complex / HL / Ex3 / 528 / OKT3 / ANTITUMOR PROTEIN / ANTITUMOR PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


regulation of lymphocyte apoptotic process / gamma-delta T cell receptor complex / T cell anergy / positive regulation of T cell anergy / CD4-positive, alpha-beta T cell proliferation / positive regulation of cell-cell adhesion mediated by integrin / gamma-delta T cell activation / negative thymic T cell selection / positive regulation of CD4-positive, alpha-beta T cell proliferation / positive thymic T cell selection ...regulation of lymphocyte apoptotic process / gamma-delta T cell receptor complex / T cell anergy / positive regulation of T cell anergy / CD4-positive, alpha-beta T cell proliferation / positive regulation of cell-cell adhesion mediated by integrin / gamma-delta T cell activation / negative thymic T cell selection / positive regulation of CD4-positive, alpha-beta T cell proliferation / positive thymic T cell selection / signal complex assembly / alpha-beta T cell receptor complex / multivesicular body, internal vesicle lumen / negative regulation of cardiocyte differentiation / Shc-EGFR complex / positive regulation of protein kinase C signaling / Inhibition of Signaling by Overexpressed EGFR / epidermal growth factor receptor activity / EGFR interacts with phospholipase C-gamma / regulation of peptidyl-tyrosine phosphorylation / epidermal growth factor binding / response to UV-A / ubiquitin-dependent endocytosis / PLCG1 events in ERBB2 signaling / positive regulation of cell-matrix adhesion / T cell receptor complex / morphogenesis of an epithelial fold / smoothened signaling pathway / PTK6 promotes HIF1A stabilization / ERBB2 Activates PTK6 Signaling / digestive tract morphogenesis / ERBB2-EGFR signaling pathway / Signaling by EGFR / Translocation of ZAP-70 to Immunological synapse / Phosphorylation of CD3 and TCR zeta chains / positive regulation of interleukin-4 production / eyelid development in camera-type eye / intracellular vesicle / cerebral cortex cell migration / dendrite development / ERBB2 Regulates Cell Motility / establishment or maintenance of cell polarity / Developmental Lineage of Mammary Gland Myoepithelial Cells / protein insertion into membrane / alpha-beta T cell activation / Respiratory syncytial virus (RSV) attachment and entry / protein tyrosine kinase activator activity / FCGR activation / Generation of second messenger molecules / Signaling by ERBB4 / negative regulation of epidermal growth factor receptor signaling pathway / PI3K events in ERBB2 signaling / immunological synapse / Co-inhibition by PD-1 / positive regulation of peptidyl-serine phosphorylation / positive regulation of phosphorylation / hair follicle development / MAP kinase kinase kinase activity / Estrogen-dependent nuclear events downstream of ESR-membrane signaling / Role of phospholipids in phagocytosis / GAB1 signalosome / embryonic placenta development / positive regulation of G1/S transition of mitotic cell cycle / salivary gland morphogenesis / xenobiotic transport / positive regulation of interleukin-2 production / positive regulation of epidermal growth factor receptor signaling pathway / T cell costimulation / T cell receptor binding / Signaling by ERBB2 / TFAP2 (AP-2) family regulates transcription of growth factors and their receptors / GRB2 events in EGFR signaling / transmembrane receptor protein tyrosine kinase activity / SHC1 events in EGFR signaling / EGFR Transactivation by Gastrin / GRB2 events in ERBB2 signaling / positive regulation of T cell proliferation / positive regulation of calcium-mediated signaling / SHC1 events in ERBB2 signaling / FCGR3A-mediated IL10 synthesis / epithelial cell proliferation / cell surface receptor protein tyrosine kinase signaling pathway / ossification / cellular response to epidermal growth factor stimulus / cerebellum development / basal plasma membrane / positive regulation of DNA replication / T cell activation / positive regulation of epithelial cell proliferation / positive regulation of DNA repair / Signal transduction by L1 / positive regulation of protein localization to plasma membrane / FCGR3A-mediated phagocytosis / negative regulation of smoothened signaling pathway / cellular response to estradiol stimulus / apoptotic signaling pathway / NOTCH3 Activation and Transmission of Signal to the Nucleus / sperm end piece / cellular response to amino acid stimulus / phosphatidylinositol 3-kinase/protein kinase B signal transduction
Similarity search - Function
CD3 gamma/delta subunit, Ig-like domain / T-cell surface glycoprotein CD3 delta chain / Ig-like domain on T-cell surface glycoprotein CD3 epsilon chain / CD3 protein, epsilon/gamma/delta subunit / Immunoreceptor tyrosine-based activation motif / Phosphorylated immunoreceptor signalling ITAM / ITAM motif mammalian type profile. / Immunoreceptor tyrosine-based activation motif / : / Epidermal growth factor receptor transmembrane-juxtamembrane segment ...CD3 gamma/delta subunit, Ig-like domain / T-cell surface glycoprotein CD3 delta chain / Ig-like domain on T-cell surface glycoprotein CD3 epsilon chain / CD3 protein, epsilon/gamma/delta subunit / Immunoreceptor tyrosine-based activation motif / Phosphorylated immunoreceptor signalling ITAM / ITAM motif mammalian type profile. / Immunoreceptor tyrosine-based activation motif / : / Epidermal growth factor receptor transmembrane-juxtamembrane segment / Tyrosine protein kinase, EGF/ERB/XmrK receptor / Growth factor receptor domain 4 / Growth factor receptor domain IV / Receptor L-domain / Furin-like cysteine-rich domain / Receptor L-domain superfamily / Furin-like cysteine rich region / Receptor L domain / Furin-like repeat / Furin-like repeats / Growth factor receptor cysteine-rich domain superfamily / : / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, active site / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Immunoglobulin-like domain superfamily / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Immunoglobulin-like fold / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Epidermal growth factor receptor / T-cell surface glycoprotein CD3 epsilon chain / T-cell surface glycoprotein CD3 gamma chain
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsSato K / Uehara S / Tsugita A / Matsui T / Asano R / Makabe K / Yokoyama T / Tanaka Y
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Cell Rep / Year: 2025
Title: Bispecific antibody-antigen complex structures reveal activity enhancement by domain rearrangement.
Authors: Kyohei Sato / Shiro Uehara / Atsushi Tsugita / Mayuka Ishii / Shieru Ishiyama / Atsushi Maejima / Ishin Nakahara / Misae Nazuka / Takashi Matsui / Gatsogiannis Christos / Takeshi Yokoyama / ...Authors: Kyohei Sato / Shiro Uehara / Atsushi Tsugita / Mayuka Ishii / Shieru Ishiyama / Atsushi Maejima / Ishin Nakahara / Misae Nazuka / Takashi Matsui / Gatsogiannis Christos / Takeshi Yokoyama / Izumi Kumagai / Koki Makabe / Ryutaro Asano / Yoshikazu Tanaka /
Abstract: Bispecific antibodies (BsAbs) have been developed as anti-cancer drugs that accumulate activated T cells on cancer cells by bridging the antigens present in each cell. Ex3 is a diabody-type BsAb ...Bispecific antibodies (BsAbs) have been developed as anti-cancer drugs that accumulate activated T cells on cancer cells by bridging the antigens present in each cell. Ex3 is a diabody-type BsAb composed of an anti-epidermal growth factor receptor (EGFR) antibody and an anti-CD3 antibody. In the design of Ex3, the LH-type domain order (Ex3LH) is shown to have more than 100-fold greater anti-cancer activity than the HL-type domain order (Ex3HL). To understand this phenomenon of activity enhancement by domain-order rearrangement, we report here cryoelectron microscopy (cryo-EM) structures of both Ex3HL and Ex3LH in complex with EGFR and CD3. A structural comparison of the HL and LH types reveals that the domain rearrangement leads to drastic structural changes and that the avoidance of steric hindrance by a favorable bridging angle on the cell surface is the fundamental mechanism for this activity enhancement.
History
DepositionJul 10, 2024-
Header (metadata) releaseMay 28, 2025-
Map releaseMay 28, 2025-
UpdateAug 6, 2025-
Current statusAug 6, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60767.png

Downloads & links

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Map

FileDownload / File: emd_60767.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.79 Å/pix.
x 384 pix.
= 302.592 Å		0.79 Å/pix.
x 384 pix.
= 302.592 Å		0.79 Å/pix.
x 384 pix.
= 302.592 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.788 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0165
Minimum - Maximum-0.057960916 - 0.14088903
Average (Standard dev.)0.00010101207 (±0.0024833472)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 302.59198 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A

Fileemd_60767_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_60767_half_map_2.map
Annotationhalf map B
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in t...

EntireName: HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilon
Components
  • Complex: HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilon
    • Complex: Soluble Epidermal Growth Factor Receptor (sEGFR)
      • Protein or peptide: Epidermal growth factor receptor
    • Complex: HL-type bispecific diabody Ex3
      • Protein or peptide: HL-type bispecific diabody Ex3
    • Complex: CD3 gamma-epsilon
      • Protein or peptide: T-cell surface glycoprotein CD3 gamma chain,T-cell surface glycoprotein CD3 epsilon chain

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Supramolecule #1: HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in t...

SupramoleculeName: HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilon
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Soluble Epidermal Growth Factor Receptor (sEGFR)

SupramoleculeName: Soluble Epidermal Growth Factor Receptor (sEGFR) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: HL-type bispecific diabody Ex3

SupramoleculeName: HL-type bispecific diabody Ex3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Supramolecule #4: CD3 gamma-epsilon

SupramoleculeName: CD3 gamma-epsilon / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Epidermal growth factor receptor

MacromoleculeName: Epidermal growth factor receptor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: receptor protein-tyrosine kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 69.496062 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: LEEKKVCQGT SNKLTQLGTF EDHFLSLQRM FNNCEVVLGN LEITYVQRNY DLSFLKTIQE VAGYVLIALN TVERIPLENL QIIRGNMYY ENSYALAVLS NYDANKTGLK ELPMRNLQEI LHGAVRFSNN PALCNVESIQ WRDIVSSDFL SNMSMDFQNH L GSCQKCDP ...String:
LEEKKVCQGT SNKLTQLGTF EDHFLSLQRM FNNCEVVLGN LEITYVQRNY DLSFLKTIQE VAGYVLIALN TVERIPLENL QIIRGNMYY ENSYALAVLS NYDANKTGLK ELPMRNLQEI LHGAVRFSNN PALCNVESIQ WRDIVSSDFL SNMSMDFQNH L GSCQKCDP SCPNGSCWGA GEENCQKLTK IICAQQCSGR CRGKSPSDCC HNQCAAGCTG PRESDCLVCR KFRDEATCKD TC PPLMLYN PTTYQMDVNP EGKYSFGATC VKKCPRNYVV TDHGSCVRAC GADSYEMEED GVRKCKKCEG PCRKVCNGIG IGE FKDSLS INATNIKHFK NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF ENLE IIRGR TKQHGQFSLA VVSLNITSLG LRSLKEISDG DVIISGNKNL CYANTINWKK LFGTSGQKTK IISNRGENSC KATGQ VCHA LCSPEGCWGP EPRDCVSCRN VSRGRECVDK CNLLEGEPRE FVENSECIQC HPECLPQAMN ITCTGRGPDN CIQCAH YID GPHCVKTCPA GVMGENNTLV WKYADAGHVC HLCHPNCTYG CTGPGLEGCP TNGPKIPSHH HHHH

UniProtKB: Epidermal growth factor receptor

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Macromolecule #2: HL-type bispecific diabody Ex3

MacromoleculeName: HL-type bispecific diabody Ex3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 56.781754 KDa
Recombinant expressionOrganism: Brevibacillus choshinensis (bacteria)
SequenceString: MAFAAQVQLV QSGGGVVQPG RSLRLSCKAS GYTFTRYTMH WVRQAPGKGL EWIGYINPSR GYTNYNQKVK DRFTISRDNS KNTAFLQMD SLRPEDTGVY FCARYYDDHY SLDYWGQGTP VTVSSAGGGG SDIVMTQSPL SLPVTPGEPA SISCRSSQNI V HNNGITYL ...String:
MAFAAQVQLV QSGGGVVQPG RSLRLSCKAS GYTFTRYTMH WVRQAPGKGL EWIGYINPSR GYTNYNQKVK DRFTISRDNS KNTAFLQMD SLRPEDTGVY FCARYYDDHY SLDYWGQGTP VTVSSAGGGG SDIVMTQSPL SLPVTPGEPA SISCRSSQNI V HNNGITYL EWYLQKPGQS PQLLIYKVSD RFSGVPDRFS GSGSGTDFTL KISRVEAEDV GVYYCFQGSH IPPTFGQGTK VE IKRAAAA GGGGSGGGGS GGGGSGGGGS QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMHWVRQA PGQGLEWMGN IWP GSGGTN YAEKFKNRVT MTRDTSISTA YMELSRLRSD DTAVYYCARS GGPYFFDYWG QGTLVTVSSA GGGGSDIQMT QSPS SLSAS VGDRVTITCS ASSSVSYMNW YQQTPGKAPK RWIYDTSKLA SGVPSRFSGS GSGTDYTFTI SSLQPEDIAT YYCQQ WSSN PFTFGQGTKL QITRAAAAEQ KLISEEDLNL GGGMRGSHHH HHH

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Macromolecule #3: T-cell surface glycoprotein CD3 gamma chain,T-cell surface glycop...

MacromoleculeName: T-cell surface glycoprotein CD3 gamma chain,T-cell surface glycoprotein CD3 epsilon chain
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.940545 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQSIKGNHLV KVYDYQEDGS VLLTCDAEAK NITWFKDGKM IGFLTEDKKK WNLGSNAKDP RGMYQCKGSQ NKSKPLQVYY RMGSADDAK KDAAKKDDAK KDDAKKDGSD GNEEMGGITQ TPYKVSISGT TVILTCPQYP GSEILWQHND KNIGGDEDDK N IGSDEDHL ...String:
MQSIKGNHLV KVYDYQEDGS VLLTCDAEAK NITWFKDGKM IGFLTEDKKK WNLGSNAKDP RGMYQCKGSQ NKSKPLQVYY RMGSADDAK KDAAKKDDAK KDDAKKDGSD GNEEMGGITQ TPYKVSISGT TVILTCPQYP GSEILWQHND KNIGGDEDDK N IGSDEDHL SLKEFSELEQ SGYYVCYPRG SKPEDANFYL YLRARV

UniProtKB: T-cell surface glycoprotein CD3 gamma chain, T-cell surface glycoprotein CD3 epsilon chain

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.9 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 56837
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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